Abstract: Objective To determine if the co-culture of rabbit articular chondrocytes and hUC-MSCs in vitro can affect differentiation of hUC-MSCs into cartilage-like cells, especially chondrocytes, and if so, what the optimal ratio of the two cell types is. Methods To Co-culture rabbit articular chondrocytes and hUC-MSCs at a chondrocyte: hUC-MSCs ratio of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4 for 21 days and cultured in DMEM high glucose medium. Type Ⅱcollagen (COL2-A1) and glycosaminoglycan (GAG) were analyzed qualitatively by toluidine blue and immunofluorescence technique, respectively. The contents of COL2-A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2-A1 were assayed by real-time fluorescence quantitative PCR. Results The expression of COL2-A1 and GAG on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in other the experimental group or the induced hUC-MSCs group. Also on day 21, the expression of COL2-A1 and GAG proteins in the 4:1 group was much higher than that in all other groups. Conclusion The optimal cell ratio in Transwell co-culture system appears to be 1:4 (hUC-MSCs :chondrocytes).

Key words: Transwell co-culture system, Human umbilical cord mesenchymal stem cells, Rabbit knee articular chondrocytes, Co-culture, chondrogenic induction