Hi all,

I'm currently attempting to differentiate feeder-free hiPSCs to cortical neurons, however, I'm having problems with simply expanding the cell line to an appropriate amount. The major problem I have is the first splitting after the thaw, cells detach daily and in what seems to be logarithmically over the course of 4-5 day until the dish is completely void of viable cells. Below is a comprehensive list and outline of my procedure:

(manufacturer's procedure: https://www.atcc.org/~/media/7E031EF950594BC3B85A411AE1DC9684.ashx)

Materials:

• DYS0100 hiPSC line from ATCC (https://atcc.org/~/media/PDFs/StemCellSolutions.ashx)
• Essential 8 Complete Medium and Supplement
• Geltrex (1:30)
• ROCK inhibitor- Y27
• HBSS solution
• DMSO

Procedure:

THAWING

• I coat the dish with its respective minimum volume of Geltrex
• Allow to incubate for 1 hour at 37oC
• Rapidly thaw cryovial of cells (suspended in E8/10%DMSO) in 37oC
• Withdraw volume from cryovial and add to HBSS solution in conical Falcon tube (in 3 times the volume of the total amount of cryomedium i.e. 3 ml HBSS per 1 ml of cells in E8C/10%DMSO
• Centrifuge for 3 min X 200g
• Aspirate supernatant
• Resuspend in E8C + 1/1000 Y27 and seed plate
• Allow to incubate 18-24 hr
• Aspirate ROCK Inhibitor medium and replace with fresh E8C (-Y27)
• Change medium once every day by half volume, maintaining "conditioned" medium

SPLITTING

• Aspirate completely the medium from the confluent plate
• Replace with minimal necessary volume of warmed Accutase and allow to incubate at 37oC for 30 min
• After detachment, I dilute the accutase/cell suspension in 3 times the volume of sterile 1x PBS
• Titurate gently no more than 3 times
• Collect the suspension in conical Falcon tube
• Centrifuge for 3 min X 200g
• Aspirate supernatant
• Resuspend in E8C + 1/1000 Y27 and seed plate
• Allow to incubate 18-24 hr

Aspirate ROCK Inhibitor medium and replace with fresh E8C (-Y27)

NUANCES:

• The cells arrived to our lab from a colleague who collected them in ~300,000 cells/ml (3 vial of 1ml each in total)
• I expanded successfully to ~30*10^6 cells but froze them at a rate of 1*10^6 cells/ml
• I originally followed only the manufacturer's instructions and split with 2x HBSS washes followed by 6 min Versene incubation in 37oC
• Cells never become quiescent after the thaw, only after the splitting.
• I have ~10*10^6 cells left dispersed evenly among 2 x 2ml conical vials

Any help or pointers or methods of rescue would be greatly appreciated. I'm at my wits' end after almost completely obliterating my stock..

Thanks so much and Happy Thanksgiving!