Freezing of feeder-free hPSCs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the process of freezing feeder-free human pluripotent stem cells (hPSCs) using Accutase or ReLeSR
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 .
Steps
A. Accutase
hPSCs are ready to be frozen when the culture reaches 50-80% confluency.
Before starting:
a. Prepare Freezing Medium I and II and keep on ice.
b. Pre-label appropriate number of cryovials (freeze approx. 2 vials/well of a 6-well plate)
Freezing Medium I
| A | B |
|---|---|
| Feeder-free medium* | 5 ml |
| FB essence** | 5 ml |
*The formulation of feeder-free medium can be found in below**We have used successfully frozen feeder-free hPSCs using KSR instead of FB essence. Final volume: 10ml
Freezing Medium II
| A | B |
|---|---|
| FB essence* | 8 ml |
| DMSO | 2 ml |
*We have used successfully frozen feeder-free hPSCs using KSR instead of FB essence. Final volume: 10ml
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
Wash hPSCs with DPBS.
Use 1 ml Accutase/well of a 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 200-300x g
Aspirate supernatant
Gently re-suspend the pellet in 500 µl Freezing Medium I per vial to be frozen, triturate 5-10 times to achieve single cell suspension using a P1000 tip
In each cryovial, add 500 µl pre-chilled Freezing Medium II.
Dispense 500 µl cell suspension into each cryovial using P1000, mix
Temporarily keep cryovials on ice until all cells dispensed.
Place cryovials into Styrofoam microtube freezer box or pre-cooled (4°C on ice) NALGENE™ Cryo 1°C Freezing Container filled with 250 ml of Isopropanol.
Freeze at -80°C
For long term storage, store cryovials in liquid nitrogen (-196ºC).
B. ReLeSR
hPSCs are ready to be frozen when the culture reaches 50-80% confluency.
Before starting:
a. Prepare Freezing Medium I and II and keep on ice.
b. Pre-label appropriate number of cryovials (freeze approx. 2 vials/well of a 6-well plate)
Freezing Medium I
| A | B |
|---|---|
| Feeder-free medium | 5 ml |
| FB essence* | 5 ml |
*We have used successfully frozen feeder-free hPSCs using KSR instead of FB essence . Final volume: 10ml
Freezing Medium II
| A | B |
|---|---|
| FB essence* | 8 ml |
| DMSO | 2 ml |
*We have used successfully frozen feeder-free hPSCs using KSR instead of FB essence . Final volume: 10ml
Wash the plates twice with DPBS
Add 1 ml/well of ReLeSR to a 6-well plate and incubate for 0h 1m 0s Room temperature
Remove the solution and let it sit at Room temperature for 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.
Pellet the cells at 150x g and aspirate the supernatant.
Gently re-suspend the pellet in 500 µl Freezing Solution I per vial to be frozen.
Carefully add 500 µl Freezing Medium II per vial to be frozen.
Dispense 1ml aliquots in pre-labeled cryovials and keep on ice.
Place cryovials into Styrofoam microtube freezer box or pre-cooled (4°C on ice) NALGENE™ Cryo 1°C Freezing Container filled with 250 ml of Isopropanol.
Freeze -80°C
For long term storage, store cryovials in liquid nitrogen (-196ºC).
