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

[DESCRIPTION]
This protocol describes the standard procedure of freezing human pluripotent stem cells (hPSCs), which were grown 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 freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences. 

[STEPS]
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
1. When hPSCs reach 50% confluency (usually on day 6 from last passage), change medium to 3 ml hPSCs medium + Rock inhibitor, to prepare for freezing the next day. 
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
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. Freezing of hPSCs as single cell solution using trypsin
2.1. Freezing Medium I
     hESCs medium      5 ml        FB essence      5 ml   

Freezing Medium II
     FB essence      8 ml        DMSO      2 ml   
  
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
3. Wash hPSCs 1x with DPBS 
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
4. Add 0.5 ml Trypsin to each well
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
5. Incubate  5 min 37 °C in incubator

SECTION: A. Freezing of hPSCs as single cell solution using trypsin
6. Gently shake the plate 
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
7. Check under microscope to confirm all cells have been lifted from the plate. 
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
8. Add 2 ml Wash Medium to each well to inactivate trypsin
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
9. Triturate using P1000 tips and collect all cell suspension to a 15 ml conical tube
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
10. Centrifuge at 200-300 x g, 5 min 

SECTION: A. Freezing of hPSCs as single cell solution using trypsin
11. Aspirate supernatant 
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
12. Gently re-suspend the pellet in 500 µl Freezing Medium I per vial to be frozen.
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
13. In each cryovial, add 500 µl pre-chilled Freezing Medium II.
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
14. Dispense 500 µl cell suspension into each cryovial using P1000, mix
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
15. Temporarily keep cryovials on ice until all cells dispensed 

SECTION: A. Freezing of hPSCs as single cell solution using trypsin
16. 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. Freezing of hPSCs as single cell solution using trypsin
17. Freeze at -80°C   

SECTION: A. Freezing of hPSCs as single cell solution using trypsin
18. For long term storage, store cryovials in liquid nitrogen (-196ºC).
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
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. Freezing of hPSCs as as cell aggregates using collagenase
20. Wash hPSCs (on feeders) 1x with PBS.
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
21. Use 1 ml Collagenase Solution/well of a 6-well plate.
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
22. Incubate 45 min   37 °C . Watch for edge curling of the colonies as this indicates that collagenase incubation is complete.



SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
23. Add 3 ml hPSC medium to quench collagenase
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
24. Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
25. Collect into 15 ml conical tube.
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
26. Add 4-5 ml Wash Medium.
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
27. Gravity precipitate cells  10 min 

SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
28. Reduce volume to 1-2 ml
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
29. Using a 10 ml strip pipette, triturate the colonies 5-10 times against the bottom of the tube to break up cell clusters

 

SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
30. Gravity precipitate cells 10 min 

SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
31. Resuspend cells in 10 ml hPSC medium 
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
32. Pellet the cells at 200 x g, 5 min aspirate the supernatant

SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
33. Gently re-suspend the pellet in 500 µl Freezing Medium I per vial to be frozen.
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
34. Carefully add 500 µl Freezing Medium II per vial to be frozen. 
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
35. Dispense 1 ml aliquots in pre-labeled cryovials and keep on ice 

SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
36. 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. Freezing of hPSCs as as cell aggregates using collagenase
37. Place container in a -80°C Freezer    

SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
38. After an overnight incubation, cryovials are removed from the Freezing Container and placed in Liquid Nitrogen (-196ºC) for long-term storage.
SECTION: A. Freezing of hPSCs as single cell solution using trypsin
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   
 
 hPSCs Medium + Rock Inhibitor
     hESCs medium      500 ml        Y-27632 (1,000X)      500 ul   
 
Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56 ml   

SECTION: A. Freezing of hPSCs as single cell solution using trypsin
8.1. Wash medium 
     DMEM/F12      470 ml        Newborn Calf Serum      25 ml        Penicillin & Streptomycin (100X)      5 ml   
 
SECTION: B. Freezing of hPSCs as as cell aggregates using collagenase
21.1. 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