Figure 5.

Reduction of surface antigen expression by long-term trypsin treatment had limited effects on multipotency of synovial MSCs. (A) Schematic diagrams of the experiment. One hundred thousand cells were seeded on 15-cm dishes and maintained for 2 weeks. Cell suspensions were prepared by TrypLE for 5 min (as a control, I) or trypsin treatment for 60 min (II). Cells (2.5 × 10⁵) were placed in a 15-ml polypropylene tube, centrifuged at 450 × g for 10 min, and cultured in chondrogenesis medium for 2 weeks. The rest of the cells were replated and maintained for another 2 weeks to recover surface antigen expression, detached by TrypLE for 5 min, and then chondrogenic differentiation assay was performed (III). To analyze the osteogenic and adipogenic potential of MSCs, 100 cells detached by three different methods (I, II, and III) were seeded on 10-cm dishes and maintained for 3 weeks in the differentiation medium. Mineralized nodule formation was detected by Alizarin red staining, and fat accumulation in the cell was visualized by Oil red O staining. (B) Macroscopic and histological/immunohistochemical analysis of in vitro chondrogenesis, osteogenesis, or adipogenesis assays. Scale bars: 50 μm (B), 200 μm (C). H.E., hematoxylin and eosin; Col, collagen.