Adapting hPSCs cultured on MEFs to feeder-free system
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the procedure of adapting human pluripotent stem cells (hPSCs) to feeder-free culturing conditions 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
When MEFs-cultured hPSCs reach 50% confluency, change medium to hPSCs medium + Rock inhibitor, preparing for the feeder-free adaptation on the next day.
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 (25ug/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
Y-27632 (1,000X)
| A | B |
|---|---|
| Y-27632 | 5 mg |
| DMSO | 1.56 ml |
hPSCs medium + Rock Inhibitor
| A | B |
|---|---|
| hPSCs medium | 500 ml |
| Y-27632 (1,000X) | 500 µl |
Final volume: 500ml
Coat three wells of a 6-well plate with either VTN/Matrigel/Geltrex for each cell line.
For a detailed protocol, refer to "Coating plates," which can be found in the protocol collection "Feeder-free culturing of hPSCs." This collection can be accessed using the collection link found in the title section of this protocol, located above
Wash one well of MEF-cultured hPSCs with DPBS
Add 1 ml Collagenase solution to this well.
Collagenase solution
| A | B |
|---|---|
| Collagenase type IV | 10 mg |
| KSR medium | 10 ml |
Final volume: 10ml
KSR medium
| A | B |
|---|---|
| DMEM/F12 | 385 ml |
| Knockout Serum Replacement | 100 ml |
| L-Glutamine (200 mM) | 5 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
| MEM Non-Essential Amino Acids (100X) | 5 ml |
Final volume: 500ml
Incubate 0h 45m 0s 37°C . Watch for edge curling of the colonies as this indicates that collagenase incubation is complete.
Add 2 ml DMEM/F12
Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
Collect into 15 ml conical tube.
Add 7 ml DMEM/F12.
Centrifuge at 200-300x g
Aspirate supernatant
Resuspend cell pellet in 1 ml pre-warmed Accutase
Incubate 0h 5m 0s 37°C
Add 9 ml DMEM/F12, invert to mix
Centrifuge at 200-300x g
Aspirate supernatant
Re-suspend cell pellet in 1 ml Feeder-Free Medium + Rock inhibitor, triturate 5-10 times to achieve single cell suspension using a P1000 tip
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
Feeder-free medium + Rock Inhibitor
| A | B |
|---|---|
| Feeder-free medium | 50 ml |
| Y-27632 (1,000X) | 50 µl |
Final volume: 50ml
Aspirate VTN/Matrigel/Geltrex solution from the coated plate, add 2 ml Feeder-free medium + Rock inhibitor to each well.
Dispense 20 µl, 60 µl, 200 µl cell suspension respectively into the three VTN/Matrigel/Geltrex-coated wells.
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 a 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 the well showing the best hPSCs morphology using Accutase or ReLeSR. It usually takes 5-7 days.
A detailed protocol on "Passaging of feeder-free hPSCs" can be found in the collection "Feeder-free culturing of hPSCs." This collection can be found using the collection link in the title section of this protocol, located above
It usually takes 2 passages for hPSCs to fully adapt to feeder-free culture. Differentiation and changes on growth speed are normal during the adaptation.
