The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells.

Xia Xu,William James,Zhanfeng Cui,Sally Cowley,Christopher J Flaim,Leonard Seymour

doi:10.1002/btpr.368

Abstract

Human embryonic stem (hES) cells have enormous potential for clinical applications. However, one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis, which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased, F-actin content and distribution is altered, and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However, exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

Highlights

HES cells have enormous potential for clinical applications, offering therapies for a wide range of degenerative diseases and disorders such as diabetes and Parkinson’s disease.[1]
The obstacle faced, using slow-freezing with 10% DMSO as a cryoprotectant for Human embryonic stem (hES) cells, is an extremely low cell survival rate, around 10%.2. It has been demonstrated by Heng et al.[2] that the low cell recovery rate after conventional cryopreservation is associated with apoptosis rather than necrosis induced by cryoinjury, which is consistent with our observations
We have shown that inhibition of Rho-associated kinase (ROCK) following cryopreservation results in the reduction of caspase-8 activity (Figure 1D), activation of ROCK is involved in the regulation of activation of caspase-8 in cryopreservation

Summary

Introduction

HES cells have enormous potential for clinical applications, offering therapies for a wide range of degenerative diseases and disorders such as diabetes and Parkinson’s disease.[1] Long term storage or banking is a prerequisite for hES cell applications. For potential therapeutic or commercial applications, banking requiring a large amount of hES cells is required. Slow freezing, rather than vitrification, is more practical. One of the current technical challenges is how to achieve high cell recovery rate after cryopreservation. Cryoprotectant agent (CPA), such as dimethylsulfoxide (DMSO), is added to cells to protect them during freezing.

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