DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells

Igor I. Katkov,Alexey V. Terskikh,Natalia G. Kan,Flavio Cimadamore,Brandon Nelson,Evan Y. Snyder

doi:10.4061/2011/981606

Igor I. Katkov, Alexey V. Terskikh + Show 4 more

Open Access

https://doi.org/10.4061/2011/981606

Copy DOI

Abstract

Three modes for cryopreservation (CP) of human iPSC cells have been compared: STD: standard CP of small clumps with 10% of CPA in cryovials, ACC: dissociation of the cells with Accutase and freezing in cryovials, and PLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells in ACC and PLT were frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI. Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze® concept developed by CELLTRONIX). CP of cells directly in plates in ready-to-go after thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests.

Highlights

Human pluripotent stem cells hold great potential for cell therapy and regenerative medicine and as useful tools to demonstrate in vitro embryotoxicity
We report first steps in this direction, namely, efficient cryopreservation of (1) dissociated, (2) adherent Human pluripotent stem cells (hPSCs)-derived induced PSCs (iPSCs) (3) with ethylene glycol (EG), (4) the Rho-associated kinase (ROCK) inhibitor Y-27632, and (5) CP using a programmable freezer
In a pilot study we found that ethylene glycol (1,2-ethane diol, EG) provided equal protection in cryopreservation of 293 cells so we considered this diol as a good candidate for substitution of DMSO

Summary

Introduction

Human pluripotent stem cells (hPSCs) hold great potential for cell therapy and regenerative medicine and as useful tools to demonstrate in vitro embryotoxicity. While effective CP protocols for ordinary cell lines and even murine embryonic stem cells (mESCs) are well established, this is not the case for hPSCs due to low recovery rates of viable and pluripotent cells following freezing, and the slow hESC growth rate, so the time from thawing to obtaining cultures suitable for experimentation can be weeks [1]. This problem is not merely an inconvenience because extended culture periods exert increased selective pressure on the cell population enhancing the likelihood of phenotypic variation and/or alterations in potency. We report first steps in this direction, namely, efficient cryopreservation of (1) dissociated, (2) adherent hPSC-derived iPSCs (3) with ethylene glycol (EG), (4) the Rho-associated kinase (ROCK) inhibitor Y-27632, and (5) CP using a programmable freezer

Derivation of hESC-iPSCs

Findings

Discussion

Conclusions