Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system.

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.