A simple method for effective cryopreservation of antlerogenic periosteum of sika deer

Antlerogenic periosteum (AP) is the unique tissue type that gives rise to antlers and their antecedents, the pedicles. Deer antlers are the only mammalian organ that can fully regenerate. Efficient investigation of the mechanism of antler formation and regeneration requires year-round availability of AP, but naturally AP can only be obtained less than two months in a year. In the present study we took the cryopreservation approach to store the sampled AP in ultra-low temperature to overcome the limited period of availability. First, we evaluated the suitability of vitrification and cell cryopreservation method for cryopreservation of AP, cell migration status of the AP tissue pieces confirmed that vitrification methods did not work as the only few AP cells migrated out, whereas migrated cell numbers in the cell-cryo group (conventional method for cryopreservation of cells) were comparable to those of the fresh AP group. To further evaluate the suitability of cell cryopreservation method for AP tissue, AP samples were allocated into three groups based on the different ratios of cryopreservation reagents (dimethyl sulfoxide [DMSO], dulbecco's modified eagle's medium [DMEM] and fetal bovine serum [FBS]): AP-Cell-1 (1:4:5), AP-Cell-2 (1:2:7) and AP-Cell-3 (1:0:9), the results showed that migrated cell number were again comparable to the fresh AP group. There was no significant difference between the cell-cryo groups (AP-Cell-1 and AP-Cell-3) and the fresh group: (1) in viability (p > 0.05) through trypan blue staining (91.2%, 90.8%, and 92.4%, respectively); (2) in the attachment day, and all on Day 5 after cell seeding; (3) in cell proliferation rate (p > 0.05) through Cell Counting kit 8 (CCK8) measurement; and (4) in number of the formed clones (Clonogenicity). In the in vivo trials, there was no visible difference in temporal differentiation sequence of the formed xenogeneic antlers between the fresh AP and cryopreserved AP (AP-Cell-1 and AP-Cell-3). Overall, we found that the AP tissue was well cryopreserved just using the conventional freezing and thawing methods for cells, and their viability and developmental potential comparable to the fresh AP both in vitro and in vivo. The long-term preservation of the AP tissue is of great significance for the study of the periosteum biology in general and the mechanism underlying xenogeneic generation and regeneration of deer antlers in specific.