TITLE: iPSC Cell Culture Protocol
AUTHORS: Isabelle J Fisher, Sally Salomonsson, Claire D Clelland

[DESCRIPTION]
This protocol describes regular maintenance of induced pluripotent stem cell (iPSC) cultures. 

[STEPS]
SECTION: Thawing iPSCs
1. Coat four wells of a six-well plate with matrigel solution and incubate at 37°C for at least 30 minutes.
SECTION: Thawing iPSCs
1.1. To make matrigel solution, resuspend matrigel 1:in KnockOut DMEM.

SECTION: Thawing iPSCs
2. Make thawing medium:
SECTION: Thawing iPSCs
2.1. Add ROCK inhibitor (RI) 1:1000 to mTeSR Plus media (ex: 20 mL medium with 20 μL RI). Warm medium in a bead bath.
SECTION: Thawing iPSCs
3. Frozen cells should be kept on ice until ready to thaw. Thaw rapidly in a water bath or by holding tube in hand. Transfer cells suspended in freezing media to a ml falcon tube with 2-5 ml of dPBS.
SECTION: Thawing iPSCs
4. Centrifuge cells at 150-300 rcf for 3 minutes.
SECTION: Thawing iPSCs
4.1. While cells are spinning, replace matrigel on six well plate with 2 mL warm thawing medium per well.
SECTION: Thawing iPSCs
5. Carefully aspirate supernatant from centrifuged cells by tilting the tube to the side, avoiding close contact with the pellet at the bottom of the tube.
SECTION: Thawing iPSCs
6. Gently resuspend the cell pellet in 1 mL thawing medium.
SECTION: Thawing iPSCs
7. Transfer cell solution into the six well plate with media. Optionally, make a concentration gradient between wells, for example:
SECTION: Thawing iPSCs
7.1. Add 400 μL, μL, 200 μL, 100 μL of resuspended cell solution into each well, respectively.

SECTION: Thawing iPSCs
8. Once cells are in colonies of at least 4-6 cells per colony, replace thawing media with mTeSR Plus without RI, ideally after about 24 hours and up to 48 hours (see fig. 1).
SECTION: Thawing iPSCs
9. Continue to feed every day, until 50-70% confluent.
SECTION: Thawing iPSCs
9.1. If cells are single cell or very small colonies, may opt to feed every other day until 10-20% confluency or large colonies.
SECTION: Passaging iPSCs
10. When cells are 50-70% confluent, they should be passaged (see fig. 2).
SECTION: Passaging iPSCs
10.1. tip: we ‘split’ or passage cells before their density outstrips the nutrient capacity of the culture media and while they are still in log growth phase. Once cells are approaching 100% confluency, they reach the plateau (stationary) phase where fewer than 10% of cells are actively dividing, and the cells are more susceptible to stress. This low rate of division makes it hard to maintain the line and increases the chances of unwanted mutations accumulating in the line over time.
SECTION: Passaging iPSCs
11. Coat a six-well plate with matrigel solution and incubate at 37°C for at least 30 minutes.
SECTION: Passaging iPSCs
11.1. To make matrigel solution, resuspend matrigel 1:100 in KnockOut DMEM.
SECTION: Passaging iPSCs
12. Aspirate medium from a well with healthy iPSCs at appropriate confluency.
SECTION: Passaging iPSCs
13. Wash once with dPBS.
SECTION: Passaging iPSCs
14. Add 1 mL of ReLeSR and incubate at room temperature in the hood for 30-40 seconds.
SECTION: Passaging iPSCs
15. Aspirate ReLeSR.
SECTION: Passaging iPSCs
16. Incubate dry well at 37°C for 3-9 minutes.
SECTION: Passaging iPSCs
16.1. While incubating, replace matrigel with warm mTeSR Plus on the new plate (optionally add RI at 1:1000).
SECTION: Passaging iPSCs
17. Resuspend cells with one mL mTeSR Plus.
SECTION: Passaging iPSCs
17.1. To resuspend, pipette mTeSR Plus over the well to lift cells. Only go over the well 1-2 times, leaving behind cells that do not lift without mechanical force (the cells that do not lift are the unhealthy cells).
SECTION: Passaging iPSCs
18. Distribute resuspended cells across 1-6 wells of your new plate.
SECTION: Passaging iPSCs
19. Gently rock plate back and forth and side to side to evenly distribute cells across the wells.
SECTION: Passaging iPSCs
20. Label plate with cell line, passage number, initials, and date.
SECTION: Passaging iPSCs
21. Look at your cells under the microscope to confirm even distribution and check the quantity of cells passaged.
SECTION: Passaging iPSCs
22. Leave plate in incubator.
SECTION: Passaging iPSCs
23. After 24-48 hours, replace media with fresh mTeSR Plus without RI.
SECTION: Passaging iPSCs
24. Continue to feed cells every 24 hours.
SECTION: Passaging iPSCs
25. When the cells are 50-70% confluent (typically after about three days), passage into a new plate.
SECTION: Passaging iPSCs
24.1. If cells are single cell or very small colonies, may opt to feed every other day until 10-20% confluency or large colonies.
SECTION: Freezing iPSCs
26. Freeze cells when the well is at 70% confluency (see fig. 2).
SECTION: Freezing iPSCs
27. Make freezing media:
SECTION: Freezing iPSCs
27.1. Make a solution of 90% mTeSR Plus and 10% DMSO
SECTION: Freezing iPSCs
27.2. Add rock inhibitor 1:1000
SECTION: Freezing iPSCs
27.3. Ex: 9 mL mTeSR Plus, 1 mL DMSO, 10 μL RI
SECTION: Freezing iPSCs
28. Label cryovials with cell line, passage number (freezing counts as a passage), initials, and date.
SECTION: Freezing iPSCs
29. Aspirate medium from a well with healthy iPSCs at 70% confluency.
SECTION: Freezing iPSCs
30. Wash once with dPBS.
SECTION: Freezing iPSCs
31. Add one mL of ReLeSR and incubate in the hood for 40 seconds.
SECTION: Freezing iPSCs
32. Aspirate ReLeSR.
SECTION: Freezing iPSCs
33. Incubate dry well at 37°C for 3 minutes.
SECTION: Freezing iPSCs
34. Resuspend cells with one mL freezing medium or Cryostor.
SECTION: Freezing iPSCs
34.1. To resuspend, pipette medium over the well to lift cells. Only go over the well 1-2 times, leaving behind cells that do not lift without mechanical force (cells that remain stuck down are the unhealthy cells).
SECTION: Freezing iPSCs
35. Transfer one mL of resuspended cell solution into a labeled cryovial.
SECTION: Freezing iPSCs
36. Add cryovials to a Corning CoolCell and leave in the -80°C freezer for a minimum of 24 hours and a maximum of 72 hours.
SECTION: Freezing iPSCs
37. Transfer vials to a box in the liquid nitrogen tank and update the liquid nitrogen spreadsheet.
SECTION: Spontaneous Differentiation
38. Signs of spontaneous differentiation (see fig. 3).
SECTION: Spontaneous Differentiation
38.1. Cells on the edge of the colonies look “spikey”
SECTION: Spontaneous Differentiation
38.2. Cells are growing slowly
SECTION: Spontaneous Differentiation
39. The first line of treatment is to passage very lightly, being careful to only pick up healthy cells.
SECTION: Bacterial Infection
40. Signs of bacterial infection:
SECTION: Bacterial Infection
40.1. Yellow, cloudy medium
SECTION: Bacterial Infection
40.2. Moving debris visible under the microscope, beyond Brownian motion
SECTION: Bacterial Infection
40.3. Increased cell death
SECTION: Bacterial Infection
41. Unless the plate is precious, spray all of the wells in the infected plate with 10% bleach, leave in the sink for at least 30 minutes, and throw away in the biohazard bin.
SECTION: Bacterial Infection
42. Sterilize the incubator and BSC hood. Notify the lab via email.
SECTION: Bacterial Infection
43. If the line is precious, it is sometimes possible to save other wells in the plate by treating with antibiotics (any severely infected well should be bleached as this is likely to spread to other wells and the incubator)
SECTION: Bacterial Infection
43.1. For example, make media with antibiotic-antimycotic 1:100, and feed the plate.
SECTION: Bacterial Infection
43.2. Effective antibiotics may vary.