Ceiling culture of mature human adipocytes: use in studies of adipocyte functions

Adipocytes contain large lipid droplets in their cytoplasm. When cultured, they float on top of the medium, clump together, and do not gain equal and sufficient access to the medium. Morphological changes cannot be observed a nd the majority of adipocytes undergo cell lysis within 72 h of isolation. We have used a ceiling culture method for human mature adipocytes which use s their buoyant property to allow them to adhere to a floating glass surfac e, where they remain viable for several weeks. Using confocal immunofluores cence microscopy we showed the cellular expression and subcellular localiza tion of leptin in ceiling-cultured adipocytes. The secretion of leptin was increased from ceiling cultures following tumour necrosis factor-alpha trea tment. Proliferation of mature human adipocytes in serum-containing medium was demonstrated by incorporation of bromodeoxyuridine, 2% of adipocytes sh owing positive incorporation after 4 h labelling. Proliferation was also ev ident from the budding of daughter cells. Apoptosis in the ceiling cultures was increased by 48 h serum deprivation (30-35 vs 10-15% in the control) a nd was assayed by propidium iodide staining and terminal deoxynucleotidyl t ransferase-mediated dUTP-fluorescein nick-end labelling. Lipolysis, analyse d by liquid scintillation counting, was increased by forskolin (10 mu M for 90 min) and lipogenesis, shown by autoradiography, was stimulated by insul in (10 and 100 nM for 4 h). These findings indicate that ceiling-cultured a dipocytes maintain adipocyte-specific functions and that ceiling culture, w hich overcomes the shortcomings of adipocyte suspension culture, can be use d to study adipocyte cell biology.