Thawing Human Mesenchymal Stem Cells (hMSCs)

Overview

Thawing cells properly is crucial to cell culture health. Follow this protocol for thawing cryopreserved human mesenchymal stem cells to ensure your cells will have an optimal yield for your experiments.

Materials

• Human Mesenchymal Stem Cell Vial
• Human Mesenchymal Stem Cell Growth Medium (MSCGM™)
• Water/Bead Bath (37˚C)
• Tissue Culture Flasks
• Centrifuge Tubes
• Incubator (37˚C, 5% CO₂, 90% humidity)
• Automatic Pipet Filler
• Serological Pipets
• Micropipettes
• Micropipette Tips
• 70% Ethanol
• Kimwipes
• Vacuum Bottle

Methods

1. Warm MSCGM™ medium to 37˚C in the water/bead bath;
2. Remember to spray everything down with 70% ethanol and wipe it with Kimwipes before moving into the BSC;
3. Check the number of cells that are in the cryopreserved vial and prepare the appropriate number of flasks with the corresponding amount of warm cell medium. Human mesenchymal stem cells have a recommended cell seeding density of 5,000-6,000 cells/cm². Amount of media will also vary by flask size, but it is recommended to use 0.2 mL of media per cm². Find useful formulae below;
   • Number of flasks = number of cells/cell seeding density/flask area in cm²
   • Amount of media = (flask area in cm²)*0.2 mL
4. Place these media-containing flasks into the incubator for 30 minutes before adding cells;
5. Remove cell vial from liquid nitrogen and submerge it in the water/bead bath up to the cap for no longer than 1.5 minutes;
6. Remove vial when its sides are thawed but its center remains frozen;
7. Add 5 mL of MSCGM™ to a sterile centrifuge tube;
8. Pipette the cell solution into the tube from step 7;
9. Centrifuge at 500 X g for 5 min;
10.  Aspirate supernatant;
11. You will need to add 1 mL of cell suspension to each flask, so add the corresponding amount of media to your cell pellet to have a total volume of n_flasks;
    • Example: If you are plating cells in 5 flasks, add 5 mL of media.
12.  Pipette solution up and down to ensure homogenous cell distribution;
13.  Add 1 mL of cell suspension to each flask you prepared on step 3;
14.  Gently rock flasks;
15.  Label your flasks. It is good practice to include the following information;
    •  Your initials and date of passage (e.g.: if your initials are YN, and the date is MM/DD/YYYY, label it as YNYYYMMDD)
    • Cell line (hMSCs) and passage number (if your vials were P_n, they will now be P_n+1).
16.  Feed cells every three to four days;
17.  Passage cells following this protocol.