TITLE: Human pluripotent stem cell culture
AUTHORS: Jiuchun Zhang, Harper JW

[DESCRIPTION]
This protocol is about human pluripotent stem cell culture.


Some facts about human pluripotent stem cells
They attach to several extra cellular matrices, including Matrigel, laminin, vitronectin, fibronectin and high-density RGD peptides. The best and most natural ECM for human PSC is Laminin 511 or 521.
Human PSCs don’t like to be in single cells. They always grow the best in colonies.
The doubling time of human PSC in E8 medium is 16-20 hours.
Cells are normally split every 4-5 days.
One well of a 6-well plate at 90% confluency will give you roughly 1.5 to 2 million cells.

[STEPS]
SECTION: Thaw frozen cell from liquid nitrogen tank
1. 
 

SECTION: Thaw frozen cell from liquid nitrogen tank
2. Take a vial of frozen cells from liquid nitrogen tank. If you have many vials you need to take out of the tank and thaw at a time, you can put them on dry ice while you are looking for other vials.
SECTION: Thaw frozen cell from liquid nitrogen tank
3. Put the vials in 37 °C water bath with gentle shaking. Do not at any point let water get above 2/3 the height of the vial to prevent water from getting into the vial. Check the vial periodically. Take vial out when there is only a small chunk of ice left. 
 

SECTION: Thaw frozen cell from liquid nitrogen tank
4. Spray the vial with 70 124. Wipe dry. In the hood, open the vial and take the cells out of the vial with a P1000 into a 15 ml conical tube.
SECTION: Thaw frozen cell from liquid nitrogen tank
5. Slowly add 5 mL to 10 mL to the conical tube with gentle agitation.
Add the first ml drop wise. This is to reduce the osmotic pressure the cells will experience. Do not add thawed cells directly to fresh media!
SECTION: Thaw frozen cell from liquid nitrogen tank
6. Pellet cells in the conical tube at 150 x g, 3 min. Take off supernatant.
Resuspend the cell pellet in appropriate amount of media.
SECTION: Thaw frozen cell from liquid nitrogen tank
7. Plate the cells onto a matrigel coated plate and put the plate back into the incubator.
SECTION: Thaw frozen cell from liquid nitrogen tank
8. Cells should attach within an hour of plating.
SECTION: Thaw frozen cell from liquid nitrogen tank
9. Check cells the next day. Some floating dead cells are OK. The survival is normally 40-70% depending on the freeze.
SECTION: Regular maintenance of human pluripotent stem cells
10. Feed cells with E8 medium everyday. It is OK to double feed (feed twice the amount of media) and let the cells go without feeding for a couple days only when the cells are not too dense (eg. Right after a split).
SECTION: Regular maintenance of human pluripotent stem cells
11. When cells are 70-90% confluent, they need to be split.
SECTION: Regular maintenance of human pluripotent stem cells
12. To split cells, follow the steps below. The volume is for one well of a 6-well plate. Change the volume of solution according to your culture vessel.
SECTION: Regular maintenance of human pluripotent stem cells
13. Take off media and rinse with 1 mL. Take off the PBS+EDTA and add another 1 mL. Let the plate sit in the hood for 3 min-5 min.

SECTION: Regular maintenance of human pluripotent stem cells
14. Look under the microscope. The cells within a colony should start to separate from each other. If they are still attached to each other, wait for another 2 min -3 min  and check again.

SECTION: Regular maintenance of human pluripotent stem cells
15. Carefully take the PBS+EDTA off the plate. If the cells are incubated too long with EDTA, they might come off the plate at this step. So be careful not to suck up your cells.
SECTION: Regular maintenance of human pluripotent stem cells
16. Resuspend cells with 3 mL using a 5 ml pipette. Dislodge the cells only with media. DO NOT scrape cells off the plate with pipettes. Pipette cells up and down several times to break up cell clumps.
SECTION: Regular maintenance of human pluripotent stem cells
17. Aliquot resuspended cells into another plate with fresh E8.
SECTION: Regular maintenance of human pluripotent stem cells
18. I normally split cells at series of different ratios (1:6, 1:12 and 1:20).
 

SECTION: Freezing cells
19. Split cells at 1:3 or 1:6 two days before freeze. Cells 2 days after split will give the best survival after thaw. You can still freeze cells 4 or 5 days after split if you have to. But the survival will be significantly reduced especially if the cells are confluent.
SECTION: Freezing cells
20. Label appropriate number of cryovials you need for freezing. Also make sure you have appropriate number of Mr. Frosty.
SECTION: Freezing cells
21. Treat cells with EDTA just like what you would do when you split cells.
Resuspend cells with fresh E8 in half the volume you want to freeze your cells in.
SECTION: Freezing cells
22. Prepare 20 124 by mixing 8 parts of E8 with 2 parts of DMSO.
SECTION: Freezing cells
23. Slowly add the same volume of 20 124 to the resuspended cells with gentle mixing. This will give 10 124 final concentration in the freezing solution.
SECTION: Freezing cells
24. Aliquot the cells into appropriate number of cryovials.
SECTION: Freezing cells
25. Put vials into Mr. Frosty and place the Mr. Frosty in a -80 °C freezer.
SECTION: Freezing cells
26. The next day, transfer all the vials to liquid nitrogen tank for long-term storage. Human pluripotent stem cells are good for several months at -80 °C. But it is general not recommended to keep your frozen cells at -80 °C for an extended period of time.
SECTION: Freezing cells
27. (Optional). Thaw one vial of cells to check on the survival rate. Repeat the freezing if survival is not optimal.
SECTION: Freezing cells
28. Generally, a 70-80% confluent well in a 6-well plate can give you 2-3 vials cells.