RESUMEN
INTRODUCTION: 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. SUBJECTS AND METHODS: 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-ßgal. 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. RESULTS: 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. CONCLUSIONS: 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.
RESUMEN
PURPOSE: Umbilical cord blood-derived platelet-rich plasma (UCB-PRP) containing growth factors has attracted attention as a biomaterial useful for regenerative medicine. The osteoblastic differentiation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) can be induced by UCB-PRP. MATERIALS AND METHODS: Nine samples of UC and UCB were used to conduct an in vitro study that determined the contents of three growth factors (i.e., platelet-derived growth factor, transforming growth factor ß-1, and vascular endothelial growth factor) and that examined, by staining with Alizarin red, their ability to induce the osteoblastic differentiation of UC-MSCs at the baseline, 3 months, and 3 years of cryopreservation. RESULTS: The contents of growth factors in cryopreserved UCB-PRP were markedly elevated compared to those found in UCB at baseline. The samples of UCB that were added with cryopreserved UCB-PRP and those with bone morphogenetic protein-2 were stained granularly with Alizarin red, thus indicating the presence of calcium. The samples of UCB that were not added with UCB-PRP were not stained with Alizarin red. The above-mentioned contents and ability were maintained at 3 years of cryopreservation. Cryopreserved UCB-PRP possibly and advantageously induced the osteoblastic differentiation of UC-MSCs. CONCLUSION: The potential clinical application of cryopreserved UCB-PRP to regenerative medicine was suggested.
