Differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation －Comparison among cells stored for 1, 5, 10, 15, and 20 years

We investigated bone differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation. We determined the presence of any morphological and characteristic changes due to freezing to identify issues that need to be solved for future clinical applications. A total of 15 samples of hBT-MSCs that had been cryopreserved for different lengths of time, ranging from one year to 20 years (n = 3 each), were thawed and recultivated after being collected from excess iliac cancellous bone specimens of patients who underwent secondary alveolar bone grafting for cleft lip and palate in our department. We determined viability by observing calcein/EthD-stained cells under a confocal microscope, and the cell proliferation experiment was performed for one week using the Water Soluble Tetrazolium salts (WST) assay method. A confocal microscope was also used to identify any excessively accumulated senescence-associated growth factor SA-βgal. Differentiation potency was assessed in the following three groups: bone differentiation, adipocyte differentiation, and nondifferentiation induction. We examined bone/adipocyte differentiation potencies using Alizarin Red staining, Ca quantitation, and Oil Red staining after continuously culturing cells for four weeks. Viability test results indicated that the proportion of viable cells decreased as the number of years of cryopreservation increased. The cell proliferation experiment showed that cells cryopreserved for a shorter duration multiplied exponentially. In the aging test, cells cryopreserved for ≥5 years showed similar positive reactions independent of the number of years of cryopreservation. In the cell proliferation test, there was no statistically significant difference between the years of cryopreserving. We compared bone differentiation and adipocyte differentiation ability with the non-induction group, and the induction group was confirmed to have a statistical advantage. However, there was no significant difference in the induction group pertaining to different ages. Samples cryopreserved for 20 years remained competent in bone and adipocyte differentiation. However, their differentiation direction tended to skew to either bone or adipocyte differentiation. Our results suggest that freezing does not accelerate aging, and samples cryopreserved for a long time are useful in future clinical applications.