Assessment of the Safety of Chondrocyte Sheet Implantation for Cartilage Regeneration

Abstract

We have previously studied the effects of chondrocyte sheets on the repair and regeneration of articular cartilage by using temperature-responsive culture inserts. On the basis of this work, we succeeded in rapid fabrication of chondrocyte sheets with the use of a coculture method in which inserts were placed between synoviocytes and chondrocytes. Treatment of cartilage defects using layered chondrocyte sheets promotes repair and regeneration; this method is compatible with in vivo osteoarthritis models that reproduce partial-thickness defects. In human stem cell clinical research guidelines, the Ministry of Health, Labour and Welfare (MHLW) approved several applications related to this technology. Indeed, its translation to a clinical setting is already yielding favorable results. In this study, we evaluated the risk of tumorigenesis associated with this treatment and characterized the dynamics of biological processes associated with the posttransplantation cell sheets in vivo. Furthermore, we also confirmed the safety of the procedure by using array comparative genomic hybridization (array CGH) and G-band staining to screen for deleterious genetic aberrations during prolonged subculture of cells. The safety of chondrocytes that were cultured for longer than normal was confirmed by the array CGH and G-band staining results. In addition, tumorigenicity testing confirmed that culture chondrocyte sheets are not tumorigenic. Furthermore, from the evaluation of bioluminescence imaging following implantation of the cell sheets, it was confirmed that the transplanted chondrocytes and synoviocytes remained in the knee joint and did not transfer elsewhere over time. We believe that the technique used in this study is a highly useful method for evaluating the safety of not only chondrocytes but also extensive subculturing in general.

Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets

“…Additionally, this model places the cell sheets in contact with healthy superficial layers of cartilage, rather than deeper calcified cartilage, which would generally be the interface in pre-clinical and clinical applications. Previously, chondrocyte sheets have been shown to successfully adhere to healthy superficial layers of cartilage in vitro 77 , as well as adhere and survive in vivo within the knee environment through spontaneous attachment to calcified cartilage and subchondral bone through pre-clinical and small cohort clinical focal chondral defect studies 46,48,49,78,79 . Based on this precedent, our attachment study aimed to first assess if post-differentiation MSC sheets retain this established cell sheet adhesion ability with healthy superficial cartilage, in addition to standard culture-plastic dishes, before moving forward with more complex transplantation studies that will fully recapitulate the cartilage defect environment.…”

Sustainable Tumor-Suppressive Effect of iPSC-Derived Rejuvenated T Cells Targeting Cervical Cancers

“…To assess tumorigenesis within the cells generated, oncogenes and tumor suppressor genes of original peripheral blood-derived HPV-CTLs, T-iPSCs, and iPSC-derived HPV-rejTs were examined by comparative genomic hybridization microarray. 40 The 12,055 probe contents were selected from 1,675 oncogenes and tumor suppressor genes (Agilent SurePrint G3 human comparative genomic hybridization (CGH) microarray 8×60K; Agilent Technologies, Santa Clara, CA). Sample labeling and array hybridization were performed according to a two-color microarray-based protocol (Agilent Technologies).…”

Cell Therapy Approaches for Articular Cartilage Regeneration

“… 42 Extensive in vivo work assessing the efficacy of chondrocyte cell sheets has been conducted in partial cartilage defect rabbit models, 43 osteochondral defects in rats, 44 rabbits, 45 , 46 and minipigs. 47 Furthermore, investigations regarding performed tumorigenicity and the monitoring of genetic mutations and abnormal chromosome emergence 48 have allowed for the application of cell sheet technology in clinical trials in Japan. 49 …”