Development of scaffold-free tissue-engineered constructs derived from mesenchymal stem cells with serum-free media for cartilage repair and long-term preservation.

Synovial mesenchymal stem cells (sMSCs) have great potential for cartilage repair, but their therapeutic design to avoid adverse effects associated with unknown factors remains a challenge. In addition, because long-term preservation is indispensable to maintain high quality levels until implantation, it is necessary to reduce their fluctuations. This study aimed to investigate the properties and feasibility of novel scaffold-free tissue-engineered constructs using serum-free media and to develop long-term preservation methods. sMSCs were cultured in serum-free media, seeded at high density in a monolayer, and finally developed as a sheet-like construct called "gMSC1". The properties of frozen gMSC1 (Fro-gMSC1) were compared with those of refrigerated gMSC1 (Ref-gMSC1) and then examined by their profile. Chondrogenic differentiation potential was analyzed by quantitative real-time polymerase chain reaction and quantification of glycosaminoglycan content. Xenografts into the cartilage defect model in rats were evaluated by histological staining. gMSC1 showed nearly similar properties independent of the preservation conditions. The animal experiment demonstrated that the defect could be filled with cartilage-like tissue with good integration to the adjacent tissue, suggesting that gMSC1 was formed and replaced the cartilage. Furthermore, several chondrogenesis-related factors were significantly secreted inside and outside gMSC1. Morphological analysis of Fro-gMSC1 revealed comparable quality levels to those of fresh gMSC1. Thus, if cryopreserved, gMSC1, with no complicated materials or processes, could have sustained cartilage repair capacity. gMSC1 is a prominent candidate in novel clinical practice for cartilage repair, allowing for large quantities to be manufactured at one time and preserved for a long term by freezing. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-024-00637-y.