Expansion and maintenance of human induced pluripotent stem cells (iPSCs)
Richard Wade-Martins, Kaitlyn ML Cramb, Quyen Do
Abstract
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
Expansion of iPSCs by thawing onto Matrigel
Day -1: Preparing plates for replating
Add 1 mL/well of Matrigel to each well of a 6-well plate one day prior to thawing the iPSCs.
Place at 37ºC overnight (for at least 1 hour before using or up to 48 hours).
Day -1: Preparing iPSC Maintenance Medium (iMM) for thawing
Thaw mTesR 5X Supplement (sold in a single package with mTesR basal medium) at 4oC overnight.
Prepare iPSC Maintenance Medium (iMM; see Materials ) and store at 4ºC.
Day of thawing: Preparing spinning tubes for thawing
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).
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).
Add 9 mL of KO DMEM basal medium to a 15 mL falcon (spinning tube) for each vial to be thawed and prewarm.
Thawing of iPSCs
Thaw cryovial containing iPSCs in water bath until only a small component remains frozen.
Carefully transfer contents of cryovial to pre-warmed spinning tubes.
Centrifuge at 350g for 5 min.
While spinning, aspirate Matrigel from each well and replace with 1mL iMM media + 10 μM ROCKi (1:1000).
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.
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.
Maintenance of iPSCs
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.
Day before split: Preparing for split
Matrigel wells of a 6-well plate as previously described in step 1.1.
Splitting iPSCs by single-cell passaging
Aspirate media from wells and rinse with 1mL PBS.
Aspirate PBS and add 1 mL of room-temperature TrypLE per well of 6-well plate.
Incubate at 37°C for ~3 minutes, until cells come off by pipetting.
Neutralize TrypLE by collecting well contents and adding to pre-warmed spinning falcon.
Spin at 350g for 5 minutes and replate cells as previously described, following steps 4.3. to 4.6.
