Phenotypic Characterization of Human Chondrocyte Cell Line C-20/A4: A Comparison between Monolayer and Alginate Suspension Culture

Abstract

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.

The Phenotypic Characterization of A13/BACii, a Novel Bovine Chondrocytic Cell Line with Differentiation Potential

“…The expression of MMP-3, MMP-9 and MMP-13 in A13/BACii chondrocytes suggested a highly metabolic cell line additionally capable of the synthesis of the ECM component, aggrecan. Conversely, C-20/A4 tested positive for MMP-3 unlike previously shown [Finger et al, 2004], with more modest levels of aggrecan compared to A13/BACii. The expression of MMP-3, MMP-9 and MMP-13 has been associated with chondrosarcomas [Kawashima et al, 1997;Soderstrom et al, 2001;Kuroki et al, 2002;Zyada and Shamaa, 2008], and correlates with their grading and capacity to metastasize to the lungs [Sakamoto et al, 1999;Hou et al, 2011], coincidentally, the main target for B[ ␣ ]P tumourgenesis in vivo.…”

“…Moreover, primary cultures undergo cellular senescence, and thus a regular and sustained source of fresh cartilage explants is required for research purposes. Therefore, the development of an immortal cell line with characteristics as a phenotypically similar alternative to human cultured chondrocytes is desirable [Finger et al, 2004].…”

Development and characterization of a human articular cartilage‐derived chondrocyte cell line that retains chondrocyte phenotype

“…These clones while exhibiting significantly lower expression than the primary showed comparatively higher expression than the remaining 114 clones. Although the expression was numerically less in clones compared to primary chondrocytes, it is still better than that observed in previously characterized human chondrocyte cell lines (Finger et al, 2003(Finger et al, , 2004. We also analyzed gene expression of other chondrocyte markers including aggrecan, COMP, ADAMTS5, and MMP-13 although no significant difference in the expression profile of these genes were noted.…”

“…This study confirms the long held belief that alginate cultures are better suited to maintain chondrocyte phenotype. Finger et al, 2003Finger et al, , 2004Saas et al, 2006), we analyzed RNA from selected clones on arrays. Because most of the primary human chondrocyte studies in the literature have been performed in monolayers, we wanted to understand the characteristics of the clones under this condition.…”

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1

“…The C-20/A4 phenotype and gene expression profiling in both monolayer and 3D alginate culture systems, confirmed their suitability as an appropriate cell line to use in the study of chondrocyte biology 39 . Furthermore, C-20/A4 cells display the polygonal cobblestone morphology and the differentiated phenotype typical of human chondrocytes such as the expression of various cartilage specific molecules including collagen type II, IX and XI 40 .…”