S&XFM TM MSC XF Medium has been developed for the growth and expansion of human mesenchymal stem cells (MSCs) under completely animal serum-free and, antibiotics-free, xeno-free conditions. Using S & XFM TM MSC XF Medium. Human MSCs can be expanded for multiple passages while maintaining their multipotent phenotype (i.e. ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages).
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S&XFMTMMSC XF Basal Medium
S & XFM TM MSC XF Supplement
* Note: S & XFM TM MSC XF Medium , its components are not available separately.
For research use only. CAUTION Not intended for human or animal diagnostic or therapeutic uses.
Thaw S & XFM TM MSC XF Supplement overnight at 2oC to 8oC prior to use (thawed supplement will have a slightly cloudy appearance). Use thawed material immediately or aliquot (i. e. 1mL) unused material and store at-20 oC 0 to-50 oC. Avoid additional freeze-thaw cycles. S & XFM TM MSC XF Medium (Basal Medium and Supplement) is stable for 2 weeks when stored in the dark at 2oC to 8oC.
Standard physical growth conditions for human mesenchymal stem cells in S & XFM TM MSC XF Medium are 37 oC in a humidified atmosphere of 5% CO. Using standard aseptic conditions. Ensure that proper gas exchange is achieved in cultures vessels. Avoid overexposure of cultures to light.
1. For 500mL S & XFM TM MSC XF Medium, aseptically add 50mL of S & XFM TM MSC XF Supplement to S & XFM TM MSC XF Basal Medium 450mL.
2. If desired, add 50 µL of 50 mg/mL gentamicin reagent solution to the S & XFM TM MSC XF Medium .
1. Rapidly thaw a frozen vial of human MSCs in a 37oC water bath until a small amount of ice remains.
2. Pipet the entire content of the cryovial into a 50mL conical tube.
3. Carefully add 5-10 mL of pre-warmed (37oC) S & XFM TM MSC XF Medium to the conical tube at an approximate rate of 3 to 5 drops per seconds and gently swirl after every addition.
4. Centrifuge the tubes at 300-4000 x g for 5 minutes at room temperature.
5. Resuspend the cell pellet in pre-warmed (37oC) S & XFM TM MSC XF Medium. And add the cell suspension to an appropriate S & XFM TM MSC XF Medium flask at a density of 2x 103 cells/cm2.
6. Incubate at 37oC in a humidified atmosphere of 5% CO2 in air.
7. Replace the medium in the flasks every 2 days.
1. observe the stock culture flask (cells growing in current medium formulation or in S & XFM TM MSC XF Medium) under the microscope and confirm that the cells are ready to be subpassaged (60-90% confluent).
2. Pre-warm trypsogen and S & XFM TM MSC XF Medium to 37oC before use.
3. Remove the spent medium from the T-75 flask and discard.
4. Wash the cell surface with 10 mL of DPBS without Ca2+and Mg2+, remove and discard,
5. Add 3-5 mL of 0. 05% Trypsogen to the T-75 flask, tilt the flask in all directions to evenly distribute. Incubate the cell in Trypsogen for 2 to 10 minutes in the incubator.
Note: Cells coming out of serum-containing medium may require a longer incubation time (5 to 10 minutes), while cell growing under serum-free conditions should detach more readily (2 to 5 minutes).
6. After incubation, check the flask under the microscope for cell detachment, Firmly tap the flask as necessary to facilitate complete cell detachment.
7. Add 7 mL of pre-warmed S & XFM TM MSC XF Medium to each flask and collect the cell suspension in the 50 mL conical lube containing complete medium. Firmly tap the flask, re-wash with 10 mL S & XFM TM MSC XF Medium and collect.
Note: The addition of S & XFM TM MSC XF Medium to harvested cells is critical for preventing the cell from adhering to the wall of the conical tube during centrifugation.
8. Centrifuge the tubes at 300-400 X g for 5 minutes at room temperature.
9. Resuspend the cells in a minimal volume of pre-warmed S & XFM TM MSC XF Medium for cell counting.
10. Add 15 mL of S & XFM TM MSC XF Medium. Mix or swirl the cell suspension to ensure even distribution.
11. Place the culture flask (s) in the incubator at 37oC with a humidified atmosphere of 5% CO2.
12. Replace the spent culture medium every 2-5 days with pre-warmed S & XFM TM MSC XF Medium.
1. Prepare cryopreservation solution (2X) by supplementing S & XFM TM MSC XF Medium.
with 20% Dimethyl Sulfoxide (DMSO), store at 4oC until use; make cryopreservation medium on the day of intended use.
2. Reconstitute the harvested cell pellet to twice the desired final cell. Concentration (i.e., 1x 106 cells/mL) in pre- warmed S & XFM TM MSC XF Medium.
3. Slowly add cryopreservation solution to the cell suspension, and gently mix to ensure even cell distribution.
4. Immediately add the desired volume of cell suspension (i.e., 1mL) to pre-chilled (2oC to 8oC ) cryovials.
5. Place the cryovials at-70oC in a cryogenic freezing container (e. g."Mr. Frosty”(1oC) Freezing Container).
6. After 24 hours, transfer the frozen cells to liquid nitrogen (vapor phase); storage at-200oC to -125oC is recommended.