TITLE: Adapting hPSCs cultured on MEFs to feeder-free system
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the procedure of adapting human pluripotent stem cells (hPSCs) to feeder-free culturing conditions using mTeSR-plus or StemFlex 

General Notes
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.
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]
1. When MEFs-cultured hPSCs reach 50% confluency, change medium to hPSCs medium + Rock inhibitor, preparing for the feeder-free adaptation on the next day.




2. Coat three wells of a 6-well plate with either VTN/Matrigel/Geltrex for each cell line. 

For a detailed protocol, refer to "Coating plates," which can be found in the protocol collection "Feeder-free culturing of hPSCs." This collection can be accessed using the collection link found in the title section of this protocol, located above



3. Wash one well of MEF-cultured hPSCs with DPBS
4. Add 1 ml Collagenase solution to this well.
5. Incubate 45 min 37 °C . Watch for edge curling of the colonies as this indicates that collagenase incubation is complete. 

6. Add 2 ml DMEM/F12
7. Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
8. Collect into 15 ml conical tube.
9. Add 7 ml DMEM/F12.
10. Centrifuge at 200-300 x g, 5 min 

11. Aspirate supernatant 
12. Resuspend cell pellet in 1 ml pre-warmed Accutase
13. Incubate  5 min 37 °C 

14. Add 9 ml DMEM/F12, invert to mix
15. Centrifuge at  200-300 x g, 5 min 

16. Aspirate supernatant
17. Re-suspend cell pellet in 1 ml Feeder-Free Medium + Rock inhibitor, triturate 5-10 times to achieve single cell suspension using a P1000 tip

18. Aspirate VTN/Matrigel/Geltrex solution from the coated plate, add 2 ml Feeder-free medium + Rock inhibitor to each well.
19. Dispense 20 µl, 60 µl, 200 µl cell suspension respectively into the three VTN/Matrigel/Geltrex-coated wells.



23. Change 2 ml pre-warmed Feeder-free medium for each well every other day.


25. It usually takes 2 passages for hPSCs to fully adapt to feeder-free culture. Differentiation and changes on growth speed are normal during the adaptation. 
1.1. 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   

 Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56 ml   
 

hPSCs medium + Rock Inhibitor
     hESCs medium      500 ml        Y-27632 (1,000X)      500 ul   
 
4.1. 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   
 
17.1. Feeder-free Medium (version A)
     StemFlex   basal medium      450 ml        StemFlex   supplement      50 ml   
 
Feeder-free Medium (version B)
     mTeSR-plus   basal medium      400 ml        mTeSR-plus   supplement       100 ml   
 
 


 Feeder-free medium + Rock Inhibitor
     Feeder-free   medium      50 ml        Y-27632   (1,000X)      50 ul   
 
21. Spread the cells by moving the plate in left-right, then backward-forward motion.
20. Check the cells under the microscope to get an idea of the resulting cell density.
22. Place the plate in a low oxygen incubator
24. When large colonies emerge or hPSCs density reaches 50-80%, passage the well showing the best hPSCs morphology using Accutase or ReLeSR. It usually takes 5-7 days. 

A detailed protocol on "Passaging of feeder-free hPSCs" can be found in the collection "Feeder-free culturing of hPSCs." This collection can be found using the collection link in the title section of this protocol, located above