Mesenchymal Stem Cell Medium-animal component free
(MSCM-acf)
Catalog Number: 7521
Product Description
Mesenchymal Stem Cell medium-animal component free (MSCM-acf) is designed for optimal
growth and expansion of normal mesenchymal stem cells (MSCs) in vitro. MSCs can be
expanded for several passages while maintaining their multipotential phenotype. It is a sterile,
liquid medium which contains non-animal source essential and non-essential amino acids,
vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals. The
medium is HEPES and bicarbonate buffered and has a pH of 7.4 when equilibrated in an
incubator with an atmosphere of 5% CO2/95% air. The medium is formulated (quantitatively
and qualitatively) to provide a defined and optimally balanced nutritional environment at serumfree
condition that selectively promotes growth of normal human mesenchymal stem cells in
vitro.
Components
MSCM-acf consists of 500 ml of basal medium, 5 ml of mesenchymal stem cell growth
supplement-animal component free (MSCGS-acf, Cat. No. 7572) and 5 ml of
penicillin/streptomycin solution (P/S, Cat. No. 0503).
Product Use
MSCM-acf is for research use only. It is not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Storage
Store the basal medium at 4oC, the MSCGS-acf and the P/S solution at -20oC. Protect from light.
Shipping
Gel ice.
Prepare for use
Thaw MSCGS-acf and P/S solution at 37oC. Gently tilt the MSCGS-acf tube several times
during thawing to help the contents dissolve. Make sure the contents of the supplement are
completely dissolved into solution before adding to the medium. Rinse the bottle and tubes
with 70% ethanol, and then wipe to remove excess. Remove the cap, being careful not to touch
the interior threads with fingers. Add MSCGS-acf and P/S solution into basal medium in a sterile
field, mix well and then the reconstituted medium is ready for use. Since several components of
MSCM-acf are light-labile, it is recommended that the medium not be exposed to light for
lengthy periods of time. If the medium is warmed prior to use, do not exceed 37oC. When stored
in the dark at 4oC, the reconstituted medium is stable for one month.
Caution: If handled improperly, some components of the medium may present a health hazard. Take appropriate
precautions when handling it, including the wearing of protective clothing and eyewear. Dispose of properly.
ScienCell
Research Laboratories
TM
Instruction for culturing cells in MSCM-acf
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the order:
1. Prepare a poly-L-lysine (PLL, Cat. No. 0403) coated flask (2 μg/cm2, T-75 flask
is recommended) and leave the flask in incubator overnight (minimum one hour at 37oC
incubator).
2. Prepare mesenchymal stem cell medium-animal component free (MSCM-acf Cat.
No. 7521): decontaminate the external surfaces of medium and medium supplements with
70% ethanol and transfer them to sterile field. Aseptically open each supplement tube and
add them to the basal medium with a pipette. Rinse each tube with medium to recover the
entire volume.
3. Rinse the poly-L-lysine coated flask with sterile water twice and add 20 ml of
complete medium to the flask. Leave the flask in the hood and go to thaw the cells.
4. Place the vial in a 37oC waterbath, hold and rotate the vial gently until the
contents are completely thawed. Remove the vial from the waterbath immediately, wipe
it dry, rinse the vial with 70% ethanol and transfer it to a sterile field. Remove the cap,
being careful not to touch the interior threads with fingers. Using 1 ml eppendorf pipette
gently resuspend the contents of the vial.
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated
culture vessels. A seeding density of >=10,000 cells/cm2 is recommended.
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of DMSO residue in the culture. It is
also important that cells are plated in poly-L-lysine coated culture vessels that promote
mesenchymal stem cell attachment.
6. Replace the cap or cover of flask, and gently rock the vessel to distribute the cells
evenly. Loosen caps if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. For best result, do not disturb the culture for at least 16 hours after the culture has
been initiated. Change the medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter. A healthy culture will display fibroblastlike
morphology, usually in a scattered single cells rather than a homogeneous bundle or
sheet of cells; and the cell number will be doubled after two to three days in culture.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells. For subsequent subcultures, change medium 48 hours
after establishing the subculture.
2. Change the medium every other day thereafter, until the culture is approximately 50%
confluent.
3. Once the culture reaches 50% confluence, change medium every day until the culture is
approximately 80% confluent.
Subculture cells MSCM-acf:
1. Subculture the cells when they are 90% confluent (It should be about 4-5 days after last
passage, if a seeding density of 10,000 cells/cm2 is used).
Note: Over-confluent culture may cause cell death. In addition, initiating subculture
under this condition may affect medium performance.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2).
3. Warm medium, trypsin/EDTA solution, trypsin neutralization solution, and DPBS to
room temperature. We do not recommend warming the reagents and medium at 37oC
waterbath prior to use.
4. Rinse the cells with DPBS.
5. Incubate cells with 9 ml of DPBS (Cat. No. 0303) and 1 ml of trypsin/EDTA (T/E, Cat.
No. 0103) solution (in the case of T-75 flask) until 80% of cells are rounded up
(monitored with microscope. DO NOT over-trypsinize the cells). Add 10 ml of trypsin
neutralization solution (TNS, Cat. No. 0113) to the digestion immediately and gently tap
the culture vessel.
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over-trypsinization.
6. Harvest and transfer released cells into a 50 ml centrifuge tube. Rinse the flask with
another 10 ml of growth medium to collect the residue cells. Examine the flask under the
microscope to make sure the harvesting is successful by looking at the number of cells
left behind. There should be less than 5%.
7. Centrifuge the harvested cell suspension at 1000 rpm for 5 min and resuspend cells in
growth medium.
8. Count cells and plate them in a new, poly-L-lysine coated flask with >=10,000 cells/cm2
seeding density.
Note: The optimal cell expansion was obtained at >= 10,000 cells/cm2. Reduced
seeding density may affect cell growth in serum free medium.
Caution: Handling human derived products is potentially bioharzadous. Although each cell strain testes negative
for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions
mush be taken to avoid inadvertent exposure. Always wear gloves and safety glasses when working these materials.
Never mouth pipette. We recommend following the universal procedures for handling products of human origin as
the minimum precaution against contamination [1].
[1]. Grizzle, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues. J Tissue Culture Methods. 11(4).