Efficient long-term cryopreservation of pluripotent stem cells at −80 °C

Ye Yuan,Yang Liu,Yuchen Tian,R Michael Roberts,Ying Yang,Jinkyu Park,Xu Han,Aihua Dai

doi:10.1038/srep34476

Ye Yuan, Yang Liu + Show 6 more

Open Access

https://doi.org/10.1038/srep34476

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Abstract

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures, e.g. −80 °C. This instability leads to ice recrystallization, causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (~−80 °C) of laboratory deep freezers. Moreover, a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at −80 °C for periods that extend up to at least one year, with the post-thaw viability, plating efficiency, and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2.

Highlights

Pluripotent stem cells have an ability to self-renew, yet can be induced to differentiate into a wide range of differentiated cell types
Acceptable cell cryopreservation has been achieved at low ice recrystallization inhibitors (IRI) concentrations and without using permeating cell cryoprotectant for some cell types, including human and sheep red blood cells[39], human embryonic liver cells[40], rat mesenchymal stem cells[41,42], mononuclear cells from human umbilical cord blood cells[43], among others
We have developed a method for the long term storage of pluripotent stem cells at the typical operating temperatures for standard laboratory deep freezers through use of a commonly available polysaccharide, Ficoll 70

Summary

Introduction

Pluripotent stem cells have an ability to self-renew, yet can be induced to differentiate into a wide range of differentiated cell types. Mechanical deep freezers, with their working temperature typically at −80 °C, provide an alternative to LN2 for many purposes, such as preserving stocks of microbes and viruses[30], and storage of human tissues[31], and work well as long as the freezers are equipped with power back-up and, temperature monitoring and alarm systems Even with such add-ons, deep freezers still potentially offer significantly improved operational and cost efficiencies compared to LN2 for many applications. To the best our knowledge, the use of such IRI has not been extended to the cryopreservation of cells other than RBC at −8 0 °C To overcome this shortcoming, we have developed a method for the long term storage of pluripotent stem cells at the typical operating temperatures for standard laboratory deep freezers through use of a commonly available polysaccharide, Ficoll 70

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Conclusion