Passaging of hPSCs grown on MEFs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the standard procedure of using collagenase to passage human pluripotenct 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 bulk/collagenase passaging is used for routine maintenance of hPSC cultures, manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires ( i ) the identification and discrimination of undifferentiated and differentiated hPSC colonies and ( ii ) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.
Steps
Check MEFs plates for suitability to receive passaged hPSCs prior to beginning. Refer to the "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture" collection; dx.doi.org/10.17504/protocols.io.b4pbqvin
Wash hPSCs with DPBS
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 2 ml wash medium to each well.
Wash medium
| A | B |
|---|---|
| DMEM/F12 | 470 ml |
| Newborn Calf Serum | 25 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
Final volume: 500ml
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 5- 10 min.
Aspirate Wash Medium, leave 0.5 ml. Don’t disturb the colonies that are loosely pelleted.
Repeat steps 8-10 once
Add 5 ml 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 (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
Repeat steps 9-10 once
Add 3 ml hPSCs medium
Use a 10 ml strip pipette and triturate the colonies 5-10 times against the bottom of the tube to break up cell clusters
In preparation for the hPSCs, plate 1 ml hPSCs medium onto each MEFs well.
Add more hPSCs medium to the triturated colonies suspension to a proper dilution so that each MEFs wells will receive 2 ml. Splitting ratio varies between different cell lines and different operators. It’s usually within a range of 1:6 to 1:20.
Mix well, distribute 2 ml to each MEFs wells.
Place the plate in the low oxygen incubator
From day 3, change 2-3 ml pre-warmed hPSCs medium for each well daily.
When hPSC density reaches 50-70% confluency, passage again. We usually adjust the splitting ratio to passage every 7 days.
