TITLE: Freezing of feeder-free hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the process of freezing feeder-free human pluripotent stem cells (hPSCs) using Accutase or ReLeSR 

Protocol overview:
A. Accutase
B. ReLeSR

General Notes:
For this protocol, hPSCs refers collectively to 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]
SECTION: A. Accutase
1. hPSCs are ready to be frozen when the culture reaches 50-80% confluency. 
SECTION: A. Accutase
2. 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)
SECTION: A. Accutase
3. Wash hPSCs with DPBS.
SECTION: A. Accutase
4. Use 1 ml Accutase/well of a 6-well plate.
SECTION: A. Accutase
5. Incubate 5 min 37 °C 

SECTION: A. Accutase
6. Add 2 ml DMEM/F12 to each well.
SECTION: A. Accutase
7. Collect all cells into 15 ml conical tube.
SECTION: A. Accutase
8. Add 7 ml DMEM/F12.
SECTION: A. Accutase
10. Aspirate supernatant 
SECTION: A. Accutase
11. Gently re-suspend the pellet in 500 µl Freezing Medium I per vial to be frozen, triturate 5-10 times to achieve single cell suspension using a P1000 tip
SECTION: A. Accutase
12. In each cryovial, add 500 µl pre-chilled Freezing Medium II.
SECTION: A. Accutase
13. Dispense 500 µl cell suspension into each cryovial using P1000, mix
SECTION: A. Accutase
14. Temporarily keep cryovials on ice until all cells dispensed. 
SECTION: A. Accutase
15. 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.
SECTION: A. Accutase
16. Freeze at -80 °C   

SECTION: A. Accutase
17. For long term storage, store cryovials in liquid nitrogen (-196ºC).
SECTION: B. ReLeSR
18. hPSCs are ready to be frozen when the culture reaches 50-80% confluency. 
SECTION: B. ReLeSR
19. 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)
SECTION: B. ReLeSR
20. Wash the plates twice with DPBS
SECTION: B. ReLeSR
21. Add 1 ml/well of ReLeSR to a 6-well plate and incubate for  1 min Room temperature 

SECTION: B. ReLeSR
22. Remove the solution and let it sit at Room temperature  for  2 min 

SECTION: B. ReLeSR
23. Add 2 ml of Feeder-free medium/well.
SECTION: B. ReLeSR
24. Gently scrape the cells from the bottom of the well by using a Corning cell lifter.
SECTION: B. ReLeSR
25. Collect the cells in a 15 ml conical tube and add 3 ml of Feeder-free medium. 
SECTION: B. ReLeSR
26. Gently mix by inversion and gravity precipitate for 5 min 

SECTION: B. ReLeSR
27. Remove the supernatant down to 1 ml and add 5 ml of Feeder-free medium. 
SECTION: B. ReLeSR
28. Pellet the cells at  150 x g, 4 min  and aspirate the supernatant.

SECTION: B. ReLeSR
30. Carefully add 500 µl Freezing Medium II per vial to be frozen. 
SECTION: B. ReLeSR
31. Dispense 1ml aliquots in pre-labeled cryovials and keep on ice.
SECTION: B. ReLeSR
32. 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.
SECTION: B. ReLeSR
33. Freeze   -80 °C 

SECTION: B. ReLeSR
34. For long term storage, store cryovials in liquid nitrogen (-196ºC).



SECTION: B. ReLeSR
19.1. Freezing Medium I
     Feeder-free   medium      5 ml        FB essence*      5        5   ml   
 

Freezing Medium II
     FB essence*      8 ml        DMSO      2 ml   
 
SECTION: A. Accutase
2.1. Freezing Medium I
     Feeder-free   medium      5 ml        FB essence*      5        5   ml   
 

Freezing Medium II
     FB essence*      8 ml        DMSO      2 ml   
 

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   
 
 
  
 
SECTION: A. Accutase
9. Centrifuge 200-300 x g, 5 min 

SECTION: B. ReLeSR
29. Gently re-suspend the pellet in 500 µl Freezing Solution I per vial to be frozen.