TITLE: Duplicating 96-well plate-cultured hPSCs clones
AUTHORS: Hanqin  Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes a standard procedure for duplicating 96-well plate-cultured human pluripotent stem cells (hPSCs).

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

[STEPS]
1. Change medium to hPSCs medium + Rock inhibitor one day ahead of plate duplication.
2. Prepare 11 ml DPBS, 3 ml Trypsin, 15 ml hPSCs medium + Rock inhibitor and 3 ml 2x Crude lysis buffer (with proteinase K) into separate reservoirs. 
3. Aspirate the medium from plates, wash by dispensing 100 µl DPBS per well using a Multichannel pipette. Gently swirl plates to wash the content of the wells.
4. Aspirate DPBS from the plates using multichannel aspirator.
5. Add 25 µl Trypsin to each well using a Multichannel pipette. 
6. Incubate at  37 °C 5 min 

7. Take a 96-well PCR plate and dispense 25 µl of 2X Crude lysis buffer with Proteinase K per well onto the plates. Label the plates according to your tissue culture plates. 
8. Take a new 96-well MEFs plate. Aspirate MEF medium and add 75 µl of hPSCs medium + Rock inhibitor to the wells. 
9. Take the Trypsin-dissociated cells out from 37°C . Dispense 25 µl hPSCs medium + Rock inhibitor onto each well using a Multichannel pipette to inactivate trypsin. 


 

10. Use a fresh 200 µl filter tip box for this step: 

Set the Multichannel at 25 µl, load it with pipette tips, break down the trypsinized wells by pipette at least 7-8 times, focusing mostly on the center of the well, also pipette 2-3 times near the circumference of the wells. 
11. Using the same tips, take out 25 µl of the dissociated cells, and dispense them over the new MEFs plate from step 8. 
12. Take the same tips to pipette out the remaining approx. 25 µl cell suspension to the PCR plate from step 7. This becomes the gDNA plate. The location for all wells on tissue culture plate should correspond with their particular gDNA collection plate. 


 

13. After adding all samples to tissue culture and gDNA plates, transfer the tissue culture plates to 37 °C . Do not shake. Medium change every other day. 

14. Seal the gDNA plate using an adhesive plate seal. Ensure they are tightly covered to prevent evaporation.
15. Place the gDNA plate at  50 °C   

16. Heat inactivate at  95 °C 5 min in a thermocycler

17. Chill  on ice 

18. Proceed to PCR, NGS and analysis to identify properly edited clones. 
19. Genotyping needs to be done in a week before the duplicated cells overgrow.


1.1. hPSC 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   
 

hPSC medium + Rock inhibitor
     hESCs medium      500 ml        Y-27632   (1,000X)      500 ul   

2.1. Crude lysis buffer (2x)