|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|
Required Equipment / Consumables:
Prepare ECM Plates:
- Aliquot Matrigel according to manufacturer’s instructions. Freeze aliquots at -80°C.
- To prepare tissue culture plates, take 1 aliquot of matrigel and thaw on ice or overnight at 4°C.
- Add the thawed matrigel in cold DMEM-F12 and plate in the appropriate sized cell culture vessel.
- For a 6-well plate, we use 1mL of matrigel/DMEM-F12 per well.
- For a 24-well plate, we use 250μl of matrigel/DMEM-F12 per well.
- For a 96-well plate, we use 50μl of matrigel/DMEM-F12 per well.
- Allow ECM to attach to plate by incubating at 37°C for at least an hour. Plates can be left overnight at 37°C.
- 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.
- 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.
- Once incubation is complete, add the desired medium to neutralize the Accutase, and collect cells to be passaged 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.
- Resuspend the cell pellet 1mL of media.
- Take 20μl of resuspended cells for cell counting.
- Note: If cells are cultured in mTeSR originally, dilute the cells in an mTeSR solution containing Rock inhibitor.
- Make the appropriate dilution for desired number of cells per well.
- Remove basal media from Matrigel plates and add the appropriate volume of resuspended cells to each well.
- Gently shake the plate back and forth and front to back to evenly distribute the cells—avoid circular motions to prevent concentrating cells in the middle of the well.
- Place culture vessel into 10% CO2 incubator.
- Feed cells after 2 days, then after 4 days, then after another 4 days until cells are ready to be passaged.
- After 2 days (2 days total): Add 80μl media/well.
- After 4 days (6 days total): Add 100μl media/well.
- After 4 days (10 days total): Add 100μl media/well.
- Note: If cells were diluted in mTeSR containing Rock inhibitor, feed the cells with mTeSR only without Rock inhibitor.