Thawing of hPSCs grown on MEFs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the standard procedure of thawing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
General notes
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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.
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Until otherwise indicated, 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.
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While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.
Steps
Prepare one 6-well MEFs plate for each vial of frozen hPSCs
Place vial of frozen hPSCs in 37°C water bath with constant agitation.
Pipette thawed cell suspension into 10 ml pre-warmed hPSCs medium.
hPSCs Medium
| A | B |
|---|---|
| DMEM/F12 | 385 ml |
| Fetal Bovine Serum (FBS) | 75 ml |
| Knockout Serum Replacement | 25 ml |
| L-Glutamine (100X) | 5 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
| MEM Non-Essential Amino Acids (100X) | 5 ml |
| 2-Mercaptoethanol (10,000X) | 50 µl |
| Heat Stable Recombinant Human FGF2 (25µg/ml)* | 80 µl |
*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2. Final volume: 500ml
L-Glutamine (100X)
| A | B |
|---|---|
| L-Glutamine, powder | 14.6 g |
| MilliQ H2O | 500 ml |
2-Mercaptoethanol (10,000X)
| A | B |
|---|---|
| 2-Mercaptoethanol | 0.78 ml |
| MilliQ H2O | 9.22 ml |
Heat Stable Recombinant Human FGF2 (25µg/ml)
| A | B |
|---|---|
| Heat Stable Recombinant Human FGF2 | 500 µg |
| 0.1% BSA | 20 ml |
Final volume: 20ml
Centrifuge 200-300x g
While cells are spinning, aspirate the MEFs medium from the MEFs plates and add 2 ml pre-warmed hPSCs medium + Rock inhibitor to each well.
hPSCs Medium + Rock inhibitor
| A | B |
|---|---|
| hPSCs medium | 500 ml |
| Y-27632 (1,000X) | 500 µl |
Final volume: 500ml
Y-27632 (1,000X)
| A | B |
|---|---|
| Y-27632 | 5 mg |
| DMSO | 1.56 ml |
Aspirate most of the medium on the centrifuged hPSCs, being careful not to disturb the pellet
Add 1 ml hPSCs medium with or without Rock inhibitor (dependent on choice made above).
Resuspend the cells using a P1000 tip.
Pipette the cells onto the 2 ml already on the MEFs.
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
From day 3, change 2-3 ml pre-warmed hPSCs medium for each well daily.
When large colonies emerge or hPSCs density reaches 50-70%, passage using collagenase. It usually takes 7-10 days for the thawed cells to grow to this point.
