Abstract: Objective This study aimed to investigate the effects of in vitro continuous passaging on the morphological phenotype and differentiation characteristics of mouse hyaline chondrocytes, as well as on the balance of the extracellular matrix (ECM). Methods Enzymatic digestion was conducted to isolate mouse hyaline chondrocytes, which expanded over five passages in vitro. Hematoxylin-eosin stain was used to show the changes in chondrocyte morphology. Semi-quantitative polymerase chain reaction was performed to analyze the mRNA changes in the marker genes, routine genes, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) in chondrocytes. Zymography was carried out to elucidate changes in gelatinase activities. Results After continuous expansion in vitro, the morphology of round or polygonal chondrocytes changed to elongated and spindled shape. The expression of marker genes significantly decreased (P<0.05), and it was almost negatively expressed by P5 chondrocytes. By contrast, the down regulation of routine genes was insignificant. The gene expression levels of MMPs and TIMPs both decreased (P<0.05), but the change in MMP-1 and TIMP-1 was not statistically significant (P>0.05). Meanwhile, the ratio of MMPs/TIMPs was altered. At the protein level, the activities of gelatinases decreased after passaging, especially for P4 and P5 chondrocytes (P<0.05). Conclusion Serially passaged chondrocytes dedifferentiated and lost specific phenotypic characteristics during in vitro expansion culture. Simultaneously, the anabolism and catabolism of the cartilage ECM became uncontrollable and led to the imbalance of ECM homeostasis. When hyaline chondrocytes are applied in research on relevant diseases or cartilage tissue engineering, P0–P2 chondrocytes should be used.

Key words: in vitro passaging, hyaline chondrocyte, cell phenotype, extracellular matrix, tissue engineering