To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.
The effects of coating the culture surface with bovine type I collagen on the culture properties of anchorage-dependent cells were investigated. When human fibroblasts were cultured on a surface coated with collagen at 5.8 x 10(-3) mg/cm2, cell attachment and subsequent cell growth were both enhanced compared to the culture on an uncoated surface. The degrees of cell attachment and growth enhancement were numerically characterized using the time constant of cell adhesion (tau) and doubling time (t(d)) as kinetic parameters. These parameters applied to cultures of human keratinocytes and rabbit chondrocytes allowed the effects of collagen coating on the respective culture properties of both types of cells to be evaluated. In addition, the relative parameters R(tau) and R(t(d)) (defined as the ratios of the tau and t(d) values at a given collagen concentration against those without collagen coating, respectively) were employed to estimate the effects of collagen based on a standardized criterion. Similar R(tau) and R(t(d)) profiles were obtained for collagen concentrations ranging from 5.8 x 10(-13) to 5.8 x 10(-3) mg/cm2, whether the cells were fibroblasts, keratinocytes or chondrocytes. It was also revealed that coating the surface with collagen at a concentration over 5.8 x 10(-7) mg/cm2 led to reductions in both the R(tau) and R(t(d)) values, i.e. the promotion of cell attachment and growth, in the culture of each type of cells examined.
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