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Introduction: We have studied the effects of chondrocyte sheets on the repair and regeneration of articular cartilage. With approval of the Ministry of Health, Labour and Welfare in Japan, we have performed a clinical study to transplant the layered chondrocytes sheets into 8 patients with osteoarthritis. Polydactyly tissue was investigated as a source of candidate cells. Materials and Methods: Safety was assessed using array comparative genomic hybridization (ACGH), G-band straining, and tumorigenesis evaluation in NOG mice. Samples were harvested during surgeries from 12 polydactyly patients (average age 16 months), and chondrocytes were isolated from polydactyly tissue and cultured until passages 2 (P2), P4, P6, and P12. Results: ACGH of genomic DNA in chondrocytes showed no differences in copy number between P2, P4, P6, and P12 cells. G-band staining showed no abnormalities in chondrocytes listed in An International System for Human Cytogenetic Nomenclature(2013). Tumorigenesis evaluation showed no differences in the external appearance and histology at any time point up to 12 weeks of culture. After conducting these investigations and confirming the safety of the polydactyly-derived cartilage cells, we obtained approval from both the university Ethics Committee to conduct clinical research and the Ministry of Health, Labour and Welfare in Japan. Conclusions: We have developed a method to assess the safety of allogeneic chondrocytes sheets, which provide a highly useful method for evaluating allogenic human chondrocytes and other cell types. In future, we plan to use allogeneic chondrocyte sheets to treat more patients.