Chapter 1 - Worcester Polytechnic Institute

Design of a Bioreactor to Cyclically Strain
Tissue Engineered Blood Vessels
A Major Qualifying Project Report:
Submitted to the Faculty
of the
WORCESTER POLYTECHNIC INSTITUTE
In partial fulfillment of the requirements for the
Degree of Bachelor of Science
By
______________________
_____________________
Kenneth Adams
Keith Bishop
_____________________
Elizabeth Casey
Date: April 28, 2011
Approved:
1. Mechanical Conditioning
2. Tissue Engineered Blood Vessels
3. Bioreactor
______________________________
Prof. Marsha W. Rolle, Major Advisor
Table of Contents
Table of Contents .......................................................................................................................................... 1
Authorship .................................................................................................................................................... 4
Acknowledgements....................................................................................................................................... 5
Abstract ......................................................................................................................................................... 6
Table of Tables .............................................................................................................................................. 7
Table of Figures ............................................................................................................................................. 8
Chapter 1 – Introduction............................................................................................................................. 10
Chapter 2 – Literature Review .................................................................................................................... 14
2.1 Mechanical Conditioning .................................................................................................................. 14
2.1.1 Definition ................................................................................................................................... 14
2.1.2 Impact on TEBV Structure and Properties ................................................................................. 15
2.1.3 Types and Studies Demonstrating Mechanical Conditioning Techniques ................................. 18
2.1.4 Conclusions ................................................................................................................................ 21
2.2 Bioreactors ........................................................................................................................................ 22
2.2.1. Current bioreactor designs ........................................................................................................... 23
2.2.2. Areas for improvement and design opportunities ....................................................................... 26
Chapter 3 – Project Strategy ....................................................................................................................... 28
3.1 Initial Client Statement ..................................................................................................................... 28
3.2 Objectives.......................................................................................................................................... 28
3.3 Constraints ........................................................................................................................................ 30
3.4 Revised Client Statement .................................................................................................................. 31
Chapter 4 – Alternative Designs.................................................................................................................. 32
4.1 Functions (Specifications) ................................................................................................................. 33
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4.2 Conceptual Designs ........................................................................................................................... 34
4.2.1. Mechanical conditioning system .............................................................................................. 34
4.2.2. Tissue chamber ......................................................................................................................... 37
4.2.3. Design assembly ........................................................................................................................ 39
4.3 Comparison of Design Components ................................................................................................. 41
Chapter 5 – Design Verification .................................................................................................................. 54
5.1 Syringe Selection ............................................................................................................................... 54
5.2 Quantification of Displaced Volume ................................................................................................. 56
5.2.1 Experimental Procedure ............................................................................................................ 57
5.2.2 Experimental Results.................................................................................................................. 57
5.3 Uniformity of Tubing Distension ....................................................................................................... 58
5.3.1 Experimental Procedure ............................................................................................................ 58
5.3.2 Experimental Results.................................................................................................................. 59
5.4 Tissue Ring Loading ........................................................................................................................... 59
5.4.1 Experimental Procedure ............................................................................................................ 59
5.4.2 Experimental Results.................................................................................................................. 60
5.5 Preliminary Impacts of Mechanical Conditioning ............................................................................. 60
5.5.1 Experimental Procedure ............................................................................................................ 60
5.5.2 Experimental Results.................................................................................................................. 61
5.6 Histological Analysis .......................................................................................................................... 64
Chapter 6 - Discussion................................................................................................................................. 67
6.1 Bioreactor design .............................................................................................................................. 67
6.2 Economic impact ............................................................................................................................... 68
6.3 Environmental impact ....................................................................................................................... 69
6.4 Societal influence .............................................................................................................................. 69
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6.5 Ethical concerns ................................................................................................................................ 69
6.6 Health and safety issues ................................................................................................................... 69
6.7 Manufacturability ............................................................................................................................. 69
6.8 Sustainability ..................................................................................................................................... 70
Chapter 7 – Final Design & Validation ........................................................................................................ 71
Chapter 8 – Conclusions and Recommendations ....................................................................................... 75
8.1 Design Features................................................................................................................................. 75
8.2 Future Recommendations ................................................................................................................ 75
References .................................................................................................................................................. 77
Appendix A – Pairwise Comparison Chart (PCC) ......................................................................................... 83
Appendix B – A Guide to Heat Shrink Tubing.............................................................................................. 84
Appendix C – A Guide to Wiring and Sealing Motors ................................................................................. 86
Potting and Sealing Motors .................................................................................................................... 86
Wiring Motors to Power Supply.............................................................................................................. 88
Appendix D – Building our Device ............................................................................................................... 93
Appendix E – User Manual .......................................................................................................................... 97
Prepare autoclaved tools & materials: ................................................................................................... 97
Non-autoclaved, sterile materials:.......................................................................................................... 97
Tools & materials cleaned with 70% ethanol solution: .......................................................................... 97
Incubator Set-up Procedure:................................................................................................................... 98
Ring Loading onto Silicone Tube Procedure: .......................................................................................... 99
Tissue Chamber Loading Procedure: .................................................................................................... 101
Appendix F – Bill of Materials ................................................................................................................... 108
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Authorship
The final version of this paper was written in equal parts by all group members. All group members were
involved in editing all sections of the paper.
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Acknowledgements
We thank our advisor, Marsha Rolle, for her guidance and support throughout our project. We would
like to thank Lisa Wall for her continuous support in the acquisition of lab materials and equipment. We
gratefully acknowledge Tracy Gwyther for her guidance and aid in data collection and user feedback
during advisor meetings. We’d like to thank Craig Jones and Jason Hu for their assistance with lab
equipment and DVT, respectfully. Many thanks to Michael Fagan for his help machining our device and
sealing the motors for incubator use. Thanks are also due to Patrick Brodeur for his assistance wiring our
electrical components. We would like to thank Jack Ferraro and Douglas White for use of the Goddard
machine shop. We would also like to thank Sakthikumar Ambady for sharing his knowledge of cell
culture techniques and proper use of lab equipment. Finally, we would like to thank James O’Rourke and
David Comeau (President, Albright Technologies) for their aid in the design process.
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Abstract
Cyclic mechanical loading improves strength and extracellular matrix (ECM) synthesis in tissue
engineered blood vessels (TEBV). The goal of this project was to design a device to impart cyclic
circumferential stretch on cell-derived TEBV rings cultured on flexible silicone tubing. Our device
cyclically displaces water (75 μl volume) to inflate tubing at a frequency of 1 Hz. During static inflation
tests, the tubing diameter increased by 10±1.6%. TEBV rings were loaded onto silicone tubing and
subjected to mechanical conditioning for 3-7 days. Our device conditioned samples in an incubator at
10% distension at a frequency of 1 Hz. Static and conditioned tissues remained viable and
uncontaminated and exhibited high cell densities and increased thicknesses from 3 to 7 days.
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Table of Tables
Table 1. Characteristics of engineered vessels in Niklason's 1999 study. .................................................. 20
Table 2. Client and Team PCC Comparison ................................................................................................. 30
Table 3. Morphological Chart ..................................................................................................................... 34
Table 4. Summary of syringe characteristics .............................................................................................. 56
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Table of Figures
Figure 1. Forces experienced within natural blood vessels ........................................................................ 14
Figure 2. Bioreactor system used by Seliktar (adapted from 2000) ........................................................... 23
Figure 3. Kelm bioreactor (adapted from 2010) ......................................................................................... 24
Figure 4. Ali bioreactor system (2009) ........................................................................................................ 26
Figure 5. Air pressurization system (Ali, 2009) ........................................................................................... 32
Figure 6. Peristaltic pump ........................................................................................................................... 35
Figure 7. OctoPump .................................................................................................................................... 35
Figure 8. Wheel and arm motor.................................................................................................................. 36
Figure 9. Solenoid flexion device ................................................................................................................ 36
Figure 10. Motor and cam system .............................................................................................................. 37
Figure 11. Double threaded cartridge......................................................................................................... 38
Figure 12. Removable compartment assembly .......................................................................................... 38
Figure 13. Milled tissue chamber ................................................................................................................ 39
Figure 14. Wheel and arm assembly ........................................................................................................... 40
Figure 15. Motorized pyramid assembly and pinch clamp (ZManCorp.com)............................................. 40
Figure 16 Motor-cam series ........................................................................................................................ 41
Figure 17. Solenoid actuator (Edited from tpub.com) ................................................................................ 43
Figure 18. 12V DC 60 RPM high torque gear box electric motor ................................................................ 44
Figure 19. Individual removable chambers on a removable tray ............................................................... 45
Figure 20. Cam and flat arm design for motor-syringe attachment ........................................................... 46
Figure 21. Various syringe tips. ................................................................................................................... 47
Figure 22. Syringe-silicone tubing connectors ............................................................................................ 48
Figure 23. Threaded syringe-silicone tubing connectors. ........................................................................... 49
Figure 24. Using sutures and silicone glue to seal syringe tips to silicone tubing ...................................... 50
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Figure 25. Pinch clamp from World Precision Instruments and Z-Man Corp ............................................. 51
Figure 26. Ultra-high molecular weight polyethylene and polycarbonate ................................................. 52
Figure 27. 3mL luer-lock syringe, Luer-slip vs. luer-lock syringe tips .......................................................... 55
Figure 28. 1 mL push-connect or luer-slip syringe ...................................................................................... 55
Figure 29. Syringe and tubing assembly. .................................................................................................... 57
Figure 30. Required volume to achieve 10% distension ............................................................................. 58
Figure 31. Experimental set-up for uniformity trials. ................................................................................. 58
Figure 32. Uniformity results ...................................................................................................................... 59
Figure 33. Six tissue rings loaded onto silicone tubing. .............................................................................. 59
Figure 34. Tissue rings mounted on silicone tubing and loaded into the tissue chambers........................ 60
Figure 35. Ultimate tensile strength first trial. ........................................................................................... 61
Figure 36. UTS second trial ......................................................................................................................... 62
Figure 37. Thickness first trial ..................................................................................................................... 63
Figure 38. Thickness second trial ................................................................................................................ 64
Figure 39. Conditioned and static tissue rings on silicone tubes after 7 days of culture. .......................... 64
Figure 40. Tissue samples stained with hematoxylin and eosin at 20x magnification ............................... 65
Figure 41. Tissue samples stained with Fast Green/Picrosirius Red at 40x magnification ......................... 66
Figure 42. Final design (CAD) ...................................................................................................................... 71
Figure 43. Final design ................................................................................................................................ 72
Figure 44. Mechanical stimulation.............................................................................................................. 73
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Chapter 1 – Introduction
Most current technologies used to develop small diameter tissue engineered blood vessels (TEBVs) do
not meet the clinical needs for a mechanically sound graft without extensive time and conditioning in
culture. Ideally, autologous tissue engineered vessels or grafts would be cultured in vitro then applied in
vivo. This concept has motivated researchers to study engineered vessels, and is the basis for this
project. There are currently a variety of methods for the development of small diameter vasculature;
however, many current technologies present a number of concerns and limitations.
Originally, engineers developed synthetic grafts made from materials such as Dacron and
polytetrafluoroethylene (Zacharias et. al., 1987) in hopes of creating a mechanically sound replacement
for human tissue. These grafts however are limited to vessels with larger diameters. In vessels smaller
than 6 mm in inner diameter, the synthetic grafts often failed due to thrombosis (Atala et. al., 2008).
Thrombosis is the formation of blood clots that occlude the vessel. The synthetic materials used in the
vascular replacement procedures often initiated an immune response. This response would result in
high levels of inflammation and failure of the graft (O’Donnell et. al., 1984). In an attempt to improve
the biocompatibility of these materials, scientists have researched biomaterials that, when combined
with autologous tissues, could result in biointegration of the graft (Mitchell et. al., 2003). The current
standard for vessel replacement is the CABG, or coronary artery bypass graft, method. The CABG
procedure requires a type of graft called an autologous graft. These grafts are sections of blood vessels
less than 6 mm inner diameter, such as the saphenous vein, which are harvested from the patient. The
graft is then implanted into the coronary circulation system to redirect flow past the damaged or
occluded vessels. This approach is viable for ten to twelve years; any longer could result in narrowing of
the vessel, resulting in a need for a second surgery. (L'Heureux, 2003; Goldman et. al., 2004)
Tissue engineered blood vessels are a potential solution to the complications that arise from the use of
the previously mentioned vascular repair approaches. TEBVs are constructed by assembling cells taken
from the patient into tubular constructs. These constructs can then be implanted into the patient, and in
theory, should have mechanical properties similar to healthy tissue. Two-dimensional models, such as
skin grafts, have been successfully created; three dimensional models are currently limited due to the
samples’ poor structural integrity and low mechanical strength. In order to be deemed successful, TEBVs
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should be able to withstand physiological loads from blood pressure and fluid dynamics within the body.
Additionally, the samples must be able to resist tearing, especially during suturing and handling.
(Mitchell et. al., 2003)
In 2003, a tissue engineering technique was patented that used a cell sheet-based method that cultured
fibroblasts and smooth muscle cells (SMCs) around a cylindrical mandrel. The result was a fully
autologous graft that was capable of withstanding mechanical loads greater than that of blood pressure
(over 2200 mm Hg) and was tear-resistant. Since the graft was composed of living cells, it was selfrenewing resulting in an increased healing potential. Additionally, the graft was completely biological
and could be remodeled by the body during the healing process. Remodeling is a response of the cells to
the mechanical manipulations enacted by the surrounding environment. (L'Heureux, 2003)
Even though the tissue-based grafts greatly improved on past technologies, concerns and limitations
remain. This approach used SMCs to improve the graft’s strength. However, while the addition of these
cells increased the strength they limited the graft’s ability to expand and contract. Postoperative studies
showed that while implanted, in vivo remodeling of the graft resulted in higher levels of elastin
(L'Heureux, 2003). Elastin is a protein that acts like a rubber band as it contracts the vessel. In order to
overcome the stiffness resulting from high levels of SMCs and collagen, it is vital to have enough elastin
so that the vessel can return to the original diameter during pulsatile and pressurized loads. The elastin
network also contributes to the long-term success of tissue engineered grafts. If a graft is too stiff, there
will be a difference between the natural tissue and the graft causing the fluid dynamics to change, thus
impacting the stream of fluids as they pass through the engineered vessel. The increased failure rate due
to compliance differentials is seen with synthetic materials as well (Mitchell et. al., 2003). An additional
concern with the application of SMCs as a means of structural support is that it remains difficult to
regulate their proliferation once implanted. If the cells proliferate excessively, the graft can actually
occlude itself resulting in failure. (L'Heureux, 2003)
Collagen networks are the natural means of vasculature support and can be induced in TEBVs in
appropriate culture environments. The use of collagen gel-based grafts has been studied as a potential
means for the reduction of the dependency on SMCs. The collagen gel is easier to control and improves
the strength of the graft. Similar to the elastin networks, remodeling enzymes promote the synthesis of
these networks. (Mauck et. al., 2009)
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In a 2009 study which compared the effectiveness of TEBVs to that of autologous grafts of saphenous
and mammary arteries, cyclic strains were proven to increase the production of collagen networks,
which is indicative of higher tensile strength. This study specifically examined burst pressure, suture
retention strength, and compliance of mechanically conditioned TEBVs in comparison to native
saphenous veins. (Konig et. al., 2009)
The TEBVs were superior to the autologous grafts in burst pressure and suture retention, but the
compliance ratios of the TEBVs were much lower. The long-term study showed that the cells appeared
to react to their environment and maintain mechanical homeostasis. After six months of implantation,
the TEBV compliance ratio increased by over 100%, whereas the native tissue samples remained
constant. This increase in compliance validates the notion that a tissue sample exposed to mechanical
manipulation is capable of remodeling and improving its mechanical properties. (Konig et. al., 2009) In
this instance, the body functions as the ideal bioreactor, conditioning the TEBVs through physiological
cardiac loading while maintaining cell viability. This response to physiological mechanical environments
provides insight into how mechanical conditioning during culture could result in an improved graft. If
cells respond to in vivo mechanical manipulation, they should also respond to in vitro loading assuming
appropriate culturing techniques are maintained. These outcomes acted as the inspiration for the
development of the in vitro mechanical loading model presented in this paper.
The Rolle Lab at Worcester Polytechnic Institute (WPI) uses tissue engineered blood vessel rings to
model the mechanical and physiological properties of complete blood vessels. Rat aortic smooth muscle
cells (SMCs) are grown in agarose molds over 8 days to form completely cell-derived, three-dimensional
rings with an inner diameter of 2, 4, or 6 mm. It has also been shown that rings cultured in close
proximity for an extended period of time could fuse to form a single cohesive tube, demonstrating
possible future applications for in vivo studies. Furthermore, the rings’ relatively small size and short
production time allowed us to experiment with greater sample sizes. (Gwyther et. al., 2011)
The goal of this project was to design a bioreactor that would impart mechanical conditioning on 2 mm
inner diameter tissue rings from the Rolle Lab. The bioreactor also had to be capable of maintaining
basic cell culture conditions. It was then our responsibility to conduct an in-depth literature review in
order to identify the limitations of bioreactors used to mechanically condition tissue constructs. As we
developed an understanding of the problem, we began compiling a list of objectives and constraints.
Using a collection of client interview data, we applied these objectives and constraints and created a list
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of functions and means for our device. These means were then analyzed based on client input, material
biocompatibility and manufacturability, and projected expenses, and several design alternatives were
created. The designs were further scrutinized based on the plausibility of component interfaces and the
availability of materials and manufacturing tools. We were able to determine our final design, which we
prototyped and evaluated through bench top testing and culture experiments.
Additionally, sections of this paper examine the effects our device may have on the environment and
the economy. Finally, future recommendations were made so that future teams could learn from our
results and conclusions and further develop the device.
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Chapter 2 – Literature Review
2.1 Mechanical Conditioning
2.1.1 Definition
In order for tissue engineered blood vessels (TEBVs) to function successfully, they must have high
strength and endurance (Butler et. al., 2009) capable of withstanding the natural forces of the human
body. These forces include shear stresses due to blood flow, vessel wall expansion and contraction due
to pressure, as well as longitudinal tension along the vessel (Isenberg et. al., 2006). A diagram of these
forces is pictured in Figure 1. The goal of mechanical conditioning of tissue engineered blood vessels is
to mimic these natural in vivo forces while TEBVs are growing in vitro.
Figure 1. Forces experienced within natural blood vessels
Mechanical conditioning is a widely used method for treating engineered tissue constructs to enhance
their physical and mechanical properties. Techniques for creating artificial tissues have used various cell
types, such as smooth muscle and endothelial, as well as differing culture conditions to create grafts
that emulate native tissue (Isenberg et. al, 2006; Isenberg et. al., 2003).
Cells are affected by an immense variety of chemical and mechanical stimuli. Bone and muscle cells in
particular are known to respond to these mechanical stresses in a positive way (Neidlinger-Wilke et. al.,
1995), constantly remodeling based on the stresses they are exposed to. Mechanical stretching of
smooth muscle cells (SMCs) cultured in collagen-based tissue equivalents has been shown to have
immense effects on SMC orientation (Dartsch et. al., 1986; Kanda et. al., 1993; Liu, 1998), ECM
deposition (Chiquet et. al., 1996; Kolpakov et. al., 1995; Kim et. al., 1999; Niklason et. al., 1999; Kulik et.
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al., 1993), and release of cellular growth factors (Cheng et. al., 1997; Sudhir et. al., 2001). Liu’s 1998
study found that under circumferential strain, rat blood vessel SMCs arrange themselves in the
circumferential direction. Kim (1999) found that cyclic mechanical strain (7% distension at 1 Hz for 4
days) applied to smooth muscle tissue grafted onto polymeric scaffolds increased SMC proliferation and
collagen and elastin expression. This resulted in enhanced mechanical properties and long term tissue
organization. Cheng (1997) found that mechanical strain influences fibroblast growth factor-2 (FGF-2)
release from human SMCs. As the amplitude of deformation rose, more FGF-2 was released in response;
from 4% amplitude (90 cycles at 1 Hz) releasing 0.1±0.1% FGF-2 to 14% (90 cycles at 1 Hz) releasing
5.7±0.5% FGF-2.
In order for engineered musculoskeletal, vascular, and cardiac tissues to function successfully without
failure in vivo, mechanical modulation must be considered. Ideally, a combination of mechanical and
biochemical factors will lead to engineered constructs whose functionality and mechanical properties
closely mimic those of native tissue. Perfecting mechanical conditioning techniques is of the utmost
importance for cardiovascular tissue engineering before in vivo applications can be seriously considered
(Rubbens et. al., 2009; Butler et. al., 2009). Mechanical forces are becoming as important as growth
factors or cytokines in the overall growth and development of TEBVs. (Freed et. al., 2006)
2.1.2 Impact on TEBV Structure and Properties
1. Benefits of Mechanical Conditioning
Naturally occurring vascular SMCs are not exposed directly to shear stress by normal blood flow, they
are primarily affected by pulsatile distension during the cardiac cycle (Isenberg et. al., 2003). Due to this,
tissue engineered blood vessels are primarily conditioned based on the dominant stress encountered in
vivo, cyclic mechanical distension. Studies have shown that cyclic mechanical distension strengthens
engineered vessels (Niklason et. al., 1999; Freed et. al., 2006; Isenberg et. al., 2006; Syedain et. al., 2008;
Seliktar et. al., 2000; Huynh et. al., 2010) and promotes collagen production and cell alignment (Niklason
et. al., 1999; Freed et. al., 2006; Isenberg et. al., 2003; Schutte et. al., 2010; Seliktar et. al., 2000). It also
has enormous beneficial effects on SMC phenotype, ECM deposition, growth factor release, and
vascular tone (Isenberg et. al., 2003). Biomechanical factors influence tissue growth (Isenberg et. al.,
2003; Niklason et. al., 1999; Syedain et. al., 2008), development (Guilak et. al., 1997; Niklason et. al.,
1999), maintenance (Butler et. al., 2009; Syedain et. al., 2008), degeneration (Butler et. al., 2009;
Fujiwara et. al., 2003), and repair (Cheng et. al., 1997; Fujiwara et. al., 2003).
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Cells receiving mechanical stimuli will align themselves in the direction of the stretch or flow to combat
vessel weakness and increase cell length. Weakness could occur when cells are aligned in a random
pattern within the wall of a vessel versus aligning along the direction of blood flow. Collagen and elastin
fibrils will similarly align in a more compact manner in the direction of tension, increasing vessel
strength and stiffness (Isenberg et. al., 2003; Tower et. al., 2002). TEBVs also respond to cyclic distension
by producing more collagen and elastin, changing the composition of the extracellular matrix and
further strengthening and stiffening vessel walls. Mechanical stretching has been shown to promote the
expression of type I and III collagens, fibronectin, and tenascin-C in cultured ligament fibroblasts (Sahoo
et. al., 2007).
Mechanical properties can be engineered to match those of natural blood vessels. The burst pressure of
a saphenous vein typically used in coronary artery bypass grafts in patients with heart disease is 1680 ±
307 mmHg. (Konig et. al., 2009) A tissue engineered blood vessel cultured for 7 weeks with the same
diameter and length as the tested saphenous vein had a burst pressure of 2594 ± 501 mmHg (Konig et.
al., 2009). Vascular compliance ( ; ΔV=change in volume, ΔP=change in pressure), stiffness ( ; F=force
applied, δ=displacement produced by force), tensile strength (calculated based on uniaxial tensile
testing, ultimate tensile stress, UTS, at failure), and burst pressure (
; t=wall thickness (in),
S=tensile strength of blood vessel (PSI), O=outer diameter (in)) can be altered and potentially controlled
for based on the frequency and force used during conditioning.
Compliance and stiffness are important properties contributing to overall TEBV health. Compliance
refers to the ability of a blood vessel to recoil to its original shape after deformation by force. Stiffness is
the blood vessels resistance to this deformation. Tensile strength is the maximum stress that a blood
vessel can handle in a 2-D plane before failure. Burst pressure denotes the maximum stress that a blood
vessel can handle in a 3-D plane before bursting during pressurization. Vascular stiffness correlates with
increased cardiovascular risk (DeLoach et. al., 2008). By controlling these parameters, tissue engineered
blood vessel properties can be catered to the needs of individual patients by altering the specific
vascular type and size and decreasing susceptibility to cardiovascular disease.
Increased ECM synthesis is a positive effect of biomechanical conditioning (Mauck et. al., 2000; Freed et.
al., 2006; Buschmann et. al., 1995; Ku et. al., 2006; Niklason et. al., 1999; Seliktar et. al., 2000; Syedain
et. al., 2009). Mauck (2000) studied dynamically loaded tissue engineered articular cartilage seeded on
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agarose gels. Tissue disks were dynamically compressed at 10% strain at a frequency of 1 Hz; at a duty
cycle of 1 hour on, 1 hour off per day for 5 days a week and 4 weeks total. Mauck (2000) found a sixfold
increase in equilibrium aggregate modulus over controls and matrix elaboration due to heightened ECM
synthesis over time for conditioned samples.
Syedain (2009) created tissue engineered heart valves (TEHVs) by entrapping dermal fibroblasts into
molded fibrin gels left to statically incubate for 2 weeks. These TEHVs were then strained incrementally
at 5% (1st week), 10% (2nd week), and 15% (3rd week) over a 3 week period in a custom-built bioreactor
at a frequency of 0.5 Hz. Syedain found that after cyclic stretching, collagen and cellular alignment was
primarily circumferential, which correlates with native TEHVs. Collagen also appeared more organized
into mature bundles at a density 86% greater than static TEHVs. Tensile strength and elastic modulus
were also improved by over 97% and 77%, respectively, compared to static TEHVs.
Mechanical properties of tissue engineered constructs (such as tensile strength, elastic modulus, and
vessel stiffness) have been improved by mechanical conditioning (Hahn et. al., 2007; Diamantouros et.
al., 2009; Freed et. al., 2006; Fujiwara et. al., 2003; Gimbronejr et. al., 1999; Isenberg et. al., 2006;
Isenberg et. al., 2003; Konig et. al., 2009; Ku et. al., 2006; Niklason et. al., 1999; Schutte et. al., 2010).
Hahn (2007) used mouse smooth muscle progenitor cells within poly(ethyleneglycol)-based hydrogels
with adhesive ligands and a collagenase degradable sequence to create 3mm inner diameter tissue
engineered vascular grafts. These grafts were subjected to 7 weeks of mechanical conditioning under
pulsatile flow at 2 mL/sec. Results showed significantly higher collagen levels (resulting in higher tensile
strength) and improved elastic moduli compared to static samples.
Overall, TEBVs show much promise as alternatives to autografting for vascular replacements based on
their burst strength, suturability, and mechanical similarities to native vessels.
2. Drawbacks of Mechanical Conditioning
Mechanical conditioning in culture is a difficult challenge. Systems have been developed which apply
mechanical forces via piston/compression systems, substrate bending, hydrodynamic compression and
fluid shear (Dobson et. al., 2006). Problems exist with these systems, namely, long term sterility and
cells receiving adequate nutrients through the thickness of the vessel. In systems that use scaffolded
tissue constructs, the scaffold takes on a portion of the mechanical load. Therefore, the tissue in culture
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does not receive the full impact of the mechanical conditioning and as a result the production of ECM is
hindered. (Dobson et. al., 2006)
Studies have shown that excessive cyclic stretch is detrimental to cells (Fujiwara et. al., 2003; Gassman
et. al., 2010). Fujiwara (2003) modeled this phenomenon using endothelial lung tissue. Lung tissue
naturally stretches by an estimated 5% during normal breathing in vivo, but anything higher than 1722% may damage the endothelial cells of the lungs. The key is to condition cells at an “ordinary”,
tolerable percentage that won’t over-stimulate or harm them (Fujiwara et. al., 2003). Despite these
limitations, mechanical conditioning remains a widely used method to prepare tissue engineered
constructs for the rigorous environment of the human body as it improves ECM synthesis, cellular
alignment, and mechanical properties.
2.1.3 Types and Studies Demonstrating Mechanical Conditioning Techniques
Several different mechanical conditioning techniques have been shown to alter the mechanical and
physiological characteristics of tissues. These techniques include magnetic stress, dynamic compressive
loading, flow-mediated shear stress, continuous mechanical tension, stretching on membranes, and
cyclic mechanical distension. Different tissue types require different mechanical stimuli to produce
tissues with specific characteristics. For instance, mechanical conditioning that focuses on applied
compressive forces would enhance a tissue’s compressive strength, while mechanical tension or
stretching would improve elasticity and compliance. Cyclic mechanical distension is the type of
conditioning the team chose to use for the purposes of our project.
1. Cyclic Mechanical Distension
A study conducted in 2000 by Seliktar cyclically strained collagen-gel blood vessel constructs composed
of adult rat aortic SMCs mounted on etched silicone sleeves (coated in type I collagen and chitosan). The
sleeves and constructs were sutured into in a sterile, air-tight bioreactor where media was added and
0.2 µm filters served as vents for gas exchange. The tissue constructs were cycled within an incubator at
a frequency of 1 Hz and 10% cyclic strain for 4 and 8 days. The silicone sleeve was distended using a
regulated compressed air supply and a solenoid valve located outside the incubator.
The vessels showed increased contraction and mechanical strength when compared to statically grown
control constructs. The vessels conditioned for 8 days showed significant increases in ultimate tensile
stress (the maximum load divided by the initial cross-sectional area of a sample) and material modulus
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(stress divided by strain). Morphologically, the mechanically conditioned vessels showed higher
circumferential orientation of the vessel cells. (Seliktar et. al., 2000)
Another study focused on the effects of applied stretching on rabbit pulmonary arteries. They found
that after four days of stretching, the amount of pro-collagen type I-positive cells grew, along with
higher levels of protein synthesis within the smooth muscle cells (SMCs) and cell replication within the
construct. (Kolpakov et. al., 1995) A similar study by Liu in 1998 used rat blood vessels. They discovered
that continuous tensile strain on the vessels tested in 10 day increments over a period of 30 days
resulted in better alignment of the SMCs. (Liu, 1998)
Brett Isenberg and Robert Tranquillo (2003) studied the effects of long-term cyclic distension on
collagen-based media equivalents (denatured, cross-linked type I collagen) consisting of adult rat aortic
SMCs. The SMCs were grown and incubated on Teflon rod mandrels in standard cell culture medium,
changed once per week. The tissue constructs themselves were 1-3 mm in length, 0.2-0.3 mm thick, and
8 mm in inner diameter. The tissue constructs were conditioned on distensible latex mandrels inflated
by pressurized air within an incubator
The collagen-based media equivalents (MEs) were strained incrementally to the target percentage over
3 days and remained at that frequency (0.5 Hz) and strain level (12.5%) for 5 weeks. The tissues were
tested for various mechanical properties and collagen content. They found that mechanical conditioning
for 2 weeks yielded little change in elastic modulus (E) or ultimate tensile strength (UTS), but
conditioning for 5 weeks resulted in significant increases in both. Isenberg and Tranquillo found that cell
age has no influence on response to stimulation and that 2.5% and 5% strain values are best for high
UTS. They also found that a 10% strain value during mechanical conditioning is best for obtaining a high
elastic modulus and higher elastin content within samples. This study failed to confirm whether or not
an increase in cell number, collagen content, or fibril alignment was present in their tissue constructs as
many other studies have claimed to have found. (Isenberg et. al., 2003)
Niklason (1999) used pulsatile radial flow to cyclically distend silicone tubing loaded with PGA scaffolds
seeded with neonatal SMCs at 2.75 Hz and 5% strain for 8 weeks. Niklason argued that pulsatile flow
conditions (165 beats per minute and 5% radial distension) would more accurately represent conditions
seen in vivo, and thus produce vessels better suited for eventual in vivo use. This study resulted in SMCs
with an increase in modulus, collagen and elastin deposition, suture retention, and calponin and myosin
19 | P a g e
heavy chain (contractile components). Vessel wall thickness was also increased with longer culture time
and pulsatile culture conditions. After treating the conditioned SMCs with prostaglandin F2α, the
structure physically contracted as a natural blood vessel should. Table 1 summarizes the different
characteristics measured in Niklason’s study. (Niklason et. al., 1999)
Table 1. Characteristics of engineered vessels in Niklason's 1999 study.
2. Other Types of Mechanical Conditioning
Magnetic stress, dynamic compressive loading, flow-mediated shear stress, continuous mechanical
tension, and stretching on membranes are all effective methods commonly used in the tissue
engineering community to alter the physical and mechanical properties of tissue engineered constructs.
Jon Dobson (2006) developed and prototyped a unique method to mechanically condition stem cells in
vitro using biocompatible magnetic nanoparticles (MNPs) and ion channels present within the stem cells
themselves. Dobson’s device applies stress to the cell membrane using forces applied directly to
magnetic micro- and nanoparticles attached to the cell membrane via surface receptors. Quantitative
results of this bioreactor system’s effects on tissue mechanical properties do not yet exist, but are
currently being modeled in vitro. (Dobson et. al., 2006)
Dynamic compressive loading is another effective means of conditioning cell constructs. Ballyns (2010)
investigated the effects of dynamic compression loading on engineered bovine meniscal
fibrochondrocytes seeded and crosslinked in alginate hydrogels and shaped as menisci. A custom
bioreactor applied sinusoidal displacement in alternating one hour increments 3 times a week for 6
weeks total. After 2 weeks, collagen content in conditioned samples increased 1.8 fold, compressive
20 | P a g e
modulus by 1.8-2.3 fold, and ECM content by 2-3.2 fold compared to static samples. (Ballyns et. al.,
2010)
Flow-mediated shear stress is another method. In vitro models are limited in nutrient delivery due to a
lack of blood supply. All nutrient/media delivery must occur through diffusion. Dynamic media flow
within or around tissue-engineered constructs offer the best solution to enhance media delivery and
waste exchange. They also simultaneously deliver flow-mediated shear stresses to cells seeded within
the constructs, improving cell strength and structure (Butler et. al., 2009). Fluid shear stress at
physiological (rest) conditions is estimated to be 1-50 dyn/cm2 in human arterial endothelial cells
(Fujiwara et. al., 2003).
A recent study has demonstrated that placing axons under continuous mechanical tension increases
axon growth rates up to 1cm/day. This was accomplished by using a microstepper motor system to
incrementally separate two neural membranes, allowing for spontaneous growth of axons from one
membrane to another. This research is useful in the repair of nerve injuries using transplantable neurons
with 10cm long axons for central nervous system repair. (Butler et. al., 2009; Pfister et. al., 2006; Smith
et. al., 2001)
Research was conducted concerning the effects of mechanical stretching on collagen synthesis by
mesenchymal stem cells and aortic valve interstitial cells. Ku (2006) found that at 14% stretch there was
a significant increase in collagen synthesis in both types of cells, directly affecting the rate of ECM
production and valve strength. Collagen synthesis was dependent on the degree and duration of stretch
in aortic valve tissue. (Ku et. al., 2006) In another study, rat vascular smooth muscle cells were subjected
to cyclic strain induced by a vacuum below the culture plates the cells were grown on. Cyclic strain
caused an increase in total cell count by 40% compared to control tests. (Wilson et. al., 1993)
2.1.4 Conclusions
The ultimate goal of blood vessel engineering is to create a nonthrombogenic, nonimmunogenic,
suturable tissue that is able to withstand arterial pressures, is compliant, and can be remodeled in
response to injury (Schutte et. al., 2010). Mechanical stimulation improves the overall structure and
function of native and tissue engineered vasculature alike; this is imperative for achieving these goals.
Biomechanical forces have been shown to enhance the physical characteristics of TEBVs. Mechanical
cues cause change in cell morphology, physiology, biochemistry, and gene expression, all of which
21 | P a g e
contribute to overall tissue function in vivo. (Butler et. al., 2009; Diamantouros et. al., 2009; Freed et. al.,
2006; Isenberg et. al., 2003; Niklason et. al., 1999; Seliktar et. al., 2000; Syedain et. al., 2009) Cyclic
mechanical distension of tissue engineered constructs has been shown to increase contraction and
mechanical strength (Seliktar et. al., 2000; Isenberg et. al., 2003), increase circumferential cellular
alignment along the vessel length (Seliktar et. al., 2000; Liu, 1998), and increase ECM synthesis and cell
growth (Kolpakov et. al., 1995).
Mechanical conditioning has become a major goal of recent tissue engineering efforts and has led to a
shift in some of the primary bioreactor design goals. Bioreactors have evolved from acting solely as
culturing devices to culturing while simultaneously conditioning tissue constructs.
2.2 Bioreactors
Bioreactors are biomimetic laboratory devices that regulate a number of environmental factors in order
to create an optimal platform for biological activity. In tissue culturing and engineering, a regulated
environment is crucial in order to promote cell growth and tissue structure and mechanics; bioreactors
can be designed to control variables including pH, temperature, hydration, nutrition, and waste
regulation and removal. Many of these conditions may be provided by an incubator. In the past decade,
the focus of these bioreactors has expanded to include devices that induce mechanical stimulation,
which improves compositional and mechanical properties.
The following sections will profile several published bioreactor designs and experiments, which will help
in identifying design intent, areas for improvement, and potential design opportunities.
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2.2.1. Current bioreactor designs
1. Seliktar’s cyclic strain bioreactor
Figure 2. The Seliktar team’s final bioreactor design houses the tissue construct on a silicon tube as an external pneumatic
control device imparts cyclic strain. Two filters installed into the device allow for gas exchange. (adapted from Seliktar et. al.,
2000.)
Seliktar recognized the importance of mechanically conditioning TEBV constructs in order to improve
their ECM production and mechanical properties. His team cultured adult rat aortic SMCs embedded in a
collagen gel scaffold; once they were mechanically stable, the tissue constructs were transferred into a
sterile bioreactor that interfaced with a controlled pneumatic flow loop to impart cyclic strain on the
tissue samples.
Seliktar’s design (Figure 2) consisted of a bioreactor chamber that contained an inlet and outlet port to
allow for the mechanical flow. The team’s TEBVs were installed on a flexible silicone sleeve and fastened
to the ports using sterile sutures. Gas exchange was made possible by two 0.2 µm filters that were
installed into the top wall of the chamber. An external compressed air system connected to the
bioreactor’s inlet and outlet ports using solenoid valves, and provided 5% and 10% cyclic strain to the
inner silicon tubing. (Seliktar et. al., 2000)
Seliktar’s design yielded TEBVs that exhibited improved cellular alignment and high collagen expression,
but experienced large decreases in volume, length, and wall thickness. Still, despite the changes in size,
the realignment of cells and collagen in response to mechanical conditioning yielded great increases in
23 | P a g e
mechanical integrity. Samples conditioned for only four days exhibited an 87% higher yield stress and
eight day samples showed a 200% higher yield stress than static samples. (Seliktar et. al., 2000)
2. Kelm improves production time using microtissues
In 2009, Kelm sought to build a bioreactor system that would yield healthy, scaffoldless, small-diameter
TEBVs while reducing the production time of previous designs. The team used myofibroblasts and
endothelial cells to construct microtissues as an initial platform for the tissue culture. Kelm suggests that
microtissues produce ECM quicker and more efficiently. This natural tendency to produce ECM,
combined with pulsatile mechanical conditioning of the tissue, would reduce the required maturation
time of the cells, and therefore expedite production of mature tissues. (Kelm et. al., 2010)
Figure 3. A cross section of the tissue mount, the Falcon tube housing unit, and the path of media flow (adapted from Kelm
et. al., 2010)
Kelm’s bioreactor design consists of three main components: a pulsatile pump, a medium reservoir, and
a structural unit. The pulsatile pump used is interfaced with control unit powered with a 1 bar inlet
pressure; it uses cell media as a flow medium. The two stated objectives of the novel assembly device
are to grow the microtissues into a three-dimensional tubular shape and to enable circumferential
mechanical stimulation. This assembly device is housed in a 50 mL falcon tube with a custom-made
stainless steel cap. The assembly component is interfaced with the rest of the system through the steel
24 | P a g e
cap using silicon tubing (Figure 3). Finally, a nutrient medium reservoir is used as a junction between the
assembly device and the pump to allow recirculation of the flow medium.
Kelm’s design demonstrates that self-assembling microtissue constructs in conjunction with a pulsatile
flow loop accelerates ECM production and tissue maturation, as the tissues showed very high collagen
expression after only 14 days. This novel concept brings the industry one step closer to the quick and
efficient production of autologous TEBV constructs. (Kelm et. al., 2010)
3. Syedain enhances ECM composition
Syedain’s team has developed a bioreactor that imparts cyclic mechanical strain on tissue engineered
heart valves in an attempt to optimize compositional properties. Syedain’s design consists of a latex
tube which houses the tissue engineered heart valves. The valves receive mechanical stimulation
through a cell media flow via a syringe pump that has been custom-made for this system. A needle valve
assists in redirecting the fluid back through the syringe. Media is also supplied to the latex chamber
through a perfusion loop that is driven by a MasterFlex peristaltic pump. Using a second loop for media
exchange allowed the mechanical frequencies and strains to be adjusted without completely altering the
nutrition and oxygen supply to the tissue. (Syedain et. al., 2009)
Syedain’s bioreactor yielded tissues that exhibited homogeneous cellularity, which proves that all of the
cells were well-nourished and that the dual-flow loop system was successful. Furthermore, the
conditioned heart valves expressed 86% more collagen than static control samples, but still fell short by
37% in comparison to native heart valves. (Syedain et. al., 2009)
4. Current device used in the Rolle lab
The current technology used in the Rolle lab at WPI consists of a polystyrene BD Falcon conical tube
(Figure 4 - A) as a tissue chamber and housing unit for a flexible silicone tube on which the vascular
constructs are installed. The cap of the tube has been modified in order to contain a threaded barb,
which acts as a platform for the junction between the compressed air chamber (which provides the
mechanical loading) and the bioreactor cartridge. The cap system is also fixed with a heat sleeve and
anti-leak o-rings in order to ensure the security of the cartridge. Furthermore, an air pressure fitting has
been installed into the cap. The final piece of the cap system is a sterile air filter that allows for gas
exchange as the tissues are being cultured and conditioned. The bottom of the silicone tubing contains a
25 | P a g e
needle end cap, onto which a small weight is secured. This grounds the tubing and, consequently, the
TEBVs, and helps control the mechanical distension of the system. (Ali et. al., 2009)
One unique feature of the air pressurization design in comparison with the other profiled current
A
B
Figure 4. The Ali design uses tube as a tissue chamber, and allows up to eight isolated bioreactor systems to operate at one
time. (Ali et. al., 2009)
technologies is the high throughput that it allows. (Figure 4 – B) The air pressurization design uses a
manifold to distribute the bursts of compressed air among up to eight bioreactor cartridges. This design
allows for several isolated tissue cultures to be active and conditioned simultaneously. (Ali et. al., 2009)
2.2.2. Areas for improvement and design opportunities
Several different bioreactor designs, both in the general industry and in the microcosm of the Rolle lab,
have exhibited the potential of enhancing ECM production and structural integrity of TEBVs through
mechanical conditioning. It is imperative to address the shortcomings of each design in order to
optimize the biological performance and economical and industrial efficiencies of these devices.
These issues were partially addressed by Kelm (2010), and they were able to reduce the total production
time for a matured vessel, the design still retained a very low throughput for the amount of work that
was required to yield a desirable tissue product. The only profiled bioreactor that exhibits the capability
to yield a high throughput is the air pressurization design that is currently being used in the Rolle lab at
WPI. Even so, the current device has sacrificed sterility (threads warp in the autoclave). It is not designed
for efficient and constant media exchange, and does not have the customizable features that are
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needed for future studies (interchangeable with a growth chamber; ability to use different tube
diameters, etc.).
The MQP team seeks to alleviate these complications by developing and prototyping a design that
allows for high throughput of isolated tissue cultures while maintaining the chemical, biological, and
mechanical conditions that optimize cell growth and alignment, and ECM production comparable to
native tissue.
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Chapter 3 – Project Strategy
Our initial client statement challenged us to design a device that overcomes limitations associated with
current bioreactors that mechanically condition tissue engineered vascular constructs. It was then our
responsibility to conduct an in-depth literature review in order to gain a full understanding of the
current technology. As we developed an understanding of the problem, we began compiling a list of
objectives and constraints. Utilizing a collection of client interview data, we then applied these
objectives and constraints and created a list of functions and means for our device, outlined in Chapter
4. These means were then analyzed and several design alternatives were created. The designs were
further scrutinized and with continued research we decided on and refined a final design, which we
evaluated through a series of quantitative and qualitative bench top tests.
3.1 Initial Client Statement
The initial statement provided by our project advisor, Marsha Rolle, was: “Bioreactors have been shown
to improve the structure and function of engineered tissues by providing conditions that simulate the in
vivo environment in which the tissue normally exists. In addition, bioreactors can facilitate seeding,
organization and culture of cells to support tissue growth and maturation. For tissue engineered blood
vessels, bioreactors that provide cyclic, circumferential mechanical loading have been shown to increase
cellular alignment and increase extracellular matrix (ECM) synthesis, leading to improved tissue
mechanical strength. The goal of this project is to create a cartridge to house a tissue engineered blood
vessel that interfaces with a pressurization system that imparts cyclic circumferential strain on the tissue
during culture. The cartridge should include an external medium flow loop to provide continuous
nutrient supply to the tissue. Ideally, the cartridge should be inexpensive and easy to manufacture, such
that multiple cartridges can be used in a single experiment to culture batches of tissue engineered blood
vessels. Finally, the cartridge should be interchangeable with the luminal flow cartridge under
development by another MQP team and members of the Rolle Laboratory.”
3.2 Objectives
Based upon our initial research of past MQPs and published literature, we developed the following
objectives:
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
Accurate: Must produce a strain magnitude within 10% ± 1%

High Throughput: The ability to “condition many rings or tubes simultaneously, run
multiple media conditions simultaneously, and/or vary culture duration.” (Ali et al.,
2009)

Adjustable: Able to condition TEBVs of different diameters and lengths at different
frequency and strain levels.

Easily Secured: TEBVs should be easily secured prior to testing.

Easily Removed: TEBVs should be easily removed after testing.

Inexpensive: Both to build (using cheap materials/parts) and to maintain (using less
media, durable and reusable materials, energy efficient, less time expense).

Easy to Use: With minimal manual input, using fewer, simpler steps.
With the completion of the literature review, the team then developed a Pairwise Comparison Chart
(PCC) which helped us determine which objectives needed to take priority when we reached the
conceptual design stage (Chapter 4). In total, nine objectives were analyzed in order to establish a set of
design priorities. Additionally, the PCCs were issued to our advisor and a graduate student, who as
actual clients of the device could further validate the design priorities. Our PCC can be found in
Appendix A – Pairwise Comparison Chart (PCC).
As seen in Table 2, three areas were determined to be a priority for both clients and the team: accuracy,
media supply and waste removal. Accuracy was defined as our device’s ability to induce 10 ± 1% cyclic
strain. As found in literature, strains of this magnitude significantly increased the mechanical and
structural integrity of TEBV samples. (Seliktar et. al., 2000) Both media supply and waste removal were
related to our device’s ability to sustain cell constructs. It was considered a necessity to maintain an
ample supply of nutrients for the cell samples during conditioning. We all agreed that our design must
provide constant nutrient supply and allow for repeated replenishment of nutrients. Additionally, all
users felt that waste removal was equally important as to maintain cellular homeostasis. These areas of
interest are highlighted yellow in Table 2. Highlighted red in Table 2 is an additional area of interest and
refers to the amount of effort required to secure tissue samples to our device. As a team we did not feel
this was as important as some of the other objectives, however after receiving the client completed PCC,
we realized that it may be more important than initially thought. Newly cultured tissue samples are very
fragile and during the process of transferring the samples from their culture molds to our device they
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can easily break. Both clients decided that our device should facilitate this process. This list of priorities
and weighted objectives functioned as the backbone for the development of design alternatives.
Table 2. Client and Team PCC Comparison
Objective
Rolle
Gwyther
Team
Average
Media Supply
8
7
7
7.333333
Waste Removal
7
5
6
6
Accurate
4
8
6
6
Easily Secured
High
Throughput
6
5
3
4.666667
2.5
4
4
3.5
5
2
3
3.333333
Programmable
0.5
1
5
2.166667
Adjustable
0.5
4
1
1.833333
Inexpensive
2.5
0
0
0.833333
Easily Removed
3.3 Constraints
In addition to the development of objectives, we also needed to determine our design constraints. Our
two most limiting constraints were time and budget. The final deadline for project completion was April
21st, 2011 on Project Presentation Day. Our budget consisted of $368.00, which would have to cover all
of our purchased materials for prototype and final design manufacturing. Safety was another priority for
all of our alternative designs. The design could not harm the user or cells in any way. The bioreactor
must be biocompatible, non-toxic to the cells within it, and contain no possible outlets for injury.
The materials we use in our design must be sterilizable in order to avoid contaminating tissue samples.
In addition to sterility, cell viability must be maintained by means of constant media supply to the TEBVs
during mechanical conditioning. Waste regulation must also be accounted for in order to guarantee
optimal cellular conditions.
After discussion with our client, we determined that the bioreactor must be transparent to allow for
imaging and observation during TEBV testing. Transparency aids in the detection of experimental failure,
such as: ring failure, culture contamination, or component failure.
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Since our device doesn’t have a means to self-regulate physiological conditions, such as temperature
and humidity, the device must fit within an incubator. Each shelf in the incubator in our laboratory is 40
cm wide x 15 cm high x 40 cm deep. This constraint impacted our designs by limiting the available space
that our device can occupy.
3.4 Revised Client Statement
After an improved understanding of the assigned problem, and an extensive knowledge of the current
technology, we were able to apply our metrics and overall project strategy, and improve upon our initial
client statement. The resulting client statement reads as follows:
To design a bioreactor used in conjunction with a pressurization system that
distends the inner diameter (2mm) of tissue engineered blood vessel rings and/or
tubes (1cm long) by 10% (± 1%) at a frequency of 1 Hz. This will be accomplished by
means of cyclic circumferential strain, which increases cellular alignment and ECM
synthesis. Multiple cartridges should allow for media regulation in a safe, sterile,
leak-proof in vitro setting that accurately simulates an in vivo environment. Finally,
the device should be inexpensive and easy to manufacture and use.
With this enhanced definition of the problem we then entered the development stage which is
completely outlined in the next chapter.
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Chapter 4 – Alternative Designs
Based on our initial client statement, we developed constraints, objectives, functions, and means by
which to formulate design alternatives. Using the air pressurization system designed by Ali, 2009 (Figure
5) as a template and the designs of other successful bioreactors as models, the team evaluated a
number of design alternatives.
Figure 5. Air pressurization system (Ali et. al., 2009)
After studying the Ali tissue chamber design outlined in yellow in Figure 5, we determined that the
design had several faults. The silicone tube fixed to the threaded cap was dangling within the BD Falcon
conical tube (Figure 4 - A). Without any effective means of fixing the free end of the silicone tube, there
was nothing to prevent it from curling or knocking against the sides of the conical tube. The only means
of holding the silicone tube taut was via an end weight attached to the needle endcap which was glued
into the free end of the silicone tube. This endcap was still not effective at preventing the free swinging
of the silicone tube during transport. The vertical orientation of the tissue chamber makes the tissue
rings susceptible to gravitational forces that can merge multiple rings together or possibly even slide
them off of the silicone tube entirely. A horizontal orientation would eliminate this problem.
Other drawbacks of the pressurization system include its size and user-friendliness. The pressurization
system makes use of compressed air, which is stored in large tanks in the lab. This takes up lots of space
in the lab and is loud and disruptive in the user’s workspace. Furthermore, a heavy aluminum base
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makes the device awkward to transport. Finally, the device was designed so large that it cannot fit in a
laboratory incubator and therefore cannot be used for culture experiments. Given these limitations of
the initial design, we chose to pursue a completely novel approach to our project.
During design conception, we made an effort to use common parts (standard sized screws, syringes,
tubes, motors, etc.) to reduce costs of manufacturing and materials, expedite the design process, and to
minimize production times.
4.1 Functions (Specifications)
With a firm understanding of our objectives and constraints we then began defining potential functions
and means which could be applied to design alternatives. We decided to separate the design functions
into two categories: functions/means for the tissue chamber and functions/means for the mechanical
conditioning system. Below is an itemized list of our proposed design functions.
Tissue Chamber Functions





Provide nutrients to cells
Remove cell waste
Allow access to and removal of tissue rings/tubes
Allow gas exchange
Compatible with a pump
Mechanical Stimulation System Functions





Cyclically distend tissue samples by 10%
Run during the duration of mechanical conditioning of samples
Consistent (must strain at 10 ± 1% per cycle)
Adjustable pressure controls (per tissue chamber)
Compatible with tissue chamber
In order to visualize these functions, after making a list, we then created a Morphological Chart (Table 3)
and continued to expand upon the potential means for each function.
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Table 3. Morphological Chart
Functions
Mean 1
Mean 2
Mean 3
Mean 4
Tissue Chamber
Media Provision
Submerge in
Media
Cell Waste Removal
Aspirator
Tissue Sample Access
Removable Lid
Gas Exchange
Rotisserie Media Pool
Porous Mandrel
Drainage Flow Loop
Individual Tissue
Chambers
Filtered Reservoir
Expose to Air
Petri lid
Humidity Regulation
HEPA Filter
Exposure to
Incubator
Pump Compatibility
Syringe Pump
Peristaltic Pump
Mechanical (CAM/Motor)
Pump
Air Pressurization
System
Mechanical Conditioning
System
Cyclic/Circumferential
Loading
Syringe Pump
Peristaltic Pump
Mechanical (CAM/Motor)
Pump
Air Pressurization
System
Consistent Work Output
Programmable
Leak Proof
Adjustable Pressure
Tissue Chamber
Compatibility
Programmable
Fluid Two-Way
Valve
TRIM Pot
Quick Connect Tubing
Couplings
Mechanically Adjustable
Bread Board w/ Variable
Resistors
Spring
Motor w/ Turn Off
Switches
Open Container
4.2 Conceptual Designs
In order to meet our outlined objectives, we broke up our bioreactor system into two major
components: a mechanical conditioning system and a tissue culture device. Each had their own set of
objectives and functional requirements.
4.2.1. Mechanical conditioning system
The purpose of the mechanical conditioning system is to use a flow medium to distend an elastic tube
on which engineered tissue samples are mounted. Distensible tubing has been used in past research as a
means of imparting mechanical strain on tissue engineered constructs (Niklason et. al., 1999). The
elastic tube is generally seeded with tissue constructs and inflated by air or liquid to distend the tube,
and thus the tissues mounted on the tube.
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1. Peristaltic pump
Using the Syedain heart valve bioreactor design
(Syedain et. al., 2009) as a model, the team
considered the use of a peristaltic pump as a
means to provide circumferential mechanical
stimulation to the TEBVs. The pump operates by
using a rotating cam (Figure 6 - 1) to flex plastic
tubing (2) that is fixed to a frame (3). The design
would incorporate inlet and outlet channels (4, 5)
in order to allow for a continuous circuit of
Figure 6. Peristaltic pump
flow medium.
Using a peristaltic pump as a design component is very appealing, as it is an existing technology and is
programmable. However, one major drawback is that existing models do not offer high throughput
capabilities.
2. OctoPump
The next design alternative was conceived by
reverse-engineering a syringe pump. The
design consists of eight syringes (Figure 7 - 1)
housed in a circular frame (2). The pump uses
an offset rotating cam (3) located in the
center of the frame to depress the plungers
of the syringes. The displacement of a
medium would inflate a plastic tube on
Figure 7. OctoPump
which the tissue rings are housed, thereby providing the desired distension.
This novel design allows for high throughput of isolated samples, and the cam device could be
manipulated to adjust flow frequencies and volumes. However, in order to remove a single sample the
user would have to deactivate the entire system.
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3. Wheel and arm motor
Another design that mimics a syringe pump is the wheel
and arm motor assembly. A small mechanical motor
(Figure 8 - 1) spins a machine wheel (2) attached to a
flat arm (3). The flat arm’s far end is attached to a
housing device that is fixed to several syringe heads (4).
As the wheel spins, there is a horizontal translation in
the syringe heads, causing them to depress and retract.
The flat arm can be fixed to the wheel at any of several
points (5), resulting in different radii and therefore
different flow volumes.
Figure 8. Wheel and arm motor
This design concept is appealing as it combines several simple existing technologies for a novel
application. It also serves as an effective, high-throughput alternative to the syringe pump, which was
deemed out of the team’s price range. Some predicted complications include size and isolating single
syringes or assembly systems.
4. Solenoid flexion device
The next preliminary design was a solenoid flexion
device. The design consists of a battery-operated
solenoid (Figure 9 - 1) suspended above the plastic
tubing (2) which contains the tissue samples. Fixed to
the solenoid is a rubber press (3). The size of press
would be chosen depending on the volume of water in
the tubing that needed to be displaced in order to
inflate the tubing and achieve the desired 10%
distension of the tubing and TEBVs.
While this design assembly eliminates several parts and
fixture points – and in turn, eliminates opportunities for the
Figure 9. Solenoid flexion device
device to fail – it was deemed unfit for this project. It was discovered that it is very difficult to program a
solenoid to function at speeds as low as 60 RPM, which is required for the mechanical conditioning to
36 | P a g e
take place at 1 Hz. Furthermore, the movements of the solenoid are abrupt, irregular, and may be
abrasive to the silicone tubing. (J. O’Rourke, personal communication, October 2010)
5. Motor and cam system
The team’s final
conceptual design for the
mechanical conditioning
system involves a small
motor (Figure 10 - 1) that
contains a cam and cam
collar (2). As the cam
spins, it remains tangent
with the head of a syringe
plunger (3) that displaces
a flow medium from a 1
Figure 10. Motor and cam system
mL syringe (4) through a series of luer fittings (5) and into a flexible plastic tube (6) that houses the
tissue rings. The tube is secured at the distal end using a pinch clamp (7). The plunger is fitted with a
compression spring (8) that makes it to retract, stay in contact with the cam, and allows for cyclic
pumping of the syringe.
One highlight of this design is that the user may choose different cams or motors to customize the
parameters of the mechanical conditioning. For example, a larger cam would displace a larger volume of
medium through the syringe, thereby causing a greater distension of the tissue rings. A motor may be
run at lower or higher voltages to increase or decrease the frequency of the conditioning.
4.2.2. Tissue chamber
All of the designs for the mechanical conditioning system involve the tissue samples being mounted on a
flexible plastic tube that is inflated in order to achieve the desired distension. The final assembly
therefore requires a unit made from biocompatible materials that safely houses the tubing and cells,
prevents contamination, allows gas exchange between the cells and the environment, and interfaces
effectively with the mechanical conditioning system.
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The current design used in the Rolle lab in conjunction with the air
pressurization system contains a number of flaws. The design fails
to secure the silicone tubing at both ends. Fixing the silicone tube
at both ends and keeping it taut prevents the tube from moving
and ensures that the displacement of water through the tube leads
to inflation and distension. Additionally, the current design offers
no outlets or means for waste removal and media exchange.
1. Double threaded cartridge
The first design alternative was based on the tissue chamber from
the air pressurization design, which uses a series of polystyrene
Falcon conical tubes. Because these units are small and create
isolated environments for each tube and set of cells, there are
Figure 11. Double threaded cartridge
many possibilities for a high throughput assembly. The design
consists of a hollow tube (Figure 11 - 1) with threaded caps on each end (2). One cap would
permanently house the distal end the silicone tube (3). The second cap would function much like the
current design, as it would be interfaced with a fitting that leads to the pump system (4). A lock-and-key
pin mechanism (5) fixes the proximal end of the silicone tube to this cap. Because the tubing is fixed to
the threaded caps before they are both screwed
onto the Falcon tube, the tubing is subjected to
torsion and possible tearing or deformation.
Therefore, the biggest challenge associated with
this design would be to not compromise the safety
of the silicone tube and the tissue sample when
fastening the threads.
Figure 12. Removable compartment assembly
2. Removable compartments
This design uses an original assembly of isolated, removable plastic compartments (Figure 12 - 1) that fit
into a containment unit (2). The frame separates the compartments, acts as a junction between the
pump system and cartridges, and provides hinged covers for each compartment (3). The hinged covers
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loosely sit atop the compartments, which allows for gas exchange and
provides easy access to the interior for aspirating and changing out cell
media. Finally, the fronts of the compartments will contain a small
opening (4) above the media pool to which the silicon tubing will be fixed
by a clamp located outside the tissue chamber.
3. Milled tissue chambers
This conceptual design consists of milled plastic tissue chambers (Figure
13 - 1) which interface with a mechanical conditioning system’s syringe (2)
through a series of luer fittings (3). The flexible plastic tubing is attached
to the luer fittings using heat shrink-wrap tubing (4). The distal end of the
silicone tubing is clamped using a pinch clamp (5) that is fixed to the
bottom of the chamber using a stainless steel dowel.
4.2.3. Design assembly
1. Wheel and arm assembly
The design is driven by the wheel and arm motor (Figure 14 - 1) as
Figure 13. Milled tissue
chamber
described in Section 4.2.1.-3. The flat arm is interfaced with a fixture that holds the heads of several BD
10cc syringe plunger heads (2). Displaced flow medium is transferred from the syringes into the tissuemounted silicone tubing located in the compartments of the removable compartment assembly (3),
which will be constructed as described in Section 4.2.2.-2. The combination of these design alternatives
allows for high throughput, isolated systems for the simultaneous operation of independent
experiments, and high customizability.
One problem that arises with this design is that a single compartment cannot be removed without
shutting off the pump for the other compartments. Also, even if a single compartment can be removed,
there is nothing stopping the water in the silicone tubing from spilling out. The team conceptualized a
system that may overcome these design barriers. Each syringe would pass the flow through a three-way
cross flow valve (4). The top end of the valve will lead into a BD Falcon polypropylene tube, which would
be fixed with a loaded silicone tube (5). When a tissue chamber needs to be removed, the flow can be
redirected using the cross flow valve into the Falcon tube, which serves as a temporary reservoir for the
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flow medium. When the tissue
chamber is put back into the
system, the flow can be directed
again back into the chamber.
When the user wants to remove
a single compartment, he or she
will simply redirect the flow to
the reservoir, stopping the flow
into that cartridge without
interrupting the others. This still
leaves the problem of water
leaking from the system into the
incubator following removal of
the compartment. The remaining
end of the three-way valve will
Figure 14. Wheel and arm assembly
be fitted with a quick-coupling
fixture that only allows one-way flow.
2. Pyramid assembly
The team conceptualized
modifications to the wheel and
arm design. In order to increase
user control over each TEBV
sample, the team sought to
create isolated systems for each
tissue chamber. In order to
achieve this, the team
concluded to use small gear-box
hobby motors (Figure 15 - 1).
Figure 15. Motorized pyramid assembly and pinch clamp (ZManCorp.com)
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These motors can be purchased off the shelf and run at 60 RPM. Again, a machine wheel (2) and flat arm
(3) would be fixed to each motor. The opposite end of the flat arm would attach to the plunger of a 1-mL
syringe (4). As the motors rotate the wheel, the flat arms would undergo a horizontal translation,
causing the plungers to depress and retract. These syringes interface with milled tissue chambers (see
Figure 13) which contain an internalized pinch clamp (5). The motors are arranged in a staggered
‘pyramid’ formation in order to minimize the amount of occupied space while maintaining the linearity
of the system and allowing the user to easily access the tissue chambers. To accomplish this, three
different sized flat arms are required (6).
In order to improve manufacturability, the team dismissed the flat arm component of the design en
route to developing the following design alternative:
2. Motor-cam series
The motor-cam series builds on the
concept of the pyramid assembly, but
simplifies it by reducing the number of
parts and fittings, improving
manufacturability, and internalizing the
mechanism in order to make a compact,
customizable, user-friendly device. The
assembly consists of a plastic base (Figure
16 - 1) which houses six 60 RPM gear-box
motors (2). Each motor is fixed with a
Figure 16 Motor-cam series
cam (3) which rotates and cyclically depresses the plunger of a 1-mL syringe (4). Again, the design
employs tissue chambers and pinch clamps as seen in Figure 13. Six of these tissue chambers (5) are
housed in a removable UHMWPE tray (6) that fits into the base.
4.3 Comparison of Design Components
In addition to comparing alternative designs as a whole, we compared individual components used
within these designs to aid in our selection of pieces for a final working assembly. We compared these
components using research, interviews with experts and clients, experimental testing, and general
knowledge obtained during the course of our project.
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1. Distensible tubing
We were given fifty feet of clean, not sterile, 1.4732 mm inner diameter x 1.9558 mm outer diameter
silicone tubing from Specialty Manufacturing Inc. (SMI) at the start of our project. We decided to build
our design around this source of distensible tubing due to the Rolle Lab’s experience handling this
particular size and brand.
Silicone tubing has been shown to be an effective means of distending tissue engineered constructs
(Niklason et. al., 1999). The inner diameter of the tissue rings being used in our project (2 mm) match
almost precisely with the outer diameter of the silicone tubing (1.9558 mm), leaving an extra 0.04 mm
to aid during tissue ring loading. Loading involves delicately placing a tissue ring or tube around the
outer circumference of a sterile distensible tube. The tube is then inflated or pressurized continuously at
10 ± 1% of its outer diameter at a frequency of 1 Hz over a 3-7 day period (in our studies). In the event
that the tissue rings adhere to the surface of the silicone tubing during distension, they can be detached
simply by stretching on either end of the silicone tube (M. Rolle, personal communication, September,
2010).
Silicone tubing is autoclavable, inexpensive (at ~2 cents per 10 cm length), disposable, can interface with
syringe tips, and will not leach when soaked in cellular media for long periods of time.
2. Mechanical conditioning device
We considered three different device designs before reaching a final decision for a means of distending
the silicone tubing. First, an air pressurization system designed by the Ali MQP in 2009 was considered
as a means of distending silicone tubing via air flow. This device used a pressure control system which
provided a constant high pressure of 26 psi which obtained the 10% strain on the silicone tubing. A
three-way solenoid valve controlled by an electrical timer switched back and forth between the system’s
high (26 psi) and low (4 psi) pressure inputs at a frequency of 1 Hz. The use of air pressurization proved
to have its drawbacks with the Ali MQP’s system. The entire system was large and complicated,
involving numerous connections between the air compressor, pressure control devices, distribution
manifold, and tissue chambers. The pressurization system shown in Figure 5 consists of every part of the
system except for the tissue chambers outlined in yellow. The overall size of this system makes it
incredibly difficult to run within an incubator, as well as incredibly noisy. While there is an option for
running tubing outside of the incubator, the size of the distributor (labeled as 1 in Figure 5) and bulk of
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the remainder of the system would make it difficult to arrange within and around an incubator. The
greater the number of connections between the system and the tissue chambers, the greater the
chance of leaks, loose fittings, and difficulty in monitoring air flow and air levels. (Ali et. al., 2009)
Our second option was to use a solenoid coil actuator to displace a specific volume of water within a
silicone tube to achieve 10±1% distension. Solenoids work by creating a magnetic field around a
solenoid coil which moves a metal armature positioned in the middle of the circular coil. The armature is
forced down by a spring, pushing the stem and base onto a silicone tube filled with water positioned
beneath the solenoid. The entire system is pictured in Figure 17. The magnetic field can be controlled to
push the armature at a rate of 1 Hz based on the number of
loops in the solenoid coil. There are several problems
associated with this system, namely that solenoids operate
in a very abrupt manner (J. O’Rourke, personal
communication, October 2010). The movement of the
armature and flexion of the spring are not smooth in
motion, which could cause damage to the silicone tube and
friction in the system. Using a solenoid in a moist, humid
environment may disrupt solenoid function as well. The
affects of electromagnetic fields on TEBV growth is also
Figure 17. Solenoid actuator (Edited from
tpub.com)
unknown and could potentially be problematic.
Our final option is a motor, pictured in Figure 18. Motors can effectively push syringe plungers when
attached to a cam and/or flatbar assembly. One motor could be used to move multiple syringes at once,
or multiple motors can be used to move syringe plungers individually. The movement of these plungers
at 60 RPM results in distension of the silicone tubes at a frequency of 1 Hz. When preloaded with water
or media, the silicone tubes can be distended by 10±1% depending on the length of the flatbar or
dimensions of the cam.
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Figure 18. 12V DC 60 RPM high torque gear box electric motor
We chose to use the motor as our method of mechanically distending the silicone tube due to its low
cost (see Appendix F for pricing), adjustability of frequency, smooth and quiet operation, size (37 mm in
diameter, 70 mm in height), and simplicity over the other two design alternatives. Using multiple motors
further improves the design by allowing for variation of separate tissue chamber conditions during
experimentation. By using multiple motors, individual chambers can be shut off or set to run at a slower
speed with the flip of a switch or the turn of a knob. We found during bench-top testing that our motor
(Error! Reference source not found.) ran very quietly and without complications for 7 days on its own.
otors can be positioned in a variety of different ways to achieve the same function, allowing for more
design flexibility.
3. Tissue chamber
We narrowed our design options down immediately when deciding between using tissue chambers
where the cells would be seeded on a vertical silicone tube (such as in the Ali 2009 MQP) versus being
seeded horizontally. We eliminated a vertical tissue chamber design based on our assessment of the air
pressurization system used in the Ali (2009) MQP introduced in the beginning of this chapter. Our final
concern with the vertical tissue chamber design of the Ali MQP is the amount of wasted cell media used
in the 50 mL conical tubes (~35 mL of media used if filled as shown in Figure 4). A horizontal design in
which enough media would be used to submerge the tissue rings would reduce the costs related to cell
media usage.
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The only stated concern of the Ali MQP team regarding horizontal tissue chamber arrangements is “the
chance that flexible tubes will not remain completely horizontal when inserted into the media” (Ali et.
al, 2009). We saw a way to overcome this problem, and so we chose to pursue horizontal tissue
chamber arrangements. Our only foreseeable challenge with horizontal chambers was in finding a way
to submerge distensible tubing (loaded with tissue rings) within the media in the chambers without
leaking. All chamber designs must allow for tissue ring submersion and interface with the motor-cam
mechanical conditioning device. We considered two different design variations for a horizontal tissue
chamber based on our decisions to eliminate vertical designs and use motors as a mechanical
conditioning device.
The first tissue chamber design involved using a solid block with separate chambers milled out of it, and
the second involved having separate removable chambers housed on a removable tray (Figure 19). Both
designs are relatively easy to manufacture through milling and sawing and both have removable
components. The tissue chamber with separate, removable chambers was deemed to be a better option
for our design. By having separate chambers, the user could have the option of removing a single
chamber from the system in the event of sample contamination
or a problem with the mechanical components. In the event
that a chamber is damaged in some way, it can simply be
replaced, eliminating unused or contaminated space in the
system. Both tissue chamber designs would interface with the
motor-cam mechanical conditioning device through a hole in
one of its walls. An air and water-tight seal would allow for the
silicone tubing to pass into the tissue chamber for submersion,
while maintaining a sterile environment within the tissue
Figure 19. Individual removable chambers
on a removable tray
chamber itself.
One function of the tissue chambers is to allow for the changing and replacing of media. Both the single
unit and individual chambers function similar to culture dishes, in which media must be manually
aspirated and added to feed the cells contained within them. The chambers would be complete with
loose fitting lids, which act as culture dish lids in permitting gas exchange and offering protection from
potential airborne contamination in non-sterile settings. Both the single unit and individual chambers
would facilitate media change quite easily with these design considerations. The single unit acts as a tray
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in itself while the individual compartments are housed on a removable tray, allowing for all chambers to
be moved at once for both designs.
We chose to use the individual, removable chambers on a removable tray to do its greater flexibility for
a variety of situations that could occur in the laboratory during testing.
4. Mechanical attachment to motor
We considered two different options for a means of mechanically pushing the syringe plunger using a
motor. The first conceptual design used a cam and flat arm design. We would manufacture a cam of
appropriate dimensions and attach and clamp it to the shaft of the motor via a D-shaped hole at the
center of the cam. In order to achieve 10% distension, the syringe plunger would have to be moved by a
certain distance depending on the size of the syringe and the volume of fluid needed to distend the
silicone tubing. The distance that the plunger must be moved is equal to the distance between the
center of the D-shaped hole and the center of the hole drilled to connect the flat arm to the cam. The
other end of the flat arm connects to a syringe plunger, the movement of which controls the distension
of a silicone tube and the tissue rings encircling it. Cams and flat arms can be manufactured relatively
easily to whatever specifications are desired. Flat arms can be as long as necessary to accommodate for
individual motor spacing, although the longer the flat arm, the larger the overall device. The cam and
flat arm design can be seen in Figure 20.
Figure 20. Top view of cam and flat arm design (left) and side view of cam design (right) for motor-syringe attachment
Our second conceptual design was based on using just the cam without a flat arm. The cam dimensions
would be calculated in the same way as for the cam and flat arm design. The cam would rotate,
depressing the plunger of the syringe with its longer side. A compression spring controls the plunger’s
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extension as the cam rotates to its shorter side. Springs will be necessary to ensure that the syringe
plunger maintains contact with the cam and that the plunger fully extends back to its original position
within the one second time frame required for running at a frequency of 1 Hz. The spring must be big
enough to fit around the plunger, yet not exceed the size of the plunger head in order to remain
positioned correctly between the syringe barrel and plunger during conditioning. By switching out the
cam for a different sized one, the volume of water displaced by the syringe can be catered to the user’s
needs. The elimination of the flat arm in this design allowed for us to shorten our design considerably. In
theory, the cam design is more compact, it allows for the motor to be inverted and anchored into the
base of the device to further save space. The use of less material for the base of the device saves
material costs, and the odds of mechanical failure are reduced by limiting the number of mechanical
connections in the overall system.
5. Syringe tips
We considered three different syringe tips as a means of connecting our syringe to the 1.9558 mm inner
diameter silicone tubing.
Blunt end metal syringe tips (2 mm diameter; Figure 21, right) were first considered due to their
similarities in diameter to the silicone tubing we were using. These tips effectively interface with a large
variety of syringes, including the 1mL luer-slip and luer-lock.
Figure 21. Pictured left to right, 1/16" inner diameter female barbed luer-lock tip, 1/16" inner diameter male barbed bayonet
tip, and 2mm blunt end metal syringe tip.
Problems arose with this type of syringe tip when trying to build it into the wall of the tissue chamber.
Any tip would have to be sealed into the wall of the tissue chamber to allow for silicone tube
submersion and easy connection from the sterile tissue chamber to the syringe and mechanical
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conditioning system. Mounting tubing connectors also helps keep the entire system in line, so fluid flows
in one direction out of the syringe and the tubing is held taut off the bottom of the tissue chamber to
prevent tissue ring damage. In the event of syringe tip or silicone tube failure, there would be no way to
remove the 2mm blunt end metal syringe tip from the tissue chamber wall without replacing the entire
tissue chamber. Similarly, if the silicone tube were to become worn out due to repeated use, damaged
during autoclaving, or otherwise harmed, the 2mm metal syringe tip would have to be removed and
replaced.
Figure 22. Syringe-silicone tubing connectors
Our plan to combat these problems was to use a two-tip, male-female luer fittings. The female tip would
be secured into the wall of the tissue chamber and fixed there permanently, while the male tip would be
permanently attached to the silicone tubing and lock onto the female tip during experimentation. The
first male-female luer parts we considered were 1/16” inner diameter barbed luer-lock male and female
tips (Figure 22) capable of interfacing with luer-slip syringes and silicone tubing. The 1/16” inner
diameter luer fittings is approximately 1.6mm, which is as close to 1.4732 mm inner diameter of the
silicone tubing as standard parts would allow. The tight fit of the silicone tubing over the luer barb
further aided in leak proofing the system. The wider opening of the luer tip leading into the narrower
inner diameter of the elastic tubing caused some problems with tube failure when distended past 20%
during testing. This gives the user the option to inflate the tube up to 120% of its original diameter in
order to increase the strain magnitudes imparted on the tissue rings. Problems arose when we
attempted to secure these luers into the wall of the tissue chambers. The luer pictured on the right in
Figure 22 was nearly impossible to permanently mount into the tissue chamber wall.
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Figure 23. Threaded syringe-silicone tubing connectors.
Due to the problems encountered with the 1/16” inner diameter barbed luers, we considered
alternative interlocking luers. We chose to use a ¼-28 threaded luer to fix into the tissue chamber wall
that would interface with a 1/16” inner diameter barbed luer-lock tip (Figure 23). These luers allowed
for permanent fixture into the tissue chamber and easily interfaced with the 1 mL syringes we had been
using throughout bench top testing. The 1/16” inner diameter barbed luer-lock tip was heat shrunk to
the silicone tubing in the same manner as described in Ali, 2009. The threaded luers were leakproof
based on bench top testing over a period of 12 hours. This was accomplished by threading the luers into
the tissue chambers, attaching a 1 mL syringe to the outside opening of the threaded luer, and filling the
chambers with water.
The fittings are manufactured from autoclavable plastics and come equipped with a barbed tip, which
aids in securing the silicone tubing onto the luer fitting and prevents fluid flow beyond the barb.
Leakproof testing involving manual fatigue testing of the luers under fluctuating silicone tube pressure
for 20 minutes as well as 16 hours of motor-cam distension confirmed the ability of interlocking luer
fittings to prevent fluid leakage from the system. By using both male and female luer fittings, we can
change out overused or damaged silicone tubes for new ones without having to disassemble or replace
any major components of the device. The female 1/16” inner diameter barbed bayonet fittings can be
permanently connected to silicone tubing, autoclaved in single-use packets, used for experimentation,
and then disposed of. The more easily replaceable standard parts we use in our design, the better the
durability and lifespan of our device before standard wear renders it non-functional.
6. Mechanism for attaching silicone tubing to syringe tip
When preloaded with water and clamped at its free end, the silicone tube slides off of the syringe tip
due to pressurization from depressing the syringe plunger. To attempt to correct this, we considered
three different approaches for sealing the silicone tubing to the syringe tip(s) to prevent leaking and
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detachment of the tubing. First, a suture was tied around the silicone tube above the barb on the tip of
the 1/16” female luer fitting (Figure 24). This method failed. Silicone glue was then applied to the tip
shaft of the 1/16” female luer fitting below and above the barb. The silicone tube was then forced onto
the luer tip over the barb and set to dry for 48 hours. A second sample using the 2mm blunt end metal
syringe tips was also used. Silicone glue failed to prevent leakage at the syringe tip-silicone tube
interface (Figure 24).
Figure 24. Using sutures (left) and silicone glue (right) to attempt to seal syringe tips to silicone tubing
Ali (2009) found an alternative to these methods. They used biocompatible 1/8” diameter, transparent,
heat shrinkable tubing with a 2:1 shrink ratio to seal the silicone tubing to the rest of their pressurization
system. After extensive testing involving preloading different syringes, luers, and silicone tubing with
water, clamping the distal end of the silicone tubing, and pressurizing the tube past 20% distension
(Section 5.1), we determined that heat shrink tubing was our best option and proved to prevent any
leaking or detachment of the silicone tubing from the 1/16” female luer fitting.
However, one limitation to this method exists. When manufacturing these syringe tip-silicone tubing
parts, one must use a heat gun to adhere the heat shrink tubing to the syringe tip-silicone tubing
junction by heating the heat shrink tubing above 175oC (see Appendix A for specific instructions). At
such high temperatures, the plastic of the 1/16” female luer fitting begins to melt, potentially sealing the
flow opening of the luer fitting. Excess heat directly applied to silicone tubing was observed to cause
point defects along the affected section of tubing when the tubing was pressurized, causing plastic
deformation and failure of the tube. To avoid these complications, specific instructions have been
outlined in Appendix B – A Guide to Heat Shrink Tubing for manufacturing these parts. Despite these
limitations, heat shrink tubing is still the best option we tested for permanently sealing the silicone
tubing to the syringe tip.
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7. Silicone tubing sealing mechanism
We had a variety of options for sealing the free/open end of the silicone tubing to ensure pressurization.
These included endcaps, and a variety of different clamps. Endcaps are small rubber plugs fitted to the
distal end of the silicone tube and held permanently in place with glue. We considered pinch clamps for
small diameter tubing due to their ease of use, low cost, and autoclavability.
The Ali MQP (2009) had a large variety of endcap designs including a needle endcap, blunt endcap,
suture, and a combination of suture and a blunt endcap. The blunt endcap and needle endcap designs
were multi-functional. Both aided in tissue ring loading by providing a point at the end of the silicone
tube to place in the center of the tissue ring, from where you can push the ring up the silicone tube with
forceps. The two endcaps also effectively sealed the silicone tubes from leaking due to air
pressurization. Sealing the distal end of the tube may also pose a challenge in preloading the silicone
tube with liquid for distension by syringe. The tube would have to be submerged completely in water or
media to allow the air within the tube to be replaced with liquid before attaching the syringe. While
these designs would have worked well in a vertical tissue chamber setting, they are far more difficult to
accomplish in a horizontal setting. These may all seal the silicone tube effectively, but there is nothing
present to hold the silicone tube taut and steady relative to the base of the tissue chamber. Clamping or
sealing the endcap inside the tissue chamber would keep the tube taut.
Figure 25. Pinch clamp from World Precision Instruments (left) and Z-Man Corp (middle & right)
For this reason, our silicone tubing sealing mechanisms were narrowed down to various types of clamps.
We chose to clamp the tubing inside of the tissue chamber in order to prevent complications that could
arise from running the tube outside, such as media leakage due to the presence of extra outlets in the
walls of the chamber. Clamps effectively seal tubing without leaks and simultaneously can hold the
tubing taut and off the bottom of the tissue chamber, securing its position in 3D space. The first type of
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clamp we tested was a pinch clamp from World Precision Instruments (Figure 25, left). This clamp was
100% leakproof based on pressurization testing involving distending silicone tubing with a syringe past
20%, yet lost much of its flexibility post-autoclaving. This led us to a patent pending model for a pinch
clamp from Z-Man Corp (Figure 25, middle and right). These clamps are autoclavable, and more
importantly, retain their functionality post-autoclaving. The pinch-to-open mechanism for this clamp is
far more ergonomic than the previous clamp, eliminating the need to hold the clamp steady with a
second hand during opening. The seal on the silicone tube is visibly much tighter than the previous
clamp as well. The hole located in the base of these clamps also makes it possible for us to manufacture
a peg or dowel and hole to mount the clamps within the tissue chamber. This would make the clamps
removable and eliminate the need to permanently adhere the clamps to the chamber surface, which is
helpful in the event of clamp malfunction or during sterilization of individual parts. The smaller
ergonomic pinch clamp was chosen for our device due to size restrictions within the tissue chamber.
8. Tissue chamber materials
Our options for tissue chamber materials were limited by the fact that the material had to be
biocompatible, autoclavable and/or sterilized by 70% ethanol, easily machinable, and relatively
inexpensive. We needed to choose a type of plastic for our tissue chambers, base component of our
design, removable tray, and tissue chamber lids.
Figure 26. Ultra-high molecular weight polyethylene (left) and polycarbonate (right)
We decided to use ultra-high molecular weight polyethylene (UHMWPE) (Figure 26, left) for our tissue
chambers due to its’ high impact strength and durability, weather resistance (humid incubator
environment), operating temperature range of -40oF to 275oF (250oF needed for autoclaving), and that it
meets ASTM (D4020, D4976, and D6712), FDA, and USDA specifications. Although this material does not
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fulfill the need for transparency of the material to allow visualization of the sample within the chamber,
transparent tissue chamber lids will satisfy this requirement.
We decided to use transparent abrasion-resistant polycarbonate (PC) (Figure 26, right) for the base and
removable trays of our device. Based on client interviews, we discovered that the base and tray of our
device need not be autoclaved between uses because they will not be in direct contact with tissue
samples. It would merely need to be wiped down with 70% ethanol before entering the incubator. For
this reason we chose abrasion-resistant polycarbonate to withstand harsh cleaners and solvents. PC also
has high impact strength and durability, is weather resistant, and has an operating temperature range of
-40oF to 200oF. Due to its low operating temperature, it cannot be sterilized within an autoclave (250oF),
and was thus not considered in designing tissue chambers.
Materials were selected based on several characteristics such as biocompatibility, sterilizability, size,
ease of use, and how well they interface with other components in the device. Using these material
choices, we designed and prototyped a device to mechanically condition tissue engineered blood vessels
in vitro. The efficacy of individual materials and interfaces between design components were then
tested through a series of qualitative and quantitative bench top experiments.
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Chapter 5 – Design Verification
To validate specific features of our design we categorized the desired functions into two categories:
those functions to be conducted by the tissue chamber and those to be accomplished by the mechanical
conditioning system. The experiments used to determine the appropriate means for these features are
explained in the following chapter.
5.1 Syringe Selection
We considered three different types of syringes that were readily available to use as a means of
displacing specific volumes of water into the silicone tubing of our device to achieve 10 ± 1% distension.
The three syringes we examined were: sterile Monoject® 3mL luer-lock syringes, sterile 1mL luer-lock
syringes, and sterile Monoject® 1mL “push-connect” or luer-slip syringes, all pictured in Figure 27. The
volume of water required to achieve ~10% distension for a 10 cm long, 1.9578 mm inner diameter
autoclaved silicone tube was found to be approximately 0.075 mL through extensive bench top testing
of multiple samples. This volume was measured through observing the specific volume displaced by the
1 ml syringe that resulted in 10% distension of the outer diameter of the silicone tube. The experiment
was performed by filling a 1 ml syringe with water completely, tapping all air bubbles out of the syringe
barrel, and attaching two interlocking male and female luers. The female luer was heat shrunk to 10cm
of silicone tubing (see Appendix # for manufacturing instructions). The system was flushed with water
until the plunger read 0.3mL and clamped at its distal end with a pinch clamp. We used approximately
0.3 mL of water to preload the silicone tube and connectors prior to sealing the distal end of the tube
and pressurizing it. Due to using such a small volume of water (0.3mL + 0.075mL + a safety factor of
0.225mL = 0.6mL), we limited our syringe options to between 1 and 3 mL syringes. The safety factor was
used in the event that the user accidentally pushed the syringe plunger too hard and ejected too much
water. It also allows for a larger cam to be used in our design further on, leaving room on the syringe
barrel for larger volumes of water to be displaced by larger cam sizes in future studies.
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Figure 27. 3mL luer-lock syringe (left), Luer-slip vs. luer-lock syringe tips (right)
The 3mL Monoject® syringes came pre-packaged and sterilized. The barrel of the syringe was marked at
0.1mL increments, 0.1mL corresponding to 0.06 mm of space between graduations. The overall length
of the syringe from luer-tip to plunger base is 80 mm when the syringe plunger is fully depressed, and
84.5 mm when at a pre-distension volume of 0.3mL, the safe minimum volume used for distension
studies. It would require 1.5mm (0.075mL on a 3mL syringe ≈ 1.5mm) of syringe plunger movement to
achieve ~10% distension in the attached silicone tubing. The tip of the syringe is fitted with a luer-lock
which secures luer-based syringe tips to the syringe by twisting the detachable tips onto the barrel of
the syringe. This ensures that the syringe tips remain attached during use and prevents the syringe tips
from being pulled from the syringe when a force is applied.
Figure 28. 1 mL push-connect or luer-slip syringe
The 1mL luer-lock syringe is identical to the 1mL “push-connect” or luer-slip syringe with the exception
of the luer fitting at the tip of the syringe. A side-by-side comparison of luer-slip and luer-lock syringe
tips can be seen in Figure 28. One mL luer-slip and luer-lock syringes come pre-packaged and sterilized.
The barrels of these syringes are marked at 0.01mL increments, 0.01mL corresponding to ~0.017 mm of
space between graduations. The overall length of the syringe from luer-tip to plunger base is 91.5 mm
when the syringe plunger is fully depressed, and 109.5 mm when at a pre-distension volume of 0.3mL.
Further fatigue testing indicated that the shorter the syringe plunger during mechanical conditioning
with the motor, cam and spring, the less likely the plunger is to bend and permanently deform over a
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period of 2 days constantly running within an incubator. The safety factor was thus later changed to
0.125mL instead of 0.225mL so the plunger would travel from the 0.2mL point to 0.125mL instead of
from 0.3mL to 0.225mL, decreasing the overall length of plunger left unsupported by the syringe barrel.
Due to these factors (summarized in Table 4), we decided to use a 1mL syringe in our design. The 4.5
mm (0.075mL on a 1mL syringe ≈ 4.5mm) distance required by the 1mL syringe to inject 0.075mL of
water into the silicone tubing to achieve ~10% distension makes it far simpler to manufacture and
operate the cam. It is more difficult to move a syringe by 1.5mm (3mL syringe) than by 4.5mm (1mL
syringe). The larger working area over which to mechanically distend the silicone tubing is preferred,
making it possible for us to use a larger cam if necessary. The 1mL syringe also has far smaller
measurable volume increments along the barrel of the syringe, making it far simpler to measure small
volume changes in the system. Between the luer-slip and luer-lock 1mL syringes, we decided to use a
1mL luer-slip syringe as seen in Figure 28. The 1mL luer-lock syringes we viewed for comparison in
catalogs were bulkier at the tip than the luer-slip. The twist required to secure the luer-lock syringe tips
into place was trivial compared to a push-connect tip. Locking luers were an unnecessary precaution
that would add an additional complication to loading and preparing silicone tube samples during
procedural set-up. 1mL luer-slip syringes are autoclavable, reusable, and inexpensive at roughly 17 cents
per syringe.
Table 4. Summary of syringe characteristics
Syringe Type
Length of Syringe
Distance Traveled for
Syringe
Luer Attachment
(Plunger Depressed)
0.075mL Injection
Accuracy
1mL luer-slip
91.5 mm
4.5 mm
0.01 mL
Push
3mL luer-lock
80.0 mm
1.5 mm
0.1 mL
Push & Twist
1mL luer-lock
N/A
4.5 mm
0.01 mL
Push & Twist
5.2 Quantification of Displaced Volume
We tested our design alternatives to determine which approach was best suited at achieving 10%
distension. Having decided upon a water-based pressurization system, rather than compressed air, we
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needed to determine the volume of water required to distend a silicone tube. Specifically, our design
called for a silicone tube 10 cm in length for optimal tissue sample loading.
5.2.1 Experimental Procedure
To measure the volume of water displaced by the 1 mL syringe that achieved the target magnitude of
10% distension, a water-filled syringe was used to statically inflate a tube with defined volumes in
increments of 25 μL (Figure 29Error! Reference source not found.). Tubing diameters were obtained
from still images of the inflated tubes using a Leica EZ4 D microscope and Leica Application Suite. For
this experiment a total of 5 autoclaved samples were tested.
Figure 29. 1 mL luer-slip syringe connected to 1/16” barbed bayonet tip mated with a 1/16” barbed luer-lock tip that is
attached by heat shrink tubing to a 10 cm silicone tube sample. A pinch clamp at the distal end of the tube was used as a
means of fixation and sealing.
5.2.2 Experimental Results
In static inflation tests, 75 μl of water resulted in 9.80±0.23% distension of a 10 cm length of 1.96 outer
diameter silicone tubing (Figure 30Error! Reference source not found.). Therefore, a 75 μL displacement
volume was used for subsequent studies.
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Figure 30. Required volume to achieve 10% distension of silicone tubing (n = 5; 9.8±0.23%). Red lines represent desired max
and min distension values. Green line represents target value (10% distension).
5.3 Uniformity of Tubing Distension
The goal of this experiment was to determine how uniform the distension was along the length of the
silicone tube. This allowed us to visualize the amount of working space for the placement of tissue rings.
5.3.1 Experimental Procedure
We marked each sample with nine markings (numbered 0-8) with 1 cm spacing between them (Figure
31). Then, similarly to the Quantification experiment, we statically distended the silicone tube by
injecting 75 μL and measured the induced diameter by taking an image through the Leica Software. This
was repeated for every reference point along the length of the tube.
Figure 31. A 1 mL luer-slip syringe connected to a 1/16” barbed luer lock tip fixed to 10 cm silicone tube sample by heat
shrink tubing. The 10 cm sample is marked with a black marker sealed and by a pinch clamp.
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5.3.2 Experimental Results
Static inflation of autoclaved tubing samples (n=20) suggested that 10 ± 1% distension could achieved.
Five reference points (points 2-6) were found to always be within 10±1% distension. After an injection of
75 μl of fluid, all points were within 10 ± 1.6% (Error! Reference source not found.Figure 32).
12
n = 20
% Distension
11
10
9
80
0
1
2
3
4
5
6
7
8
Length Along Tube (cm)
Figure 32. Uniform distension along silicone tubing (n=20). Red lines represent desired max and min distension values. Green
line represents target distension (10%).
5.4 Tissue Ring Loading
5.4.1 Experimental Procedure
To verify the desired objectives of a high throughput and easy sample loading, four tubing samples were
each loaded with 4-6 TEBV rings (Figure 33) and secured within our tissue chamber.
Figure 33. Six tissue rings loaded onto silicone tubing.
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The chambers were filled with culture media and placed in the tissue chamber housing unit (Figure 34).
For a complete procedure, see Appendix E.
B
A
C
Figure 34. Tissue rings (A) mounted on silicone tubing (B) and loaded into the tissue chambers (C).
5.4.2 Experimental Results
Tissue ring samples (n=45) were successfully mounted (45/53; 85% success rate) onto 4 silicone tubes
(n=4-6 per tube). After 3 and 7 day conditioning periods, samples remained viable and uncontaminated.
5.5 Preliminary Impacts of Mechanical Conditioning
5.5.1 Experimental Procedure
Using the experimental procedure outlined in Appendix E, four tissue chambers were prepared. In two
of the four chambers, rotating cams were turned on to induce mechanical conditioning while the other
two were left off and represented static culture control samples. The four chambers where then placed
into the bioreactor system and run for two different time periods. One set, consisting of a static and
conditioned chamber, was allowed to culture for 3 days while the other ran for a total of 7 days. To
ensure that the cell constructs received proper nutrients, the media (DMEM, 1X with 4.5 g/L glucose,
L-glutamine, & sodium pyruvate – modified with 10% FBS, 1% Penicillin/Streptomycin - 60 mL
per chamber) was changed every two days. Following the end of the culture periods, the tissue rings
were harvested for uniaxial tensile testing and histology to measure ultimate tensile stress (UTS),
thickness, and cell density. Thickness was measured using DVT Imaging Software while UTS was
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determined through the use of an Instron machine. The static samples were compared to the
conditioned samples to determine the effectiveness of our device.
5.5.2 Experimental Results
Two individual trials were conducted, each consisting of two time points (3 and 7 day), in which both
static (control) and mechanically conditioned (experimental) samples were examined for UTS, thickness,
and cell density.
For the first trial (Figure 35) the average UTS of unconditioned samples after a three day culture period
was 555.92 ± 129.78 kPa, whereas conditioned samples after 3 days were found to have a mean UTS of
429.60 ± 107.33 kPa (p = 0.19, therefore not statistically different). For the 7 day samples,
unconditioned rings were found to have a UTS of 82.79 ± 13.29 kPa while the conditioned rings were
measured to have a UTS of 122.42 ± 32.96 kPa (p = 0.052, therefore not statistically significant). The
UTS for both static and conditioned ring samples were significantly stronger at 3 days compared to 7
days (p<0.001).
Figure 35. Ultimate tensile strength first trial. N-values are 2, 3, 2, and 4, respectively.
These calculations were repeated for the second trial (Figure 35). After three days of culture, the static
samples exhibited an average UTS of 128.23 ± 36.8 kPa, whereas the conditioned samples were found
be able to withstand 160.27 ± 31 kPa before failing (p = 0.18, therefore not statistically different). After
seven days of culture, the static samples experienced a UTS of 84.19 ± 19.4 kPa compared to the
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conditioned samples which had a mean UTS of 84.9 ± 8.9 kPa (p = 0.48, therefore not statistically
different). All day 3 samples were compared to all day 7 samples; this statistical comparison yielded a pvalue of 0.004, therefore confirming a statistical difference between the strengths of the samples. As
before, the only conclusion that could be made was that the strength statistically decreased from 3 to 7
days. The p values for the other groupings were too low to determine statistical differences.
Figure 36. UTS second trial. N-values are 3, 3, 2, and 3, respectively.
In addition to UTS, we also measured the mean thickness of the unconditioned and conditioned
samples. Displayed in Figure 37 are the results from these experiments. The average thickness of static
samples after 3 days in culture was 0.491 ± 0.069 mm, and the 3 day conditioned samples’ average
thickness was measured as 0.548 ± 0.045 mm (p = 0.16, therefore not statistically different). After 7 days
in culture the static samples were found to have an average thickness of 1.045 ± 0.19 mm, whereas the
conditioned rings had a mean thickness of 1.041 ± 0.078 mm (p = 0.47, therefore not statistically
different). All day 3 samples were compared to all day 7 samples; this statistical comparison yielded a pvalue of 3.43 E-6, therefore confirming a statistical difference between the thicknesses of the samples.
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Figure 37. Thickness first trial. N-values are 2, 3, 2, and 4, respectively.
Thicknesses were also measured for the second trial (Figure 38). After three days of culture, the average
thicknesses for static and conditioned samples were calculated to be 0.7018 ± 0.05 mm and 0.72 ± 0.02
mm, respectively (p = 0.20, therefore not statistically different). After being cultured for seven days, the
thicknesses of the rings increased to 1.08 ± 0.08 mm for the static samples and 1.154 ± 0.09 mm for the
conditioned samples (p = 0.10, therefore not statistically different). All day 3 samples were compared to
all day 7 samples; this statistical comparison yielded a p-value of 9.6 E-10, again confirming a statistical
difference between the thicknesses of the samples.
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Figure 38. Thickness second trial. N-values are 5, 6, 6, and 5, respectively.
5.6 Histological Analysis
Rat aortic smooth muscle tissue rings (WKY 3M-22) were harvested at 3 and 7 days (Figure 39) for both
sample sets.
A
B
Figure 39. Conditioned (A) and static (B) tissue rings on silicone tubes after 7 days of culture.
The tissues were sectioned, mounted, and stained with hematoxylin and eosin and imaged under 20x
magnification. Cell count and area of the tissue in square millimeters were calculated using ImageJ
software. Mechanically conditioned samples yielded a density of 12.28 cells/mm2 after 3 days (Figure
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40- A) and 18.26 cells/mm2 after 7 days (B). Static samples yielded densities of 22.16 cells/mm2 (C) and
15.87 cells/mm2 (D) after 3 and 7 days, respectively. Data is based on sample sizes of 2 for each time
period and culture condition with the exception of conditioned 7 days, which had 1 sample. This
preliminary analysis suggests that cell density increased by 49% from three to seven days for
conditioned samples, while static samples exhibited a decrease of 29% cell density according to ImageJ
calculations.
Figure 40. 3 and 7 day tissue ring samples stained with hematoxylin and eosin at 20x magnification.
Collagen staining was also conducted using Fast Green/Picrosirius Red for all tissue ring samples (Figure
41). Upon review under 20x and 40x magnification, little to no collagen appeared to be present. In order
to definitively make a conclusion, the samples should be recut deeper into the tissue rings, or use a
biochemical assay to quantify the amount of collagen in each ring sample.
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Figure 41. Tissue samples stained with Fast Green/Picrosirius Red at 40x magnification.
Overall, all tissue rings maintained a high cell density, remained viable, and were uncontaminated for all
sample sets.
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Chapter 6 - Discussion
Each component of the final assembly was designed to meet or help meet a project objective or
constraint, including being easy to use and assemble, achieving the desired distension along the silicone
tube, providing a sterile culture environment for tissue samples, and offering a number of customizable
features to the user. Also, as our device promotes advances in medicine and patient wellness, it
addresses several greater social and ethical concerns.
6.1 Bioreactor design
6.1.1. Successfully achieved 10±1% distension
The silicone tube was statically inflated and changes in outer diameter were measured in order to
calculate the percent distension. It was determined that the displacement of 75µL of water by the 1 mL
syringe would yield an average distension of 9.8 ± 0.2%.
6.1.2. Uniformity of distension leads to high throughput
The preloaded 10 cm silicone tube was marked at every centimeter and subjected to inflation of +75µL.
While the tube exhibited inconsistent distension values at the proximal and distal ends, 10±1%
distension was consistently achieved at markers 2-8. This suggested that the tube achieves uniform
distension across at least 6 cm; thereby defining the length of usable tubing on which tissue rings may
be loaded and conditioned. In a client interview, it was determined that six tissue samples could be
loaded on each silicone tube. Therefore, the bioreactor may culture and condition a total of 30 tissue
samples simultaneously.
6.1.3. Customizability
The bioreactor is designed so the user can run up to five isolated systems at one time. Each unit has its
own mechanical conditioning system; the main components of this system are the motor and cam. The
motors interface with a set of power switches so some motors may run at 60 RPM while others are shut
off. This also presents the possibility of conditioning individual samples in intervals (e.g., one hour on,
one hour off). The size of the cam may also be changed in order to adjust the volume of medium
displaced by the syringe. A smaller cam will displace a smaller flow volume, and likewise a larger cam
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will displace a larger volume. The individual tissue chambers provide isolated culture environments for
each set of tissue rings. The user may adjust the type of culture media and additives in each system.
6.1.4. Device and materials are biocompatible
After 3 and 7 day culturing periods, cells remained viable and healthy without evidence of
contamination. This is partially due to the biocompatible and sterilizable material choices (UHMWPE,
sterile syringes, stainless steel dowels, polypropylene pinch clamps). Additionally, the tissue chamber’s
loose-fitting Petri dish-style lids allow gas exchange, and the removable tray allows the samples to be
easily transported to a biosafety cabinet where cell culture media can be replenished. Finally, the
bioreactor meets the defined size constraints and is able to fit in an incubator.
6.1.5. Easy to secure and remove samples
During the loading phase of cell testing, 88% (21 of 24) of tissue engineered blood vessel rings were
successfully loaded onto the silicone tube. This confirms that the 10 cm silicone tube (OD=1.968,
ID=1.9558) successfully houses the tissue rings and provides a simple, user-friendly assembly platform.
Furthermore, the luer fittings allow the user to access the tubing and rings which facilitates the loading
and unloading of culture samples into and out of the tissue chambers.
6.1.6. Time and budget met
The entire bioreactor was designed and manufactured within the 21 week time constraint, and the team
produced a working prototype for less than the $368 budget.
6.2 Economic impact
This bioreactor design has the potential for economic impacts in the research community and in clinical
use. The bioreactor was manufactured for less than $400. For comparison, the Harvard 22 Syringe Pump
which uses two syringes compared to five and may only contribute as a single component of other
bioreactor designs is advertised as the industry standard and costs over $3000 (Harvard Pump 22, 2010).
The production of our high-throughput bioreactor would supplement the production rate of cell culture
and research. This may lead to earlier FDA approved TEBV grafts that could be used to treat
cardiovascular disease. As these types of grafts would consist of tissues that are derived from the
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patient, fewer instances of rejection can be expected. This will ultimately result in the user enduring
fewer costs associated with pharmaceuticals, follow-up procedures, and hospital stays.
6.3 Environmental impact
The bioreactor is composed primarily of durable plastics that are abrasion resistant and may be
sterilized in an autoclave, with the only exceptions being bulk products such as pinch clamps and luer
fittings. The use of sterilizable, reusable materials minimizes the waste production associated with the
device.
6.4 Societal influence
According to the World Health Organization (WHO, 2010), heart disease is the second leading cause of
death in the world, killing about 2.5 million people annually. The development of patient-derived TEBV
grafts could therefore have a positive impact on a tremendous number of patients worldwide.
6.5 Ethical concerns
The use of laboratory animals as models for experimental purposes is a common topic of debate. Tissue
engineered constructs grown in vitro offer an alternative to animal models during research and
development. Furthermore, this device and all relevant research may lead to saving lives and improving
the conditions of millions of patients.
6.6 Health and safety issues
Our bioreactor facilitates research that may develop usable, patient-derived blood vessel grafts. Selfderived grafts are generally safer than synthetic grafts or donor transplants, where mechanical failure,
infection, compliance mismatches, and rejection of the graft are prominent issues. The production of
this bioreactor and the resultant TEBVs will ultimately lead to advances in the medical industry and
improved patient care.
6.7 Manufacturability
The bioreactor primarily consists of components that can be machined using high-speed milling
techniques and a series of machining files. Most other components in the bioreactor are standard parts
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that can be purchased from catalogs, such as luer fittings, silicone tubing, and 12V hobby motors.
Minimal assembly is required by the user, and all assembly only requires simple common tools such as a
screwdriver.
6.8 Sustainability
The plastics that are used in the body of the bioreactor (UHMWPE and polycarbonate) are both
recyclable materials. Machining scraps and even the device itself can be reprocessed and used for other
applications, thereby minimizing the amount of waste produced in the production and use of the
bioreactor.
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Chapter 7 – Final Design & Validation
A computer-generated drawing of the final design assembly is shown in Figure 42. A photograph of the
final design assembly is show in Figure 43.
The primary architecture of the assembly consists of a polycarbonate base (Figure 42 & Figure 43 - 1).
The surface of the base contains four corner barriers (2) to hold a polycarbonate tray (3). The tray has
been machined to contain five wells in which milled ultra high molecular weight polyethylene
(UHMWPE) tissue chambers sit (4). Each chamber houses a series of threaded luer fittings that are fitted
with a 10 cm, 1.968 mm outer diameter silicone tube. The distal end of the tube is clamped shut using a
polypropylene pinch clamp, which is secured to the bottom of the chamber using a 1/8” stainless steel
dowel (see Section 4.2.2 - 3).
Figure 42. Final design (CAD)
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6
7
5
2
3
4
1
Figure 43. Final design
The base also contains five hose clamps (5) and a milled section in which five 12V 60 RPM hobby motors
are mounted (6). Each motor contains a cam and interfaces with a reverse-engineered 1 mL syringe
pump (7) as described in 4.1.1-4. The design’s mechanical conditioning component consists of five 12
volt, 60 RPM gear-box hobby motor (Figure 42 & Figure 43Error! Reference source not found. - 8) which
s fixed with a circular cam (9). As the cam rotates, it depresses the plunger of the fixed 1-mL syringe
(10). It has been determined that the distance the plunger needs to be depressed in order to displace
enough water to provide a 10% distension in the silicone tubing is 4.5 mm. Therefore, the axis of
rotation for the cam is offset from the center by 4.5 mm.
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9
10
11
8
Figure 44. Mechanical stimulation.
Between the plunger head and the syringe body is a spring (11) which allows the plunger to retract as it
is in contact with the short leg of the cam. This essentially creates a syringe pump. When interfaced with
the silicone tubing through the luer fittings in the new tissue chamber design, the displacement of the
syringe plunger will cause the silicone tubing in the tissue chamber to distend (the amount of distension
depends on the size of the cam).
Both the tray and the base can be manufactured using high speed milling techniques. This assembly was
chosen as the final design because its materials and architecture helps maintain the stability of the
assembly. Also, the base internalizes most of the mechanical components in the design. Furthermore,
the removable tray provides a definable housing unit for the tissue chambers and allows the user to
easily remove and transport all tissue chambers at once.
These design aspects are appealing to both the designer and user because the small, individualized
motors minimize the size of the entire design while allowing for customizability and individual shutoffs.
Also, by having isolated a single mechanical failure is not likely to compromise the whole system. The
removals of the wheel and flat arm are also expected to decrease failure rates as there are fewer
mechanical interfaces in the system. Finally, this component of the design allows for increased
customizability; while the cam has been designed to displace only enough water to distend the silicone
tubing (and therefore the TEBV samples) by 10%, this cam may simply be changed out with another of a
larger diameter to provide greater distension of the tubing.
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The final design component is the tissue chamber compartment. This design is preferred to the other
design alternatives because it completely separates the silicone tubing and pinch clamping from the
mechanical system and the environment, and therefore significantly reduces the risk of contamination.
Also, the use of the luer fittings allows for easy removal and attachment of silicone tubes and TEBV
samples.
Our device offers several unique features that overcome the limitations of current devices. Our device
offers high throughput options by allowing for the conditioning of up to 30 tissue rings simultaneously.
The device has also been designed so the cam, syringe, and silicone tubing can be removed and replaced
with different sized components in order to adjust the flow volume and frequency, and to accommodate
different sized tissue rings. Tissue chambers are isolated from one another and the motors run
independently. This allows several different culture conditions and experimental variables to be altered
during a single experiment. Standard replaceable parts have been incorporated into the design in order
to allow the user to easily replace damaged or contaminated components. Internalizing the motors in
the polycarbonate base helps align and secure several design components. Internalized motors keep
mechanical systems separate from tissue chambers so they may remain within the incubator while the
tissue samples are transported for feeding using the removable tray.
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Chapter 8 – Conclusions and Recommendations
Through component verification and bench top tests, both aspects of the bioreactor were validated: the
mechanical conditioning system and the tissue chamber. This validation is proof-of-concept suggesting
that the bioreactor could be used to successfully impart mechanical conditioning on tissue engineered
ring samples.
8.1 Design Features
The design of a novel bioreactor which cyclically imparts mechanical conditioning was accomplished.
The preliminary tests in conjunction with the bench top validations lead to the conclusion that a 10 cm
long silicone tube sample could uniformly be distended by ~10% and therefore can impose cyclic
circumferential strain on tissue engineering ring constructs.
Compared to other devices currently available, our bioreactor design is beneficial because it allows for a
great deal of variability and customizable features. Changes in culture conditions can be varied by
altering the dimensions of the easily replaceable cam component of the mechanical stimulation system.
Additionally the individual tissue chambers allow several culture media conditions to be altered at once.
Motors run independently from one another so several static and mechanically conditioned samples can
be cultured simultaneously and for different periods of time.
Another benefit to our device is that with the total number of tissue chambers, a high throughput is
achieved. Our device is capable of housing up to six tissue rings per chamber and the entire system is
capable of maintaining five chambers yielding in total 30 tissue rings being conditioned in vitro during a
single experiment.
8.2 Future Recommendations
Having conducted one successful full system experiment the team has developed the following
recommendations.
The current hose clamps being used as syringe holders are very stiff and have been shown to crack over
time with repeated use. We suggest that an alternative be designed or purchased. Even though the hose
clamps successfully support the syringe during the conditioning cycles, it is very difficult to insert and
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remove the syringe barrels. Metal syringe holders would be an appropriate alternative to the current
holders. Additionally, the syringe plunger was found to be prone to deformation due to the high levels
of shear stress created at the plunger and cam interface. We suggest that either a metal syringe or a
stiffer syringe be used.
Small amounts of leaking were also observed in two of the four chambers during testing. It was
determined that an adjustment of the threaded luer located in the wall of the tissue chamber resolved
this issue. However, in order to avoid this from occurring during future experiments we suggest that a
rubber o-ring be placed around the thread of the luer. Another alternative could be to permanently fix
the threaded luer into the wall of the tissue chamber, preventing the possibility of user error during
assembly.
Even though the team experienced no contamination during our week long experiment, we suggest that
tissue chamber lids be machined with overhang rather than the current design’s lids which simply rest
on top of the tissue chambers. We feel that this will aid in the prevention of spilling during transport
from biosafety cabinet to incubator and will further reduce the chance of contamination.
During the manufacturing stage we learned that the UHMWPE sheets used for the tissue chambers was
a relatively soft plastic compared to other materials available in the market. For our purposes, the
material was sufficient, however it was not the easiest to machine due its softness. We recommend that
alternative plastics be considered to avoid issues with milling, tapping, and cutting.
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Appendix A – Pairwise Comparison Chart (PCC)
Bioreactors
Accurate
(10 ±1%
Adjustable
size of
ring/tube
High
Throughput
Programma
ble
TEBVs
easily
secured
TEBVs
easily
removed
Media
supply
Waste
removal
Inexpensive
Accura
te (10
±1%
Adjusta
ble size
of
ring/tub
e
1
0
Programma
ble
TEBVs
easily
secur
ed
1
0.5
1
1
0
0.5
1
6
0
0
0
0
0
0
1
1
1
0.5
0.5
0
0
1
4
1
1
0
0.5
1
4.5
2.94%
11.76
%
13.24
%
0.5
0
0
1
3
8.82%
0
0
1
3
1
1
7
1
5.5
8.82%
20.59
%
16.18
%
0
0.00%
High
Through
put
TEBVs
easily
remov
ed
Medi
a
supp
ly
Waste
remov
al
Inexpensi
ve
Tota
ls
0
1
0.5
1
0
0
1
0.5
0
0
1
0.5
0
0.5
1
1
1
1
1
1
0.5
1
1
0.5
1
1
0
0
0
0
0
0
0
0
0
Perce
nt
Total
17.65
%
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Appendix B – A Guide to Heat Shrink Tubing
Goals:

Effectively seal silicone tubing to 1/16” inner diameter male barbed bayonet luer fittings
Materials:







Scissors/scalpel
Heat gun (preferably with low and high heat settings)
Tweezers/forceps
Gloves
Biocompatible heat shrinkable tube (1/8" diameter)
Silicone tubing
Male barbed bayonet luer fitting for 1/16" inner diameter tubing
Procedure:
1: Using gloves to handle the silicone tubing, cut the tubing to desired length (10cm)
Note: For more accurate, clean cuts, use a scalpel on a flat surface alongside of a measuring device
2: Cut heat shrink tubing to desired length (3-5mm or long enough to cover the barb and silicone tubing
on the luer fitting) (see Figure below for approximate scaled sizing)
3: Using gloves to handle the silicone tubing, attach the silicone tubing to the barbed end of the luer
fitting, ensuring that the tubing is secured above the barb (see Figure below)
4: Using gloves, cover the silicone tubing and barbed tip overlap with heat shrink tubing (see Figure
below)
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5: Remove your gloves (in case of heat exposure) and use tweezers/forceps to pick up the luer fitting by
its’ hollow end (see Figure below)
6: Position the heat gun pointing away from yourself and turn it on (depending on the type of heat gun
used, this may require an additional person to hold the heat gun while the original person completes
steps 7-9) (see Figure below for model heat gun we used)
7: Hoist the silicone tubing—heat shrink tubing—luer fitting combination approximately 1.5 inches from
the mouth of the heat gun using the tweezers/forceps and spin continuously for 10-30 seconds to
adhere the heat shrink tubing to all sides of the luer fitting (*BEWARE of melting the luer fitting due to
positioning the parts too closely to the heat gun) (see Figure below)
8: Repeat for multiple tubing assemblies
9: Switch the heat gun to the cool setting for a minimum of one minute before shutting the heat gun off
and storing it
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Appendix C – A Guide to Wiring and Sealing Motors
Potting and Sealing Motors
Materials:











12 V 60 RPM DC motor (x5) mounted into motor mount
Epoxy, hysol M-121hp medical device epoxy adhesive (50 mL)
Hysol adhesive for encasing, urethane, (50 mL)
Potting gun
Easy-ID low voltage cable, 24/2 AWG, 0.11” W x 0.06” thick, 12 VDC, 50’ L
Wire cutters
Heat gun
Soldering kit
Vice
Moisture-seal polyolefin heat-shrink tubing, 1.5” ID before, 0.75” ID after – cut into 2.5” lengths
(x5)
Small diameter heat shrink tubing for wiring
Procedure (Note – do everything listed for each motor individually):
1. Cut wire to 6 foot lengths
2. Strip wire and fix to motor leads
3. Solder wire to motor leads
4. Heat shrink positive and negative wires near soldering point to prevent further separation of zip
wire
5. Secure motor mount into vice facing upside down with wires coming out the top
6. Pot motor holes with epoxy hysol M-121 medical device epoxy and potting gun
7. Once all motors have been potted, flip motor mount in vice so epoxy drips down wires (and not
into motor)
8. Let dry for 6 hours
9. Attach moisture-seal heat-shrink tubing to outside of motors (encasing both the gearbox and
motor)
10. Use heat gun to permanently attach tubing around motor
11. Fill remaining area within heat shrink tubing with hysol adhesive for encasing
12. Position wires in the same direction perpendicular to the long side of the motor mount and
pinch the heat shrink tubing and hysol adhesive encasing closed around the wire
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13. Let dry for 24 hours
Before:
After:
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Wiring Motors to Power Supply
Materials:







Motors sealed into motor mount and base
5-hole 22mm button enclosure, plastic
Selector switch, 22mm, plastic (x5) + contact blocks
Easy-ID low voltage cable, 24/2 AWG, 0.11” W x 0.06” thick, 12 VDC, 50’ L
Wire cutters
Wire connectors (male and female pins) (x10)
16 AWG power cable
Procedure:
1. Assemble the button enclosure as per instructions given with purchase
2. Measure, cut, and strip (all wires should look like this when stripped) the ends of four 5 cm long
pieces of positive (red) 24/2 AWG 12 VDC wire (see Figure below)
3. Attach and screw the pieces of wire between the 5 selector switches (see blue arrows in Figure
below)
Wire
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4. Measure, cut, and strip the ends of five pieces of positive (red) 24/2 AWG 12 VDC wire so all
wires feed out of the same side of the 5-hole 22mm button enclosure and end at approximately
the same point (see Figure below)
5. Attach and screw one length of wire to each contact block (see Figure below)
Screw in Here
6. Strip the other end of the same lengths of wire and crimp push-in wire connector pieces to the
ends of each wire and fit into plastic alignment case, ensuring wires of the same color are all
secured on the same side
7. Cut and strip 5 lengths of negative (black) 24/2 AWG 12 VDC wire to equal lengths
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8. Crimp push-in wire connector pieces to the ends of these lengths of wire and fit into the same
plastic alignment case, ensuring wires of the same color are all secured on the same side (see
Figure below)
9. Cut and strip a 60 cm length of wire (still zip connected together) and attach one end of the
negative (black) wire and positive (red) wire to the power supply and secure with a screw (see
orange box in Figure below)
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10. Cut and strip a 5 cm long piece of negative (black) wire and twist together with all other free
ends of negative (black) wires, securing together with tape (see Figure below)
11. Attach the free end of the positive (red) wire to the contact block nearest the 22 mm button
enclosure’s exit hole (see orange arrow in the Figure in step 3)
12. Attach the 16 AWG power cable to the power supply (see green box in Figure in step 9) and
cover all wires with electrical tape
13. Strip free ends of 6 foot long wire sealed into 60RPM 12V DC motors and crimp push-in wire
connectors onto lengths of wire and fit into plastic alignment case, ensuring wires of the same
color are all secured on the same side (see Figure below)
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Final Product:
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Appendix D – Building Our Device
The following engineering drawings profile the geometry and dimensions of the components of the
device that were machined. These include the tissue chamber, base, motor mount, and removable tray.
All of these components were manufactured from raw plastic sheets using high-speed milling
techniques in a HAAS automated mill. The following models and dimensions were programmed using
SolidWorks and FeatureCAM softwares, and were uploaded into the mill’s computer system.
HAAS Milling Machine
Partially Milled Tissue Chamber
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Tissue Chamber
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Base & Tray Holders
Motor Mount
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Tray
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Appendix E – User Manual
Prepare autoclaved tools & materials:
-
Forceps (narrow tip, 450 and/or 900)
-
Forceps (blunt curved tip)
-
Flat spatula (for grease)
-
Aspirator tips
-
10 cm silicone tube heat shrunk to 1/16” inner diameter female barbed polypropylene luer (x5)
-
Tissue chamber (x5) with stainless steel dowel pin fitted into pinch clamp hole and
polypropylene female ¼-28 threaded luer mounted into chamber wall
-
Tissue chamber lids (x5)
-
Compression spring (number dependent on number of mechanically conditioned tissue
chambers in use)
-
Silicone-based high vacuum grease (Dow Corning, Midland, MI) (as needed)
Non-autoclaved, sterile materials:
-
Latex gloves (as needed)
-
10 & 25 mL setological pipets (as needed)
-
Sterile Monoject® 1mL TB syringe (x5)
-
25 cm2 tissue culture flask (x2)
-
90mm (or larger if preferred) tissue culture dish (x2)
-
140mm tissue culture dish
-
Culture media (DMEM, 1X with 4.5 g/L glucose, L-glutamine, & sodium pyruvate) (~50 mL)
-
Complete culture media (DMEM, 1X with 4.5 g/L glucose, L-glutamine, & sodium pyruvate –
modified with 10% FBS, 1% Penicillin/Streptomycin) (~300 mL)
Tools & materials cleaned with 70% ethanol solution:
-
Pipetter
-
Aspiration hose
-
Base of device (including motors+ cams + syringe holders)
-
Tray of device
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Incubator Set-up Procedure:
1.) Spray and wipe down the base component of the device and position in the incubator with the
motors furthest from, and parallel to, the front of the incubator.
2.) Feed the wires through the hole at the back of the incubator.
3.) Arrange the switches and power supply on top of or next to the incubator near an electrical
outlet and away from any liquids.
4.) Connect the male and female wire connectors (red wires to red wires; black wires to black
wires), being very careful not to bend any of the pins in the process (if bent, motors may not
turn on – can be fixed by straightening or pulling on pins).
5.) Plug in the power supply.
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Ring Loading onto Silicone Tube Procedure:
*Note – All procedural steps take place within a sterile biosafety cabinet unless otherwise specified.
Whenever leaving the biosafety hood, be sure to apply 70% ethanol before re-entering to control
contamination risk.
1.) Fill base of 140mm tissue culture dish with ~ 25mL culture media.
2.) Aspirate media out of tissue ring culture molds.
3.) Carefully remove tissue rings from culture molds using narrow 90o forceps and submerge in
tissue culture dish media.
4.) Repeat for all tissue rings.
5.) Open autoclaved silicone tubing and place within tissue culture dish with tissue rings.
6.) Using narrow forceps, place tissue ring (arrow) around one open grip of the blunt curved forceps
and angle blunt curved forceps upwards in media to prevent ring sliding.
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7.) Use narrow forceps to place the open end of the silicone tubing tightly over the open grip
containing the tissue ring.
8.) Use narrow forceps to slide the tissue ring (arrow) onto the silicone tube and position it
approximately 2 cm from the luer.
9.) Let silicone tube and tissue rings remain submerged in culture media while loading other rings
and samples.
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10.) Repeat steps 6-9 until 5-6 tissue rings have been successfully loaded ~1 cm apart on the silicone
tube.
11.) Repeat steps 5-10 until 5 silicone tubes have been loaded with 5-6 tissue rings each (Note – if
using less tissue chambers, use fewer silicone tubes, lids, and syringes.
Tissue Chamber Loading Procedure:
*Note – All procedural steps take place within a sterile biosafety cabinet unless otherwise specified.
Whenever leaving the biosafety hood, be sure to apply 70% ethanol before re-entering to lessen
contamination risk.
1.) Set up biosafety cabinet as seen in Figure # below, adding more tissue chambers if necessary.
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2.) Fill tissue culture flask with ~25 mL of culture medium using pipetter.
3.) Remove syringe plunger, place compression spring around plunger, and reinsert plunger into the
syringe barrel.
4.) Depress syringe plunger until spring is fully compressed, insert syringe into tissue culture flask,
and fill syringe to the 0.9 mL mark on the syringe barrel.
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5.) Invert the syringe and tap out any extra air bubbles that may exist within the syringe barrel.
6.) Depress the plunger to remove the excess air that floats to the syringe tip during tapping and
place the syringe in a sterile petri dish.
7.) Fill the tissue chamber with ~25 mL of complete culture medium (with FBS and P/S added).
8.) Using foreceps, remove a tissue ring loaded silicone tube from the 140 mm culture dish and
carefully place it into the tissue chamber.
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9.) Using foreceps, grip the luer attached to the silicone tube and fit in tightly over the luer
threaded into the chamber wall.
10.) Insert the 1 mL push-connect syringe into the outer opening of the threaded luer in the tissue
chamber wall.
11.) Depress the syringe plunger until the compression spring is flush with the plunger head and
syringe barrel.
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12.) Use a pair of forceps to hold a pinch clamp in one hand, use the other hand to push the free end
of the silicone tube through the pinch clamp opening.
13.) Hold the pinch clamp sideways onto the base of the tissue chamber with a pair of forceps with
one hand, and with your other dominant hand, grip the inside of the silicone tube using narrow
tipped forceps and pull or slide the silicone tube beyond the clamping point. Be very careful not
to damage the tubing or pull it more than half a centimeter past the clamping point.
14.) Place the pinch clamp onto the steel dowel at the base of the tissue chamber.
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15.) Use one hand to pressurize the syringe plunger down to 0.2 mL and the second hand to press
the pinch clamp closed using a pair of forceps.
16.) Fill the tissue chamber with an additional ~35 mL of complete culture medium (with FBS and P/S
added).
17.) Cover the tissue chamber with a sterile lid.
18.) Position the tissue chamber within the tray with the clamp-end nearest to the plastic screws and
secure the tissue chamber into the base by hand-tightening the screw (can further tighten once
removed from the biosafety cabinet).
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19.) Repeat steps 2-19 until all tissue chambers are secured and ready for transport from the
biosafety cabinet to the incubator.
20.) Carefully lift the tray by its handles with the syringes pointing away from you and transport it to
the incubator.
21.) Position the tray within the tray holder, with syringes on top of the syringe holders in the
incubator.
22.) Depress the syringe plunger so that it’s flush with the cam, and snap the syringe(s) into place.
23.) Close the incubator doors and switch on the mechanically conditioned tissue chambers.
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Appendix F – Bill of Materials
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