2002 Volume 73 Issue 3 Pages 431-439
In adult animals, the enlargement of white adipose tissue mass is due to increases in both adipocyte size and number. Recent reports have predicted that an increase in adipocyte number, indicating growth and adipogenic differentiation of immature cells, is controlled not only by systemic circulating hormones but also by factors existing locally. To investigate such local control mechanisms, it is necessary to develop a culture system, in which cell-cell interactions mediated by paracrine factors are demonstrated. We have developed a co-culture system in which both mature adipocytes and immature cells isolated from adipose tissues are maintained in a culture medium ; we have used this system to examine effects of mature adipocytes on the growth and adipogenic differentiation of the immature cells. Epididymal and perirenal white adipose tissue was dissected from adult male rats, and immature cells and mature adipocytes were obtained by collagenase digestion followed by centrifugation. Immature cells were collected from the pellet and seeded into 12-well multiwell culture plates at a density of 3×104 cells/well. Mature adipocytes were collected from the floating layer and suspended in 0.2ml of collagen gel, and clotted on the membrane filters of culture inserts at a density of 4×104 cells/insert. The two cell types were cultured separately for one day in DMEM supplemented with 10% fetal bovine serum. On the second day, co-cultures were initiated by placing the culture inserts containing mature cells into the wells containing immature cells. Culture inserts containing 0.2ml of collagen gel with no cells, were used as controls. The culture medium was changed once every 2 days, and the cells were co-cultured for 12 days. Immature cells co-cultured with mature adipocytes for 12 days contained 1.3-fold the amount of DNA compared to immature cells that were not co-cultured. Up to 20% of co-cultured immature cells accumulated lipid droplets that were visualized by oil red O stain, while only 1.3% of immature cells without co-culture accumulated. Similarly, co-cultured immature cells accumulated 1.9 times as much triglyceride as did immature cells without co-culture. Activity of glycerol-3-phosphate dehydrogenase was assayed as a marker of adipocyte differentiation ; co-culture increased the activity of immature cells 1.8-fold. In conclusion, using a new co-culture system, we have found that both growth and adipogenic differentiation of immature cells are promoted by the paracrine effect of mature adipocytes. Our co-culture system is useful to investigate mechanisms of cell-cell interactions mediated by paracrine factors in adipose tissues.
