Freezing of hPSCs grown on MEFs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the standard procedure of freezing human pluripotent stem cells (hPSCs), which were grown 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
A. Freezing of hPSCs as single cell solution using trypsin
When hPSCs reach 50% confluency (usually on day 6 from last passage), change medium to 3 ml hPSCs medium + Rock inhibitor, to prepare for freezing 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
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 |
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 |
|---|---|
| hPSCs medium | 5 ml |
| FB essence* | 5 ml |
*We have successfully used FB essence and FBS to freeze hPSCs and have not observed obvious difference. Final volume: 10ml
Freezing Medium II
| A | B |
|---|---|
| FB essence* | 8 ml |
| DMSO | 2 ml |
*We have successfully used FB essence and FBS to freeze hPSCs and have not observed obvious difference. Final volume: 10ml
Wash hPSCs 1x with DPBS
Add 0.5 ml Trypsin to each well
Incubate 0h 5m 0s 37°C in incubator
Gently shake the plate
Check under microscope to confirm all cells have been lifted from the plate.
Add 2 ml Wash Medium to each well to inactivate trypsin
Wash medium
| A | B |
|---|---|
| DMEM/F12 | 470 ml |
| Newborn Calf Serum | 25 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
Final volume: 500ml
Triturate using P1000 tips and collect all cell suspension to a 15 ml conical tube
Centrifuge at 200-300x g
Aspirate supernatant
Gently re-suspend the pellet in 500 µl Freezing Medium I per vial to be frozen.
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. Freezing of hPSCs as as cell aggregates using collagenase
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)
Wash hPSCs (on feeders) 1x with PBS.
Use 1 ml Collagenase Solution/well of a 6-well plate.
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 3 ml hPSC medium to quench collagenase
Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
Collect into 15 ml conical tube.
Add 4-5 ml Wash Medium.
Gravity precipitate cells 0h 10m 0s
Reduce volume to 1-2 ml
Using a 10 ml strip pipette, triturate the colonies 5-10 times against the bottom of the tube to break up cell clusters
Gravity precipitate cells 0h 10m 0s
Resuspend cells in 10 ml hPSC medium
Pellet the cells at 200x g aspirate the supernatant
Gently re-suspend the pellet in 500 µl Freezing Medium I per vial to be frozen.
Carefully add 500 µl Freezing Medium II per vial to be frozen.
Dispense 1 ml 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.
Place container in a -80°C Freezer
After an overnight incubation, cryovials are removed from the Freezing Container and placed in Liquid Nitrogen (-196ºC) for long-term storage.
