Thawing of feeder-free hPSCs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex
General Notes
- Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
- Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions.
- We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it.
- We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.
Steps
Prepare one 6-well VTN/Matrigel/Geltrex-coated plate for each vial of frozen hPSCs.
A detailed protocol on "Coating plates" can be found in the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above
Place vial of frozen hPSCs in 37°C water bath with constant agitation.
Pipette thawed cell suspension into 10 ml pre-warmed DMEM/F12.
Centrifuge 200-300x g
While cells are spinning, aspirate the VTN/Matrigel/Geltrex solution from the coated plates and add 2 ml pre-warmed Feeder-free medium + Rock inhibitor to each well.
Feeder-free Medium (version A)
| A | B |
|---|---|
| StemFlex basal medium | 450 ml |
| StemFlex supplement | 50 ml |
Final volume: 500ml
Feeder-free Medium (version B)
| A | B |
|---|---|
| mTeSR-plus basal medium | 400 ml |
| mTeSR-plus supplement | 100 ml |
Final volume: 500ml
Y-27632 (1,000X)
| A | B |
|---|---|
| Y-27632 | 5 mg |
| DMSO | 1.56 ml |
Feeder-free medium + Rock inhibitor
| A | B |
|---|---|
| Feeder-free medium | 50 ml |
| Y-27632 (1,000X) | 50 µl |
Final volume: 50ml
Aspirate most of the medium on the centrifuged hPSCs, being careful not to disturb the pellet
Add 1 ml Feeder-free medium + Rock inhibitor
Re-suspend the cells using a P1000 tip.
Dispense the cells onto the VTN/Matrigel/Geltrex-coated plate, adding 160 µl per well.
Check the cells under the microscope to get an idea of the resulting cell density.
Spread the cells by moving the plate in left-right, then backward-forward motion.
Place the plate in the low oxygen incubator
Change 2 ml pre-warmed Feeder-free medium for each well every other day. When large colonies emerge or hPSCs density reaches 50-80%, passage using Accutase or ReLeSR. It usually takes 5-7 days for the thawed cells to grow to this point.
A detailed protocol on passaging using Accutase or ReLeSR can be found in the "Passaging of hPSCs" protocol, in the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above
