Abstract: Objective To investigate the effect of cryopreservation on cell viability,proliferation,and in vitro/in vivo cartilage formation abilities of human auricular chondrocytes using a scaffold-free cartilage sheet model.Methods Human auricular cartilage was obtained from patients with microtia.Chondrocytes were isolated through collagenase digestion.The chondrocytes were cultured and expanded into passage 1(P1)before being divided into the experiment and the control groups.In the experiment group(Exp),the chondrocytes were collected,suspended with FBS and DMSO,then froze according to the cell cryopreservation steps and thawed after storage for 1 month in liquid nitrogen before being used to generate the cartilage sheet.In the control group(Ctrl),the cells were not cryopreserved and continued to be expanded until passage 3(P3)before being used for cartilage sheet generation.Cell morphology,viability,proliferation and cartilage specific extracellular matrix(ECM)production were examined by light microscopy,CCK-8 assay,live/dead,HE and Alcan blue staining.The in vitro scaffold-free cartilage sheets in each group were examined through wet weight,volume and biochemical contents to determine the in vitro cartilage formation abilities.Then the scaffold-free cartilage sheets were diced into neocartilage fragments(injectable cartilage)for nude mouse injection(n=5).After 8 weeks of injection,the explants were examined again by gross view,HE staining and biochemical contents to determine the in vivo cartilage formation abilities of the chondrocytes in each group.Results No significant differences were observed in morphology,viability,proliferation and cartilage specific GAG production abilities of the chondrocytes between the two groups.Compared with the Ctrl group,the wet weight and collagen content were slightly higher in the Exp group.However,there was no significant difference in the in vivo cartilage-forming ability between the two groups.Conclusion Cryopreserving and thawing process have no significant effect on the viabili

Key words: Cryopreservation, Human auricular chondrocytes, Sc