TITLE: iPSC Cell Culture – Maintenance and Expansion
AUTHORS: Jacob Marsh, Celeste Karch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about Maintenance and Expansion of induced pluripotent stem cells.</div></div>

[STEPS]
?. [Matrigel Coating]
To resuspend, thaw aliquot .
on ice
?. [Matrigel Coating]
Add  cold DMEM/F12.
12.5 ml
?. [Matrigel Coating]
Pipette up and down twice.
?. [Matrigel Coating]
Add  of Matrigel per well of 6 well plate.
1 ml
?. [Matrigel Coating]
Store diluted Matrigel at .
4 °C
?. [Thawing iPSC]
Prior to thawing cells, coat plate with Matrigel for .
1 vial of iPSC should be thawed into 1 well of a 6 well plate.
?. [Thawing iPSC]
Add  DMEM/F12 to a 15 ml conical tube labeled with the iPSC line name and passage number.
9 ml
?. [Thawing iPSC]
Remove cells from liquid nitrogen storage.
?. [Thawing iPSC]
Quickly thaw cells in  water bath and/or in hands.
37 °C
?. [Thawing iPSC]
Just prior to complete thaw, remove vial from water bath.
?. [Thawing iPSC]
Transfer the contents of the cryo-vial (~ ) into the 15 ml conical tube.
1 ml
?. [Thawing iPSC]
Spin at  for  at .
Centrifuge: 750 33
37 Room temperature
?. [Thawing iPSC]
Aspirate media.
?. [Thawing iPSC]
Resuspend cells in  mTesR1 (supplemented with  —  Rock Inhibitor) by pipetting two times.
2 ml
?. [Thawing iPSC]
Transfer the cell solution to one well of a 6-well plate.
?. [Thawing iPSC]
Incubate at  in 6 % CO2.
37 °C
?. [Thawing iPSC]
Replace the media daily until cells are ready to split or analyze.
?. [Cell Growth/Maintenance]
Aspirate media.
Media should be changed daily. It is okay to skip a media change one time each week if double feeding is performed; however, this is largely dependent on the density of the cells and volume of media (do not double feed if cells are more than 70% confluent).
?. [Cell Growth/Maintenance]
Gently add fresh mTesR1 to cells (volume depends on cell density and well size). per well to 24 well plate —  per well to 6 well plate —  to 10 cm2 plate
0.5 ml
2 ml
4 ml
5 ml
10 ml
?. [Cell Growth/Maintenance]
Incubate at  in 6 % CO2.
37 °C
?. [Cleaning]
Under microscope, remove differentiated cells with p20 or p200 tip (depending on the amount of differentiation). Transfer the cells/media to a biohazard bag.
When differentiating cells appear in the culture, it is important to remove all the cells promptly.Repeated cleaning may be necessary over the course of several days to remove all the material. If differentiation is excessive and line is precious, perform subcloning.
?. [Cleaning]
Gently wash cells with 1x PBS.
?. [Cleaning]
Add fresh mTesR1. per well to 24 well plate —  per well to 6 well plate —  to 10 cm2 plate
0.5 ml
2 ml
4 ml
5 ml
10 ml
?. [Cleaning]
Incubate at  in 6 % CO2 until cells are 60 — 80 % confluent. Change mTesR1 media daily untilcells are needed. Repeat cleaning as necessary.
37 °C
?. [Splitting/Expanding]
iPSCs grow on Matrigel. Plates should be coated with Matrigel at least 1 hour prior to plating and nolonger than 24 hours prior to plating cells: in 12 well plate in 6 well plate in 10 cm2 plate
0.5 ml
1 ml
4 ml
It is critical to keep Matrigel on ice while coating. Prior to plating cells, ensure Matrigel has notevaporated from well.
?. [Splitting/Expanding]
Aspirate media.
?. [Splitting/Expanding]
Gently wash cells with 1x PBS (2 — 3 ml/well).
?. [Splitting/Expanding]
Add Accutase (Gibco A11105-01) directly to the cells and incubate at  for  — .6 well plate, add  —  per well24 well plate, add 10 cm2 dish, add
37 °C
0.75 ml
1 ml
0.5 ml
3 ml
?. [Splitting/Expanding]
Tap dish to aid in dislocation of cells.
?. [Splitting/Expanding]
Add DMEM/F12 directly to cells and scrape gently to remove all cells (use p1000 for 24 well plate, and cell scraper for 6 well plate and 10cm2 dish).6 well plate, add  —  per well24 well plate, add 10 cm2 dish, add
2 ml
4 ml
1 ml
9 ml
?. [Splitting/Expanding]
Collect cells in conical tube (15 ml/50 ml depending on volume).
?. [Splitting/Expanding]
If necessary, add  —  DMEM/F12 to dish to remove all cells from the dish and add to conical tube.
2 ml
5 ml
?. [Splitting/Expanding]
Centrifuge cells at  for  at .
Centrifuge: 750 33
37 Room temperature
?. [Splitting/Expanding]
Carefully aspirate supernatant.
To avoid aspirating cell pellet, it is OK to leave a small amount of media (  —  ).
?. [Splitting/Expanding]
Resuspend cell pellet with mTesR1 (Rock Inhibitor addition varies, see below). mTesR1 per well of a 6 well plateOur goal is to maintain iPSC lines without using Rock Inhibitor; however, this must be done through careful weaning off Rock InhibitorAll cells should be thawed in Rock Inhibitor:    -  concentration for new iPSC lines, lines thawed from 96 well after editing.    -  concentration if thawing from a line without knowledge of its Rock sensitivity.    -  concentration for all other lines (for lines still exposed to Rock Inhibitor, use. Otherwise, do not use Rock Inhibitor.)
2 ml
?. [Splitting/Expanding]
Pipet cells 2 times only to preserve clumps.
?. [Splitting/Expanding]
Transfer cell suspension to appropriate plate (pre-coated with Matrigel for at least ).For maintenance, dilute cells 1:3 in mTesR1For expansion, plate all cells
?. [Splitting/Expanding]
Incubate at  in 6 % CO2 until cells are 60 — 80% confluent. Change mTesR1 media daily until cells are needed.
37 °C
?. [iPSC Freezing]
Aspirate media.
?. [iPSC Freezing]
Gently wash cells with 1x PBS (Use  —  per well in 6 well plate).
2 ml
3 ml
?. [iPSC Freezing]
Add Accutase (Gibbco A11105-01) directly to the cells and incubate at  for  — .6 well plate, add  —  per well10cm2 dish, add
37 °C
0.75 ml
1 ml
3 ml
?. [iPSC Freezing]
Tap dish to aid in dislocation of cells.
?. [iPSC Freezing]
Add DMEM/F12 directly to cells.6 well plate, add —  per well10cm2 dish, add
2 ml
4 ml
9 ml
?. [iPSC Freezing]
Collect cells in conical tube (15 ml/50 ml depending on volume).
?. [iPSC Freezing]
Add  —  DMEM/F12 to dish to remove all cells from the dish and add to conical tube.
2 ml
5 ml
?. [iPSC Freezing]
Centrifuge cells at  for  at .
Centrifuge: 750 33
37 Room temperature
?. [iPSC Freezing]
Carefully aspirate supernatant.
To avoid aspirating cell pellet, it is OK to leave a small amount of media (  —  ).
?. [iPSC Freezing]
Resuspend cell pellet with mTesR1 (No Rock Inhibitor).Use volume appropriate for freezingAssume   per cryovial total and add ½ total volume of mTesR1Pipet cells 1 — 2 times only to preserve cell clumpsAssume  per cryovial total and add ½ total volume of mTesR1
1 ml
Example: to freeze 10 tubes, you will need   total and will add   mTesR1 to cell pellet (and   of 2x Freezing Media below)
?. [iPSC Freezing]
Add an equal volume of cold 2x Freezing Media (20 % DMSO, FBS). Pipet cells 1 time only to preserve cell clumps.
?. [iPSC Freezing]
Transfer cell suspension to pre-labeled cryovials ( per cryovial).Ensure that cryovials are labeled with the following:Cell TypeLine NamePassage #DateYour Name
1 ml
?. [iPSC Freezing]
Freeze vials at  in foam racks for  — .
-80 °C
?. [iPSC Freezing]
Transfer vials to liquid nitrogen for long-term storage.