Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide

Abstract

Background: Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed.

Results: The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a 'straight freeze' protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods.

Conclusion: A 5% DMSO / 5% HES solution cryopreservation solution using a 'straight freeze' approach can be recommended for rat MSC.

Figure 1

MSC phenotyping. Level of CD-expression in MSCs cryopreserved in different solutions directly after thawing (day 0) and after 3 days with protocol 1 (A) and protocol 7 (B).

Figure 2

The effect of cryopreservation on MSC viability immediately after thawing and after 3 days at various HES/DMSO combinations and freezing rates. Viability of MSCs after cryopreservation with either different freezing rates (A, C) or different concentrations of DMSO and HES 450 (B,D) measured directly after thawing (A, B) and after 3 days by MTT (C, D).

Figure 3

Cryopreservation of MSCs using HES/DMSO combinations. Viability of MSCs after cryopreservation with different concentrations of DMSO and HES 450, measured by 3 days after thawing in normal media (A) and after 14 days of osteogenic differentiation (B). Alkaline phosphatase activity of cryopreserved MSCs after osteogenic differentiation for 14 days (C).

Figure 4

Effect of HES with different molecular weight distributions on MSCs cryopreservation. Viability of MSCs after cryopreservation with 10% HES of different molecular weight distributions (109–609) in combination with DMEM or FCS, measured 3 days after thawing (A) and by methylene blue staining after osteogenic differentiation for 14 days (B). Alkaline phosphatase activity of cryopreserved MSCs after osteogenic differentiation for 14 days (C).