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

[DESCRIPTION]
This protocol describes the process of passaging human pluripotent stem cells (hPSCs) for feeder-free culturing of hPSCs using Accutase or ReLeSF

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.
We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences. 


[BEFORE_START]
Prior to passaging, prepare VTN/Matrigel/Geltrex-coated plates. A detailed protocol for this procedure can be found in the protocol "Coating plates," which is located in the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above

[STEPS]
SECTION: A. Accutase
1. Wash hPSCs with DPBS
SECTION: A. Accutase
2. Add 1 ml Accutase/well to the 6-well plate.
SECTION: A. Accutase
3. Incubate  5 min 37 °C 

SECTION: A. Accutase
4. Add 2 ml DMEM/F12 to each well.
SECTION: A. Accutase
5. Collect all cells into 15 ml conical tube.
SECTION: A. Accutase
6. Add 7 ml DMEM/F12.
SECTION: A. Accutase
7. Centrifuge at  200-300 x g, 5 min 

SECTION: A. Accutase
9. Re-suspend cell pellet in 1 ml Feeder-Free Medium + Rock inhibitor. Use a P1000 tip and triturate 5-10 times to achieve single-cell suspension 
SECTION: A. Accutase
10. Aspirate VTN/Matrigel/Geltrex solution from the coated plate, add 2 ml Feeder-Free Medium + Rock inhibitor to each well.
SECTION: A. Accutase
11. Dispense the proper amount of cells to VTN/Matrigel/Geltrex-coated plates. The splitting ratio differs between cell lines but is usually within a range of 1:6 to 1:30. 
SECTION: A. Accutase
15. When hPSCs density reaches 50-80% confluency, passage again or freeze. It usually takes 5-7 days. 

A detailed description for freezing can be found in the "Freezing of feeder-free hPSCs" protocol within the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above
SECTION: A. Accutase
14. Change 2 ml pre-warmed Feeder-free medium for each well every other day.
SECTION: A. Accutase
12. Spread the cells by moving the plate in left-right, then backward-forward motion.
SECTION: A. Accutase
13. Place the plate in the low oxygen incubator
SECTION: B. ReLeSR
16. Wash the feeder-free hPSCs twice with DPBS 
SECTION: B. ReLeSR
17. Add 1 ml/well of ReLeSR to a 6-well pate and incubate at Room temperature for 1 min 

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

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

SECTION: B. ReLeSR
23. Remove the supernatant down to 1 ml and add 5 ml of Feeder-free medium. 
SECTION: B. ReLeSR
24. Gently mix by inversion and gravity precipitate for 5 min 

SECTION: B. ReLeSR
25. Remove the supernatant down to 1 ml. 
SECTION: B. ReLeSR
26. Dilute the cells with the desired volume and carefully plate cell aggregates on VTN/Matrigel/Geltrex-coated plates in Feeder-free medium. The splitting ratio differs between cell lines but is usually within a range of 1:6 to 1:30. 
SECTION: B. ReLeSR
30. When hPSCs density reaches 50-80% confluency, passage again or freeze. It usually takes 5-7 days. 

A detailed protocol for freezing can be found in the "Feeder-free culturing of hPSCs" collection. A link to this collection can be found in the title section of this protocol, located above.
SECTION: B. ReLeSR
29. Change 2 ml pre-warmed Feeder-free medium for each well every other day or if necessary earlier.

SECTION: B. ReLeSR
28. Place the plate in the low oxygen incubator.
SECTION: B. ReLeSR
27. Spread the cells by moving the plate in left-right, then back-fourth motion.
SECTION: A. Accutase
9.1. 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   

 

Y-27632 (1,000X)
     Y-27632      5 mg        DMSO      1.56 ml   
 
 
 Feeder-free medium + Rock inhibitor
     Feeder-free   medium      50 ml        Y-27632   (1,000X)      50 ul   
 
SECTION: A. Accutase
8. Aspirate supernatant