TITLE: Passaging of hPSCs grown on MEFs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure of using collagenase to passage human pluripotenct stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).

General notes
1. 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.
2. 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. 
3. 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]
2. Wash hPSCs with DPBS
3. Use 1 ml Collagenase solution/well of a 6-well plate.
4. Incubate 45 min 37 °C . Watch for edge curling of the colonies as this indicates that collagenase incubation is complete.

5. Add 2 ml wash medium to each well.
6. Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
7. Collect into 15 ml conical tube.
8. Add 4-5 ml Wash Medium.
9. Gravity precipitate cells 5- 10 min.
11. Repeat steps 8-10 once
12. Add 5 ml hPSCs medium
13. Repeat steps 9-10 once
14. Add 3 ml hPSCs medium
15. Use a 10 ml strip pipette and triturate the colonies 5-10 times against the bottom of the tube to break up cell clusters
 

3.1. Collagenase solution
     Collagenase type IV      10 mg        KSR medium      10 ml   
 
KSR medium
     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   
 
5.1. Wash medium
     DMEM/F12      470 ml        Newborn Calf Serum      25 ml        Penicillin & Streptomycin (100X)      5 ml   
 
10. Aspirate Wash Medium, leave 0.5 ml. Don’t disturb the colonies that are loosely pelleted.
16. In preparation for the hPSCs, plate 1 ml hPSCs medium onto each MEFs well.
17. 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.
18. Mix well, distribute 2 ml to each MEFs wells. 

 

19. Place the plate in the low oxygen incubator

 

20. From day 3, change 2-3 ml pre-warmed hPSCs medium for each well daily.
21. When hPSC density reaches 50-70% confluency, passage again. We usually adjust the splitting ratio to passage every 7 days.
1. 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
12.1. hPSCs Medium
     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 ul        Heat Stable Recombinant Human FGF2 (25ug/ml)*      80 ul   
 

L-Glutamine (100X)
     L-Glutamine,   powder      14.6 g        MilliQ H2O      500 ml   
 
2-Mercaptoethanol (10,000X)
     2-Mercaptoethanol      0.78 ml        MilliQ H2O      9.22 ml   
 
 
Heat Stable Recombinant Human FGF2 (25µg/ml)
     Heat Stable Recombinant Human FGF2      500 ug        0.1% BSA      20 ml