Apr 21, 2023

Public workspaceExpansion and maintenance of human induced pluripotent stem cells (iPSCs) V.1

DOI
dx.doi.org/10.17504/protocols.io.36wgqjew3vk5/v1
  • 1Oxford Parkinson's Disease Centre and Department of Physiology, Anatomy and Genetics, University of Oxford, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Cláudia C. Mendes
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.36wgqjew3vk5/v1
Protocol Citation: Quyen Do, Kaitlyn ML Cramb, Richard Wade-Martins 2023. Expansion and maintenance of human induced pluripotent stem cells (iPSCs). protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjew3vk5/v1Version created by Cláudia C. Mendes
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 22, 2023
Last Modified: April 21, 2023
Protocol Integer ID: 77448
Keywords: iPSCs, Single-Cell Passaging
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020370
Abstract
This protocol describes the maintenance and expansion of iPSCs in the adherent culture via single-cell passaging.
Materials
Reagents:

Preparing iPSC Maintenance (iMM) Media:
  • 100 mL of mTesR 5X Supplement
  • 400 mL of mTesR basal medium
  • 5 mL of 100x Penicillin-Streptomycin


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.

Expansion of iPSCs by thawing onto Matrigel
Expansion of iPSCs by thawing onto Matrigel
Day -1: Preparing plates for replating
Note
Geltrex can be substituted for Matrigel at any stage of the protocol.

Matrigel should be stored at -80ºC and prepared in KO DMEM basal medium on manufacturer’s dilution instructions. Once prepared it should be kept cold at all times.

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.
Note
Swirl the vial to thaw the iPSCs and check frequently to ensure the vial do not get completely thawed while in the water bath.

Be very careful to wipe down the vial with ethanol after removing from the water bath, and try to not place lid underwater to remove the risk of contamination.

If inexperienced, do 1 or maximum 2 vials at a time as a time delay will affect the viability of your cells.

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
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.

Note
Check cells daily under a light microscope to track confluency and health.

These cells are particularly temperature sensitive and mTesR basal medium should be warmed briefly to at least room temperature before use.

Single cell passaging of iPSCs is not recommended for more than 2 weeks unless regularly karyotyped and SNPs.

Cells at 100% confluency should be split 1:2 or 1:3. If cells don’t look particularly healthy or are growing slower then split 1:2.

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.

Note
Check if cells have dissociated by tapping gently on side of plate.

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.