Systematic parameter optimization of a Me ₂SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

authored by

Denise Freimark, Constanze Sehl, Christian Weber, Klaus Hudel, Peter Czermak, Nicola Hofmann, Ralf Spindler, Birgit Glasmacher

Abstract

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me ₂SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients.Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me ₂SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me ₂SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.

Organisation(s)

Institute of Multiphase Processes

External Organisation(s)

Technische Hochschule Mittelhessen University of Applied Sciences
Martin Christ Gefriertrocknungsanlagen GmbH
Kansas State University

Type

Article

Journal

CRYOBIOLOGY

Volume

63

Pages

67-75

No. of pages

9

ISSN

0011-2240

Publication date

10.2011

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

General Biochemistry,Genetics and Molecular Biology, General Agricultural and Biological Sciences

Sustainable Development Goals

SDG 3 - Good Health and Well-being

Electronic version(s)

https://doi.org/10.1016/j.cryobiol.2011.05.002 (Access: Closed)