Jun 03, 2025
Growing iPSCs in eTeSR+
- 1SUNY Upstate Medical University
Protocol Citation: Richard Kopp 2025. Growing iPSCs in eTeSR+. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqqzxogk5/v1
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: May 23, 2025
Last Modified: June 03, 2025
Protocol Integer ID: 218842
Keywords: human ipscs in etesr media, growing ipsc, human ipsc, etesr media, protocol, stemcell technology, stemcell technologies with minor modification
Abstract
This protocol describes how to grow human iPSCs in eTeSR media. This protocol is based heavily on the protocol published by StemCell Technologies with minor modifications.
Plate Preparation
Dilute Matrigel in chilled DMEM according to lot instructions. Keep Matrigel on ice at all times.
Add 1 mL chilled diluted Matrigel to each well of 6-well plate using chilled serological pipettes. Use of chilled pipettes is essential.
Incubate plate at room temperature for 1 hour. Plates can be made in advance and stored at 4 oC sealed with parafilm for up to one week. Warm to room temperature for 1 hour prior to use.
Media Preparation
Combine eTeSR+ basal medium and supplement to make complete medium
Add CloneR 2 to eTeSR+ to make 10% solution (ex. 1.2 mL CloneR 2 in 10.8 mL eTeSR+)
Remove Matrigel and add 2 mL prepared media to each well of prepared plate
Cell Split
Wash well containing iPSCs with 2 mL room temperature D-PBS after removing media
Add 500 uL Accutase warmed to 37 oC to well
Place plate in 37 oC CO2 incubator for 10 minutes
Add 1 mL room temperature eTeSR+ to well
Pipette 3 times slowly with 1000 uL pipettor
Transfer cell suspension to 15 mL tube
Centrifuge cell suspension at 300xg at room temperature for 5 minutes
Discard supernatant, add 1 mL eTeSR+ to tube
Flick tube 5 times to resuspend cells
Perform cell count
Add 10 uL cell suspension to 20 uL tube
Add 10 uL Trypan Blue to tube
Pipette 10 times with 20 uL pipettor to mix
Transfer 10 uL mix to each side of a Countess slide
Run slide on Countess
If cell count is lower than 1 million, re-centrifuge and resuspend in less than 1
mL of media, then recount; if cell count is greater than 10 million, add media,
flick tube to mix and recount
Add 10,000-40,000 cells to each well of prepared plate with media
Move plate side-to-side and forward-to-back gently to distribute cells
Place plate in incubator
Feeding
Change media daily with room temperature eTeSR+
Cells are ready to split again when colonies have pale centers
Avoid going over 10 passages