Evaluation of Bone Marrow-Derived Mesenchymal Stem Cells After Cryopreservation and Hypothermic Storage in Clinically Safe Medium

Abstract

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable "off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe, animal product-free medium containing 2%, 5%, and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery, viability, apoptosis, proliferation rate, expression of a broad panel of MSC markers, and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO, respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO, respectively, were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity, ALP surface expression and Ca⁺⁺ deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution, also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery, respectively, less than 10% of apoptotic cells, and normal proliferation, marker expression, and osteogenic potential. Overall, our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents.

Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects

“…The final cellular product could be delivered to the patient's bedside for infusion. Alternatively, it has been shown 914 that sub-confluent cultures of MSCs that have been stored hypothermically at 4 °C for 2 to 4 915 days and then allowed to recover at 37 °C for 3 h yielded cells with normal proliferation, surface 916 marker expression and osteogenic potential[76]. Moreover, the addition of chemical modulators917 such as resveratrol and salubrinal decreased apoptosis, necrosis and the levels of cell stress 918 signaling proteins, thereby improving the viability of human MSCs following hypothermic 919 storage [41].…”

The sensitivity of human mesenchymal stem cells to vibration and cold storage conditions representative of cold transportation

“…Different transportation strategies have been investigated [30] and a significant amount of research has been carried out to understand the effects of cold storage and cryopreservation on the viability of cells [31][32][33]. The study carried out within the research reported here has investigated the effects of the combination of cold storage and mechanical stress during cold transport of an hMSC suspension without the presence of cryoprotectant.…”

The role of biopreservation in cell and gene therapy bioprocessing

“…The commercial hypothermic preservation medium solution HypoThermosol Free Radical Solution (BioLife Solutions, USA) has been used for the successful storage and preservation of a range of cell types including hMSCs [44] and differentiated human neural stem cells (hNSCs) [45]. In the case of hMSCs, the cells retained > 85% viability after 96 h when stored at 4˚C [44], and for the differentiated hNSCs, the cells were seeded onto cellular collagen constructs and placed into a range of different medium compositions for storage at 4˚C for 48 h. The cells which were preserved in the HTS-FRS medium composition demonstrated the least cell death (<10%), even lower than cells which were preserved using DMSO under cryogenic conditions [45]. Indeed, recently, HTS was used for TiGenix's phase 1 clinical trial study where expanded allogeneic adipose-derived stem cells stored in HTS were administered to patients [46].…”