OriCellP Human Umbilical Cord Blood Mesenchymal Stem Cell

Cyagen Biosciences Inc.
www.cyagen.com
OriCellTM Human Umbilical Cord Blood Mesenchymal Stem Cell
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Chondrogenic Differentiation Medium
Catalog No. HUXUB-90042
Product Description:
Human Umbilical Cord Blood MSC Chondrogenic Differentiation Medium consists of optimized Human
Umbilical Cord Blood MSC Chondrogenic Differentiation Basal Medium and supplements. This product
has been developed for the optimal differentiation of Human Umbilical Cord Blood MSC (Cat. No.
HUXUB-01001) into cartilages.
The product is intended for laboratory research use only, not for drug, house hold, or other uses.
Kit Component:
Human Umbilical Cord Blood MSC Chondrogenic Differentiation Basal Medium
97 mL
(Cat. No. HUXUB-03042-97)
Dexamethasone
Ascorbate
ITS + Supplement
10 μL
300 μL
1 mL
Sodium Pyruvate
100 μL
Proline
100 μL
TGF-β3
1 mL
Instructions:
Prepare Incomplete Chondrogenic Induction Medium
1. Disinfect with 70%v/v ethanol the external surfaces of the bottles/vials for Basal Medium and the
following component in the kit. Allow ethanol to evaporate away.
a) Dexamethasone
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b) Ascorbate
c) ITS + Supplement
d) Sodium Pyruvate
e) Proline
2. Aseptically open the above components and add the contents to Human Umbilical Cord Blood
Mesenchymal Stem Cell Chondrogenic Differentiation Basal Medium to prepare the Incomplete
Chondrogenic Induction Medium.
3. Rinse each vial with the medium. It may not be possible to recover the entire content of each
component. Small losses should not affect cell characteristics.
4. Store the Incomplete Chondrogenic Induction Medium at 2 °C to 8 °C in the dark until needed.
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Aliquot TGF-β3:
Aliquot small volumes of TGF-β3 into freezer-safe tubes and store at less than –70°C for no more than
6 months.
Note: 10 µL of TGF-β3 will convert 1 mL of Incomplete Chondrogenic Medium into Complete
Medium.
Complete Chondrogenic Induction Medium:
1. After thawing, the aliquot of TGF-β3 may need to be centrifuged briefly at low speed to pull the
small volume to the bottom of the tube.
2. Pipette the volume of Incomplete Chondrogenic Induction Medium that you intend to supplement
(e.g. 10 mL) into a tube.
3. To recover the full volume of TGF-β3, transfer 100 µL of this Incomplete Chondrogenic Medium to
the tube of TGF-β3.
4. Mix the solution by pipetting and transfer it back to the tube of Chondrogenic Induction Medium.
Repeat this process to be certain that you have recovered the TGF-β3. Cap and invert several times
to mix.
5. The Chondrogenic Induction Medium is now complete.
Note: Complete Chondrogenic Medium must be prepared fresh and used within 12 hours.
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Chondrogenesis Protocol:
1. Calculate the total number of pellet cultures required for your experiment (2.5×105 MSCs are needed
to form each chondrogenic pellet). Transfer this amount of cells to an appropriate culture tube to
wash the cells.
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2. Wash the MSCs with Incomplete Chondrogenic Medium: Centrifuge the cells at 150g for 5 minutes
at room temperature, and aspirate/discard the supernatant. Resuspend the cells in 1 mL Incomplete
Chondrogenic Medium per 7.5×105 cells, centrifuge again at 150g for 5 minutes and aspirate/discard
the medium.
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3. Resuspend the MSCs in Complete Chondrogenic medium to a concentration of 5.0×105 cells per
mL.
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4. Aliquot 0.5 mL (2.5×105 cells) of the cell suspension into 15 mL polypropylene culture tubes.
Centrifuge the cells at 150 g for 5 minutes at room temperature.
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DO NOT aspirate the supernatant or resuspend the pellet.
5. Loosen the caps of the tubes one half turn to allow gas exchange and incubate the tubes at 37°C, in a
humidified atmosphere of 5% CO 2 . Do not disturb the pellets for 24 hours.
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6. Feed the cell pellets every 2-3 days by completely replacing the medium in each tube (to avoid
aspirating the pellets when aspirating the medium, attach a sterile 1-200μL pipette tip to the end of
the aspirating pipette). Add 0.5 mL of freshly prepared Complete Chondrogenic Medium to each
tube.
7. After replacing the medium, flick the bottom of each tube to ensure that the pellet is freefloating,
loosen the caps and return the tubes to the 37°C incubator.
8. Chondrogenic pellets should be harvested after 14 to 28 days in culture. Pellets may be formalin
fixed and paraffin embedded for Alcian blue stain.
Alcian Blue Staining Procedure
1. Fixation: formalin fixed, paraffin embedded tissue sections.
2. Staining procedure:
a) Deparaffinize slides and hydrate to distilled water.
b) Stain in alcian blue solution for 30 minutes.
c) Wash in running tap water for 2 minutes.
d) Rinse in distilled water.
e) Visualize under light microscope, and capture images for analysis. Blue staining indicates
synthesis of proteoglycans by chondrocytes.
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Stability/Storage:
All products should be stored in the dark.
Human Umbilical Cord Blood Mesenchymal Stem Cell Chondrogenic Differentiation Basal Medium is
stable at 2 to 8 °C for up to one year. ITS + Supplement is stable for a minimum 3 months at 2 to 8 °C,
Other components are stable at -20 °C for up to two years. These products should be discarded beyond
the labeled expiration date.
Once prepared, the fully supplemented complete medium can be stored for 12 hours when stored in the
dark at 2 to 8 °C.
For optimal performance, repeated warming/cooling and freeze-thawing should be avoided.
Quality Control:
Human Umbilical Cord Blood Mesenchymal Stem Cell Chondrogenic Differentiation Medium is
performance tested on Human Umbilical Cord Blood Mesenchymal Stem Cells.
Standard evaluation includes:
1. Sterility test (bacteria, fungi, mold and mycoplasma)
2. pH test
3. Endotoxin
Cyagen Biosciences reserves all rights on the technical documents of its
OriCellTM cell culture products. No part of this document may be reproduced
or adapted for other purposes without written permission from Cyagen
Biosciences.
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