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

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(2)SO), 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(2)SO 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(2)SO, 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.

Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have some favorable characteristics like high plasticity, multilineage differentiation potential, and comparably easy handling in vitro, making them of interest for many clinical and therapeutic approaches including cell therapy. For routine applications, these cells have to be stored over a certain period of time without loss of cell vitality and function. An easy way to preserve cells is to store them at temperatures between -80 degrees C and -196 degrees C (liquid nitrogen). To prevent cells from the damage caused by the cryopreservation process and to achieve high cell recovery and vitality, cryoprotectants are used. Typically dimethylsulfoxide, often in combination with serum, is used as a cryoprotectant. However, for clinical approaches, the use of dimethylsulfoxide and serum in patients is problematic for several reasons. Therefore, the cryopreservation of human mesenchymal stem cells for cell therapeutic applications without dimethylsulfoxide and serum demands investigation. In this work, non-toxic alternatives to dimethylsulfoxide such as glycerol or the compatible solutes, proline and ectoin, were analyzed in a serum-free cryomedium with respect to their cryoprotective properties. Different concentrations of the cryoprotectants (1-10% (w/v) ectoin or proline, respectively, or 5-20% (v/v) glycerol) and certain incubation times (0-60 minutes) were investigated with regard to post-thaw cell vitality and cell growth. Our results showed that, in general, cryopreservation with ectoin led to high post-thaw cell survival of up to 72% whereas after cryopreservation with glycerol and proline, the hMSC cells were completely dead (glycerol) or had only poor cell survival (proline, 22%). Moreover, the morphology of the hMSC cells changed to a large and flat phenotype after cryopreservation with proline. These results indicate that glycerol and proline are not suitable for cryopreservation of hMSC. In contrast, ectoin has the potential to replace dimethylsulfoxide as a cryoprotectant in a serum-free cryomedium.