Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation.

Lei Gao,Xiaohui Wang,Liping Lv,Yulong Zhang,Ping Ma,Qianqian Zhou,Sujing Sun,Min Liu,Lei Zhang,Linsheng Zhan,Jing Yang

doi:10.3390/ijms22147445

Abstract

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.

Highlights

Theinfluence influenceofofbetaine betaineononwater water behaviors during freezing warming proThe behaviors during thethe freezing andand warming process cess was investigated by using a differential scanning calorimeter (DSC)
Fresh cell or green fluorescent protein (GFP)-Fluc-umbilical cord mesenchymal stem cells (UCMSCs) recovered from B–E cryopreservation and dimethyl sulfoxide (DMSO) cryopreservation demonstrated the same in vivo distribution manner as the GFP-Fluc-UCMSCs, which were mainly concentrated in the lungs after intravenous administration and gradually diminished over 72 h
No obvious differences were observed between the two aspects of fresh UCMSCs and UCMSCs recovered from B–E cryopreservation

Summary

Introduction

Mesenchymal stem cells (MSCs) have emerged as the ideal candidate for treatments of tissue damage, graft versus host diseases, osteoarthritis, and gynecological diseases due to their favorable properties such as self-renewal, multi-lineage differentiation, and immunoregulation [1,2]. The success of these clinical applications requires a large number of viable MSCs [3]. The current protocols for MSC cryopreservation require dimethyl sulfoxide (DMSO), which is permeable and able to protect MSCs from cryo-injuries [7]

Methods

Results

Discussion

Conclusion