Clonetics™ bovine brain microvascular endothelial cell

Clonetics™ bovine brain microvascular endothelial cell
www.lonza.com
U.S. Scientific Support: 800-521-0390
scientific.support@lonza.com
EU/ROW Scientific Support: +49-221-99199-400
scientific.support.eu@lonza.com
Document # AA-1037-6 06/11
© 2011 Lonza Walkersville, Inc
Clonetics™ bovine brain microvascular endothelial cell system
Instructions for use
should not affect the cell growth characteristics
of the supplemented medium.
4. Transfer the label provided with each kit to the
basal medium bottle being supplemented. Use it
to record the date and amount of each
supplement added. We recommend that you
place the completed label over the basal
medium label (avoid covering the basal medium
lot # and expiration date) to avoid confusion or
possible double supplementation.
5. Record the new expiration date on the label
based on the shelf life.
Safety statements
THESE PRODUCTS ARE FOR RESEARCH USE ONLY. Not
approved for human or veterinary use, for application to humans
or animals, or for use in vitro diagnostic or clinical procedures.
WARNING: CONTAINS BOVINE SOURCE MATERIAL, TREAT
AS POTENTIALLY INFECTIOUS. PREPARED FROM U.S.
SOURCE BOVINE BRAIN. Not tested for bovine viruses or BSE.
Products should be handled at the biological safety level 2 to
minimize exposure of potentially infectious products, as
recommended in the CDC-NIH manual, Biosafety in
Microbiological and Biomedical Laboratories, 5th edition. If you
require further information, please contact your site safety officer
or scientific support.
Unpacking and storage instructions
1. Check all containers for leakage or breakage.
2. For cryopreserved cells – remove cryovials from
the dry ice packaging and immediately place into
liquid nitrogen storage. Alternatively, thaw and
use the cells immediately. If no dry ice remains,
please contact customer service.
3. BulletKit™ instructions: upon arrival, store basal
medium at 2-8°C and SingleQuots™ at –20°C in
a freezer that is not self-defrosting. If
SingleQuots™ are thawed upon arrival, they can
be stored at 2-8°C and added to basal medium
within 72 hours of receipt. After SingleQuots™
are added to basal medium, use within 1 month.
Do not re-freeze. Using medium or reagents other
than what’s recommended will void the cell warranty.
Please contact scientific support if you need help
selecting media and/or reagents.
Preparation of media
For a BulletKit™, perform the following steps:
1. Decontaminate the external surfaces of all
supplement vials and the medium bottle with
ethanol or isopropanol.
2. Aseptically open each supplement vial and add
the entire amount to the basal medium with a
pipette.
3. Rinse each cryovial with the medium. It may not
be possible to recover the entire volume listed
for each cryovial. Small losses, even up to 10%,
Note: If there is concern that sterility was compromised
during the supplementation process, the entire newly
prepared growth medium may be refiltered with a 0.2 µm
filter to assure sterility. Routine refiltration is not
recommended.
Thawing of cells / initiation of culture
process
bMVEC-B cells should be seeded onto rat tail
collagen type I coated multi-well culture plates or
inserts. Polycarbonate membrane inserts are
recommended for bMVEC-B cells. Collagen coated
inserts must be over-coated with fibronectin prior to
seeding the cells. Coating materials are not
supplied with the cells, however rat tail collagen type
I and fibronectin are available commercially. Coat
culture vessels with collagen according to the
manufacturer’s procedure. We recommend coating
2
10-25 µg of collagen per cm of culture surface.
When using polycarbonate membrane culture
inserts, over-coat the collagen coating with
2
fibronectin at 5 µg/cm , using the fibronectin
manufacturer’s recommended procedure. Have
culture vessels coated and ready to use before
thawing cells.
1. The recommended seeding density for bMVEC2
B cells is 20,000 to 50,000 cells/cm . The
highest density is recommended when seeding
onto polycarbonate membrane culture inserts.
All trademarks herein are marks of Lonza Group
or its subsidiaries.
Page 1 of 2
2. To set up cultures, calculate the number of
vessels needed based on the recommended
seeding density and the surface area of the
vessels.
3. Calculate the volume of growth medium required
to set up the culture based on the
manufacturer’s recommended volumes for the
vessels. In a sterile field, remove the required
volume of medium into a sterile secondary
container. Also remove 10 ml of medium into a
sterile centrifuge tube. Warm the medium in both
the container and tube to 37°C in a water bath.
4. Wipe cryovial with ethanol or isopropanol before
opening. In a sterile field, briefly twist the cap a
quarter turn to relieve pressure, and then
re-tighten. Quickly thaw the cryovial in a 37°C
water bath being careful not to submerge the
entire vial. Watch the cryovial closely; when the
last sliver of ice melts remove it. Do not
submerge it completely. Thawing the cells for
longer than 2 minutes results in less than
optimal results.
Seeding
1. Using a 1000 µl micropipetter with a tip set to
1000 µl, transfer the contents of the cryovial into
10 ml of warm medium. Cap the tube and gently
invert twice to mix.
2. Centrifuge the tube for 10 minutes at 150 x g.
3. Without disturbing the cell pellet, remove
supernatant with a 10 ml pipette (do not aspirate
with vacuum) and transfer it into a second sterile
tube.
4. Resuspend the pellet in an appropriate volume
of warm growth medium and seed the cells into
rat tail collagen type I coated culture vessels.
5. Place the culture vessels in 37°C, 5% CO 2
incubator. Leave the culture vessels untouched
for at least 48 hours to allow the cells to anchor
to the collagen coated surface.
Maintenance
1. Change the growth medium 48 to 72 hours after
seeding and every other day thereafter.
2. Warm an appropriate amount of medium to 37°C
in a sterile container. Remove the medium and
replace it with the warmed, fresh medium and
return the flask to the incubator.
3. Avoid repeated warming and cooling of the
medium. If the entire contents are not needed
for a single procedure, transfer and warm only
the required volume to a sterile secondary
container.
Ordering information
Cryopreserved cells (pool of multiple donor
brains)
AC-2509
bMVEC-B
≥750,000 cells
Related products
™
BMVEC-B growth medium Bulletkit (EMVB
BulletKit™) (must be purchased separately):
AC-3103
EMVB
BulletKit™
Kit which contains a
500 ml bottle of EBM™
2 and EMVB
SingleQuots™
CC-3156
EBM™-2
Endothelial basal
medium-2 (no growth
factors) (500 ml)
AC-4207
EMVB
Supplements and
SingleQuots™ growth factors (b
ECGF, ascorbic acid,
heparin, platelet poor
horse serum and
penicillin, streptomycin,
fungizone).
Product warranty
CULTURES HAVE A FINITE LIFESPAN IN VITRO.
Lonza warrants its cells in the following manner only
if Clonetics™ media and reagents are used.
1. Clonetics™ cryopreserved endothelial cultures
are warranted for 1 experimental use without
prior expansion in culture.
Quality control
All cell strains test negative for mycoplasma,
bacteria, yeast and fungi. bMVEC-B test positive for
acetylated LDL uptake. The cell strains also test
positive for the drug transporter P-glycoprotein
function by calcein AM efflux assay using verapamil
hydrochloride as a P-gp inhibitor. All QC testing is
conducted using the recommended Clonetics™
medium. For detailed information concerning QC
testing, please refer to the certificate of analysis.
All trademarks herein are marks of Lonza Group
or its subsidiaries.
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