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[Introduction] We are currently preparing for a Japanese Ministry approved clinical study using allogeneic chondrocyte sheets derived from polydactyly patients.Polydactyly derived chondrocytes proliferate rapidly,and thus mass production can be realized; however,making chondrocyte sheets widely available requires proper storage systems.We have already reported an effective vitrification method for rabbit chondrocyte sheets(BMC Biotec.2013).Here,we aimed to establish effective vitrification conditions for human polydactyly derived chondrocyte sheets.[Methods] Under the approval and guidance of the Clinical Research Review Committee,cartilage tissues obtained from polydactyly patients(avg.13.3 months)were used to create chondrocyte sheets.The sheets were pre-treated with equilibration and vitrification solutions,permeated with a cryoprotectant,packaged in aluminum,and then vitrified.The pre-treatment was set at 10,20,30,or 45 minutes to determine optimal conditions.To recover,vitrified sheets were placed on a heating plate(38℃)and transferred to a sucrose solution to diffuse out the cryoprotectants.Sheets were checked for cracks,and cell viability was determined by trypan blue staining.The production of cartilage anabolic factors(TGFβ1,MIA)by vitrified sheets were measured by ELISA and compared with that of non-vitrified controls.[Results] All sheets were visibly undamaged.The number of viable cells per sheet was the highest for 10 minutes pre-treatment and comparable to that of the control group(2.41±0.45×10^6 vs.2.98± 0.69×10^6,n=8,p=0.54).TGFβ1 production(ng/ml)was equivalent to 86%of control group(2.44±0.37 vs.2.82±0.60,n=7,p=0.07).MIA production(ng/ml)decreased significantly to 67%of control group(9.38±4.21 vs.13.99±6.47,n=7,p<0.05).[Conclusion] Vitrification of polydactyly derived chondrocyte sheets was possible.The production of anabolic factors likely depends on the number of viable cells; nevertheless,MIA production was reduced at a cellular level.We will further explore such issues and optimize the vitrification method for cell viability and functionality.