Cranial suture-like gap and bone regeneration after transplantation of cryopreserved MSCs by use of a programmed freezer with magnetic field in rats.

Mesenchymal stem cells (MSCs) can be used for regeneration of various organs and tissues. A previous study revealed that cryopreserved MSCs, which were frozen by a programmed freezer with a magnetic field (Cells Alive System: CAS) and cryopreserved for 7 days in a -150°C deep freezer, can maintain high survival and proliferation rates while retaining both adipogenic and osteogenic differentiation abilities. The purpose of this study was to examine MSC viability and tissue regenerative ability after long-term cryopreservation using a CAS freezer. MSCs were isolated from rat femora bone marrow and cryopreserved in a -150°C deep freezer (CAS group) or directly cryopreserved in a deep freezer (Direct group). After 3 years, the cells were thawed and the number of viable cells was counted. Cell proliferation was also examined after 14 days in culture. For histological examination, forty 4-week-old Fischer 344 male rats received bone and sagittal suture defects with a diameter of 6.0mm, and MSCs (CAS or Direct group) cryopreserved for 1 year were grafted with membranes. Non-cryopreserved MSCs (Control group) were transplanted to an additional twenty rats. The rats were sacrificed at 4, 8, 16, and 24 weeks after surgery. The parietal bones, including the sagittal suture, were observed under a light microscope and the extent of bone regeneration was measured. Our results indicate that MSCs survival and proliferation rates were significantly higher in the CAS group than in the Direct group. In the Control and CAS groups, a large amount of new bone formation and a suture-like gap was identified 24 weeks after transplantation, whereas only a small amount of new bone formation was observed in the Direct group. These results suggest that the CAS freezer is amenable to long-term cryopreservation of MSCs, which can be applied to the regeneration of various tissues, including bone tissue with suture-like gap formation.