To obtain well characterized immortalized murine chondrocyte cell lines. The cell lines were obtained from mature articular chondrocytes, instead of embryonal cells which are used in most other studies.
Pieces of articular cartilage were cut from murine patellae and femoral heads. Chondrocytes were isolated by digestion with collagenase. These cells were cultured in monolayer and immortalized by transfection of the SV40 large T antigen gene. To preserve the differentiated phenotype, the resulting clones were cultured in three-dimensional carriers, alginate beads. The phenotypes of the cells were characterized using the following parameters: Cell morphology (light microscopy), messenger RNA (RT-PCR) and protein (immunohistochemistry) levels of extracellular matrix molecules. Moreover, responsiveness to interleukin-1(IL-1) was determined by measuring production of proteoglycans ((35)S-sulfate incorporation) and of nitric oxide (Griess reaction).
Sixteen clones were obtained, ten (P1 to P10) derived from patellar cartilage, and six (H1 to H6) from femoral head cartilage. In seven cell lines (P2, P5, H1, H3, H4, H5, H6) high production of type II collagen corresponded with high levels of mRNA of type II collagen (and prevalence of the IIB type) and with high IL-1-induced suppression of proteoglycan synthesis. Like intact murine articular cartilage, all cell lines produced type I and type X collagens, but mRNA levels of both types of collagen were never higher in the cell lines as compared with intact cartilage.
Our results demonstrate that it is possible to immortalize mature murine articular chondrocytes. Each of the obtained chondrocyte cell lines appeared to have a stable phenotype. Both relatively differentiated and relatively dedifferentiated chondrocyte cell lines could be identified.