Application

Thawing of Cells:

1. Do not thaw the cells until the recommended medium and appropriate plasticware and/or glassware are on hand.
2. Remove the vial of mesenchymal stem cells from liquid nitrogen and incubate in a 37°C water bath. Closely monitor until the cells are completely thawed. Maximum cell viability is dependent on the rapid and complete thawing of frozen cells. IMPORTANT: Do not vortex the cells.
3. As soon as the cells are completely thawed disinfect the outside of the vial with 70% ethanol. Proceed immediately to the next step.
4. In a laminar flow hood, use a 1 or 2 mL pipette to transfer the cells to a sterile 15 mL conical tube. Be careful to not introduce any bubbles during the transfer process.
5. Using a 10 mL pipette, slowly add dropwise 9 mL of Mesenchymal Stem Cell Expansion Medium (pre-warmed at 37°C) to the 15 mL conical tube. IMPORTANT: Do not add the whole volume of medium at once to the cells. This may result in decreased cell viability due to osmotic shock.
6. Gently mix the cell suspension by slow pipeting up and down twice. Be careful to not introduce any bubbles. IMPORTANT: Do not vortex the cells.
7. Centrifuge the tube at 300 x g for 2-3 minutes to pellet the cells.
8. Decant as much of the supernatant as possible. Steps 4-8 are necessary to remove residual cryopreservative (DMSO).
9. Resuspend the cells in a total volume of 10 mL of Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37°C).
10. Plate the cell mixture onto a 10-cm tissue culture plate.
11. Incubate the cells at 37°C in a 5% CO₂ humidified incubator.
12. The next day, exchange the medium with fresh Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37°C). Exchange with fresh medium every two to three days thereafter.
13. When the cells are approximately 80% confluent, they can be dissociated with Accutase^® and passaged or alternatively frozen for later use.

Subculturing:

1. Carefully remove the medium from the 10-cm tissue culture plate containing the confluent layer of mesenchymal stem cells.
2. Apply 3-5 mL of Accutase™ and incubate in a 37°C incubator for 3 minutes.
3. Inspect the plate and ensure the complete detachment of cells by gently tapping the side of the plate with the palm of your hand. 4. Apply 5 mL of Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37°C) to the plate.
5. Transfer the dissociated cells to a 15 mL conical tube.
6. Centrifuge the tube at 300 x g for 2-3 minutes to pellet the cells.
7. Discard the supernatant
8. Apply 2 mL of Mesenchymal Stem Cell Expansion Medium to the conical tube and resuspend the cells thoroughly. IMPORTANT: Do not vortex.
9. Count the number of cells using a hemacytometer.
10. Plate the cells to the desired density into the appropriate flasks, plates or wells in Mesenchymal Stem Cell Expansion. We typically plate the cells at ~2 million cells per 10-cm plate or T75 flask.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Legal Information

Accutase is a registered trademark of Innovative Cell Technologies, Inc.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Physical form

Mesenchymal Stem Cell Expansion Medium is a proprietary formulation that contains fetal bovine serum.

Quality

Each lot of medium is tested for the optimal cell growth and proliferation of mesenchymal stem cells. Sterility Testing: Negative

Storage and Stability

Mesenchymal Stem Cell Expansion Medium should be stored at -20°C until ready to use. Upon thawing, the basal medium should be stored at 2-8°C for up to one month.