Gene originating in human chondrocyte
a human chondrocyte and gene technology, applied in the field of human chondrocyte gene origin, can solve the problems of difficult to search for human chondrocyte specific gene expression, cell culture system useful in analysis, and method for monolayer culturing human chondrocytes in a differentiated state has not yet been established, so as to facilitate analysis of chondrocyte properties in a differentiated sta
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Culture of Chondrocytes in Differentiated State
[0071] Epiphyseal cartilage of the femur knee joint of a human fetus that was naturally aborted about 25 weeks of pregnancy (obtained from Norman Bethune University of Medical Sciences, Department of Pathology) was obtained. Chondrocytes were isolated from this cartilage according to the same method as that described in Shimomura et al. Calcif. Tissue Res. 19, 179-187 (1975), except that the finely cut cartilage was incubated in a α-modified Eagle's medium (α-MEM) containing 3 mg / ml collagenase (Type IA, Sigma) for 3 h. The cells were seeded at 1×105 cells per Type I collagen-coated dish and maintained in α-MEM (10 ml / dish) containing 10% fetal bovine serum, 50 μg / ml of ascorbic acid, 32 unit / ml of penicillin, and 40 μg / ml of streptomycin. Dibutyryl cAMP (dbcAMP) (1 mM) was added to the culture medium when the cells became subconfluent. Cells were cultured over two days either in the presence or in the absence of dbcAMP. Then, while th...
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(1) Materials and Methods
[0082] Chondrocytes were isolated from the rib growth plate and resting cartilage of ribs of a male Japanese white rabbit (four-weeks old) according to the method as already reported in Shimomuraet al. Calcif. Tissue Res. 19, 179-187 (1975).
[0083] These cells were seeded at 5×105 cells per 10 mm plastic culture dish and maintained in 10 ml of α-MEN supplemented with 10% FBS, 60 mg / ml of kanamycins, 250 ng / ml of amphotericin B, and 50 unit / ml of penicillin G at 37° C. in the air containing 5% CO2. After cells reached confluent, the cells were washed with PBS, transferred to a fresh α-MEN (10 ml) containing no serum, and maintained for 48 h. From 1 to 24 h before the completion of incubation, 1 mM dbcAMP or 10×x−7 M human recombinant PTH-(1-84) was added to the medium.
[0084] Human embryonic pulmonary fibroblasts (MRC-5), human cervix uteri epithelial cells (Hela), human hepatoma cells (HepG2) and canine renal epithelial cells (MDCK) were obtained from the ...
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