Mesenchymal stem cells (MSCs) in synovial fluid (SF) actively participate in the regeneration process of healthy joints and have been defined as a good source of chondrocyte cells, which can be obtained by in vitro differentiation of stem cells. This cell population derived from synovial fluid shares cellular characteristics with bone marrow and synovial membrane MSCs with respect to their differentiation potency. Current cell isolation protocols for cartilage therapy mainly focus on the isolation of chondrocytes or MSCs from biological samples. However, the isolation of chondrocytes after the in vitro differentiation from MSCs has not been described in the literature. In this context, we defined a novel method based on Ficoll-Paque density gradient centrifugation for high-throughput isolation of differentiated chondrocytes from human synovial fluid mesenchymal stem cells (hSF-MSCs) without the requirement for cell labeling. In this study, terminally differentiated chondrocytes were obtained after 21 days of chondrogenic differentiation from hSF-MSCs and were isolated using this novel protocol. The isolated chondrocytes were later analyzed for cell viability and functionality by staining with Alcian Blue and by gene expression analysis for chondrocyte markers. In conclusion, the novel chondrocyte isolation method described here is capable of achieving low-cost efficiency. According to the minimal process principles, we hope that this protocol will find use in both translational research and routine clinical applications involving the differentiation and isolation of chondrocytes in vitro.
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