Freezing and Thawing Feeder-free Cells
Reagent | Vendor | Catalog Number |
---|---|---|
Corning™ Matrigel™ hESC-Qualified Matrix | Fisher Scientific | 354277 |
Falcon™ Polystyrene Microplates | Fisher Scientific | Various |
Corning™ Cell Culture PBS (1X) | Thermo Fisher / Life Technologies | MT21040CV |
DMEM-F12 | Thermo Fisher / Life Technologies | MT10092CV |
Essential 8 Medium | Thermo Fisher / Life Technologies | A1517001 |
Stemflex Medium | Thermo Fisher / Life Technologies | A3349401 |
mTesR Medium | Stemcell Technologies | 85850 |
Rock Inhibitor | Stemcell Technologies | 72302 |
Accutase | Innovative Cell Technologies | AT104 |
Cryostor CS10 | Stemcell Technologies | 07930 |
STEM-CELLBANKER® | Thermo Fisher / Life Technologies | 11897 |
Nunc™ Biobanking and Cell Culture Cryogenic Tubes | Thermo Scientific | 12-565-164N |
Required Equipment / Consumables:
Freezing:
- Remove plate to be passaged from incubator and place it in the biosafety cabinet.
- Remove the spent medium from the wells to be passaged by aspirating with borosilicate pipet.
- Rinse each well to be passaged with 1ml room temperature PBS.
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Add Accutase to each well to be passaged.
- For a 6-well plate, add 1mL Accutase to each well.
- For a 24-well plate, add 500μl Accutase to each well.
- Incubate for 6-10 minutes at 37°C until cells detach.
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Once incubation is complete, add the desired medium to neutralize the Accutase, and collect cells to be frozen using fresh media using a 5mL pipet.
- For a 6-well plate, add 3mL media to each well.
- For a 24-well plate, add 1mL media to each well.
- Spin the cells at 120g for 3 minutes.
- Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
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Resuspend the cell pellet with an appropriate amount of freezing media for freezing.
- For a 6-well plate, add 2mL freezing media.
- For a 24-well plate, add 500μL freezing media.
- Aliquot 500μL to each cryogenic tube.
- Place cryogenic tube in freezing container and store in -80°C.
Thawing:
- Retrieve cryogenic tube from either -80°C or LN2.
- Thaw the cryogenic tube quickly in a 37°C water bath.
- Transfer thawed cells to a 15mL tube.
- Add ~500μL of media of choice (E8 or mTESR1 or Stemflex) drop by drop, swirling the tube while doing so.
- Add an additional ~2mL of media of choice to the tube.
- Spin the cells at 120g for 3 minutes.
- Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
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Resuspend the cell pellet with an appropriate amount of media for passaging (E8+ Rock inhibitor/ or mTESR1 + Rock inhibitor/ or Stemflex).
- For a 6-well plate, add 2mL media.
- For a 24-well plate, add 500μl media.
- Transfer cells to a matrigel-coated plate.
- Incubate at 37°C.