TITLE: Expansion and maintenance of human induced pluripotent stem cells (iPSCs)
AUTHORS: Quyen  Do, kaitlyn.cramb, Richard Wade-Martins

[DESCRIPTION]
This protocol describes the maintenance and expansion of iPSCs in the adherent culture via single-cell passaging.

[BEFORE_START]
Sterile working techniques are an absolute must to ensure cell viability and vitality. This includes, but not limited to, filtering of all media to be used with 0.22 μm filter, sterilisation of gloves, stripettes, falcons, or any materials to be in contact with cells or cell media.

All growth factors should be added fresh on the day of intended use, or within 48 hours. Prior to use media must be warmed preferentially to 37ºC, or room temperature at the very least, as these cells are temperature-sensitive.

Cells should be regularly checked under brightfield microscope for monitoring of normal growth and identification of potential contamination. 


[STEPS]
SECTION: Expansion of iPSCs by thawing onto Matrigel
1. Day -1: Preparing plates for replating 
 

SECTION: Expansion of iPSCs by thawing onto Matrigel
1.1. Add 1 mL/well of Matrigel to each well of a 6-well plate one day prior to thawing the iPSCs.
SECTION: Expansion of iPSCs by thawing onto Matrigel
1.2. Place at 37ºC overnight (for at least 1 hour before using or up to 48 hours). 
SECTION: Expansion of iPSCs by thawing onto Matrigel
2. Day -1: Preparing iPSC Maintenance Medium (iMM) for thawing
SECTION: Expansion of iPSCs by thawing onto Matrigel
2.1. Thaw mTesR 5X Supplement (sold in a single package with mTesR basal medium) at 4oC overnight. 
SECTION: Expansion of iPSCs by thawing onto Matrigel
2.2. Prepare iPSC Maintenance Medium (iMM; see Materials) and store at 4ºC. 
SECTION: Expansion of iPSCs by thawing onto Matrigel
3. Day of thawing: Preparing spinning tubes for thawing 
SECTION: Expansion of iPSCs by thawing onto Matrigel
3.1. Add ROCKi (1:1000) to iMM media and pre-warm in 37oC water bath (2.5 mL of media per well of a 6-well plate).
SECTION: Expansion of iPSCs by thawing onto Matrigel
3.3. Add 9 mL of KO DMEM basal medium to a 15 mL falcon (spinning tube) for each vial to be thawed and prewarm.
SECTION: Expansion of iPSCs by thawing onto Matrigel
4. Thawing of iPSCs
SECTION: Expansion of iPSCs by thawing onto Matrigel
4.1. Thaw cryovial containing iPSCs in water bath until only a small component remains frozen.
 

SECTION: Expansion of iPSCs by thawing onto Matrigel
4.2. Carefully transfer contents of cryovial to pre-warmed spinning tubes. 
SECTION: Expansion of iPSCs by thawing onto Matrigel
4.3. Centrifuge at 350g for 5 min.
SECTION: Expansion of iPSCs by thawing onto Matrigel
4.4. While spinning, aspirate Matrigel from each well and replace with 1mL iMM media + 10 μM ROCKi (1:1000).
SECTION: Expansion of iPSCs by thawing onto Matrigel
4.5. Aspirate media from cell pellet in spinning falcon and replace with 1 mL iMM media + 10 μM ROCKi (1:1000), slowly and gently resuspending the pellet. 
SECTION: Expansion of iPSCs by thawing onto Matrigel
4.6. Transfer whole 1 mL to each well with 1 mL iMM media + 10 μM ROCKi (1:1000) and swirl plate gently in a figure 8 motion. 
SECTION: Maintenance of iPSCs
5. Feeding iPSCs 
Replace iMM media daily and split (by colony passage or single cell passage) when cells have reached 100% confluency and continue until enough have been obtained to start your experiment.

 

SECTION: Maintenance of iPSCs
6. Day before split: Preparing for split 
Matrigel wells of a 6-well plate as previously described in step 1.1.
SECTION: Maintenance of iPSCs
7. Splitting iPSCs by single-cell passaging
SECTION: Maintenance of iPSCs
7.1. Aspirate media from wells and rinse with 1mL PBS.
SECTION: Maintenance of iPSCs
7.2. Aspirate PBS and add 1 mL of room-temperature TrypLE per well of 6-well plate.
SECTION: Maintenance of iPSCs
7.3. Incubate at 37°C for ~3 minutes, until cells come off by pipetting.

 

SECTION: Maintenance of iPSCs
7.4. Neutralize TrypLE by collecting well contents and adding to pre-warmed spinning falcon.
SECTION: Maintenance of iPSCs
7.5. Spin at 350g for 5 minutes and replate cells as previously described, following steps 4.3. to 4.6. 
SECTION: Expansion of iPSCs by thawing onto Matrigel
3.2. Calculate the desired volume of Phosphate-buffered Saline (PBS) media and pre-warm in 37oC water bath (1 mL of media per well of a 6-well plate).