Mesenchymal stem cells (MSCs) have recently generated great interest in the fields of regenerative medicine and immunotherapy due to their unique biologic properties. In this review we attempt to provide an overview of the current clinical status of MSC therapy, primarily focusing on immunomodulatory and regenerative or tissue repair applications of MSCs. In addition, current manufacturing is reviewed with attention to variation in practices (e.g., starting material, approach to culture and product testing). There is considerable variation among the 218 clinical trials assessed here; variations include proposed mechanisms of action, optimal dosing strategy, and route of administration. To ensure the greatest likelihood of success in clinical trials as the field progresses, attention must be given to the optimization of MSC culture.

Background Current methods of MSC cryopreservation result in variable post thaw recovery and phenotypic changes due to freezing. The objective of this investigation is to determine the influence of ex-vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods Human bone marrow aspirate was purchased from Lonza (Walkersville, MD). MSCs were isolated, and cells were assessed for total count, viability, apoptosis, and senescence over 6 passages (8–10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were re-plated for continued culture, and the other half were frozen at 1°C/min in a controlled rate freezer. Frozen samples were stored in liquid nitrogen, thawed, and reassessed for total cell count, viability, and senescence immediately and 48 hours post thaw. Results Viability did not differ significantly between samples pre freeze or post thaw. Senescence increased over time in pre freeze culture, and was significantly higher in one sample that experienced growth arrest both pre freeze and post thaw. Freezing resulted in similar initial post thaw recovery in all samples, but 48 hour post thaw growth arrest was observed in the sample with high senescence only. Conclusion High freeze senescence appears to correlate with poor post thaw function in MSC samples, but additional studies are necessary to obtain a sample size large enough to quantify results.

There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives. Postthaw recovery of mesenchymal stem cells (MSCs) in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1°C/min when the solution composition is optimized. Maximum postthaw recovery was observed in these multiple osmolyte solutions with incubation times of up to 2 h before freezing. Raman images demonstrate large ice crystal formation in cryopreserved cells incubated for shorter periods of time (*30 min), suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the postthaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted U-shape behavior, indicating that there may be a ''sweet spot'' for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed postthaw recovery. Phase change behavior (solidification patterns and glass-forming tendency) did not differ for solutions with similar osmolarity, but differences in postthaw recovery suggest that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance postthaw recovery.

Raman microspectroscopy was used to quantify freezing response of cells to various cooling rates and solution compositions. The distribution pattern of cytochrome c in individual cells was used as a measure of cell viability in the frozen state and this metric agreed well with the population-averaged viability and trypan blue staining experiments. Raman imaging of cells demonstrated that intracellular ice formation (IIF) was common and did not necessarily result in cell death. The amount of intracellular ice as well as ice crystal size played a role in determining whether or not ice inside the cell was a lethal event. Intracellular ice crystals were colocated to the sections of cell membrane in close proximity to extracellular ice. Increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF. Reducing the effective stiffness of the cell membrane by disrupting the actin cytoskeleton using cytochalasin D increased the amount of IIF. Strong intracellular osmotic gradients were observed when IIF was present. These observations support the hypothesis that interactions between the cell membrane and extracellular ice result in IIF. Raman spectromicroscopy provides a powerful tool for observing IIF and understanding its role in cell death during freezing, and enables the development, to our knowledge, of new and improved cell preservation protocols.

Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols, and small-molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and quantitative real time-polymerase chain reaction validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin, the antiapoptotic marker B cell lymphoma 2, as well as the cell surface adhesion molecules CD106 (vascular cell adhesion molecule 1) and CD54 (intracellular adhesion molecule 1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome, and retain the differentiation ability of MSCs, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.