Passaging of feeder-free hPSCs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the process of passaging human pluripotent stem cells (hPSCs) for feeder-free culturing of hPSCs using Accutase or ReLeSF
Protocol overview
A. Accutase
B. ReLeSR
General Notes:
- For this protocol, hPSCs refers collectively to 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 .
- We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences.
Before start
Prior to passaging, prepare VTN/Matrigel/Geltrex-coated plates. A detailed protocol for this procedure can be found in the protocol "Coating plates," which is located 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
Steps
A. Accutase
Wash hPSCs with DPBS
Add 1 ml Accutase/well to the 6-well plate.
Incubate 0h 5m 0s 37°C
Add 2 ml DMEM/F12 to each well.
Collect all cells into 15 ml conical tube.
Add 7 ml DMEM/F12.
Centrifuge at 200-300x g
Aspirate supernatant
Re-suspend cell pellet in 1 ml Feeder-Free Medium + Rock inhibitor. Use a P1000 tip and triturate 5-10 times to achieve single-cell suspension
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 VTN/Matrigel/Geltrex solution from the coated plate, add 2 ml Feeder-Free Medium + Rock inhibitor to each well.
Dispense the proper amount of cells to VTN/Matrigel/Geltrex-coated plates. The splitting ratio differs between cell lines but is usually within a range of 1:6 to 1:30.
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 hPSCs density reaches 50-80% confluency, passage again or freeze. It usually takes 5-7 days.
A detailed description for freezing can be found in the "Freezing of feeder-free hPSCs" protocol within the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above
B. ReLeSR
Wash the feeder-free hPSCs twice with DPBS
Add 1 ml/well of ReLeSR to a 6-well pate and incubate at Room temperature for 0h 1m 0s
Remove the ReLeSR solution and let it sit at Room temperature 0h 2m 0s
Add 2 ml of Feeder-free medium/well.
Gently scrape the cells from the bottom of the well by using a Corning cell lifter.
Collect the cells in a 15 ml conical tube and add 3 ml of Feeder-free medium.
Gently mix by inversion and gravity precipitate for 0h 5m 0s
Remove the supernatant down to 1 ml and add 5 ml of Feeder-free medium.
Gently mix by inversion and gravity precipitate for 0h 5m 0s
Remove the supernatant down to 1 ml.
Dilute the cells with the desired volume and carefully plate cell aggregates on VTN/Matrigel/Geltrex-coated plates in Feeder-free medium. The splitting ratio differs between cell lines but is usually within a range of 1:6 to 1:30.
Spread the cells by moving the plate in left-right, then back-fourth motion.
Place the plate in the low oxygen incubator.
Change 2 ml pre-warmed Feeder-free medium for each well every other day or if necessary earlier.
When hPSCs density reaches 50-80% confluency, passage again or freeze. It usually takes 5-7 days.
A detailed protocol for freezing 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.
