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

Abstract Background: Synovial mesenchymal stem cells (sMSCs) have great therapeutic potential for cartilage repair and have been used for clinical application. However, therapeutic design considering products effectively without loss efficacy and safety with eliminated unknown factors or adverse effects remains a challenge. In addition, because long-term preservation is indispensable to maintain high levels of cell-based products until implantation, factors that affect quality must be evaluated and demanded to reduce their fluctuations throughout the distribution. This study aimed to investigate the properties and feasibility of novel scaffold-free tissue-engineered constructs using serum-free media and develop long-term preservation methods considering logistic and distribution strategies. Methods: sMSCs obtained from the human synovium 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 the frozen gMSC1 (Fro-gMSC1) using a control rate freezer were compared with those of refrigerated gMSC1 (Ref-gMSC1). gMSC1s were examined by cell number, viability, and surface antigen expression profile by flow cytometry, immunostaining, and scanning electron microscopy (SEM) observation. Chondrogenic differentiation potential was analyzed by quantitative real-time polymerase chain reaction and quantification of glycosaminoglycan content in the pellet culture. Xenografts into the cartilage defects in rat knees were evaluated by histological staining. Results: gMSC1 showed nearly similar properties independent of the preservation conditions. The animal experiment demonstrated that the defect could be filled with cartilage-like tissue under both gMSC1 application groups at approximately the same level 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 the gMSC1. The morphological analysis of Fre-gMSC1 revealed comparable levels of quality against fresh gMSC1 in immunostaining and SEM observations. Thus, if cryopreserved, gMSC1, with no complicated materials or processes, could have sustained cartilage repair capacity under stable quality attributes and no adverse effects on efficacy or safety. Conclusions: 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.