O A Boubriak1, J T S Brooks, J P G Urban. 1. Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK. olga.boubriak@dpag.ox.ac.uk
Abstract
OBJECTIVE: Here we investigate whether monolayer culture or culture at 21% oxygen influences activity of cytochrome c oxidase, the terminal enzyme in the respiratory chain whose activity is essential for oxidative metabolism and whether return to three dimensional (3-D) culture restores cytochrome c oxidase activity to original levels. METHODS: Primary bovine articular chondrocytes were cultured in alginate beads (3-D) for 4 weeks or in monolayer under 1% and 21% oxygen for up to 9 days and then returned to 3-D culture for up to 4 weeks. Cells were stained to localise cytochrome c oxidase within the cells. Mitochondrial protein content and cytochrome c oxidase enzymatic activity were determined. Expression of cytochrome c oxidase subunits, COXI and COXIV, was assessed by qRT-PCR. RESULTS: Cytochrome c oxidase staining remained minimal in chondrocytes cultured in alginate for 4 weeks under 21% oxygen. Mitochondrial protein content and cytochrome c oxidase activity increased significantly during 9 days of chondrocyte expansion in monolayer, accompanied by up-regulation of the COXI mitochondrial gene but not the COXIV nuclear-encoded gene. Cytochrome c oxidase staining increased from day 5 of monolayer culture and remained high even after the cells were returned to 3-D culture for 4 weeks. CONCLUSIONS: Culture of chondrocytes in monolayer leads to a rapid increase in mitochondrial protein content and cytochrome c oxidase activity. The increase in cytochrome c oxidase activity is not reversed even after chondrocytes are returned to 3-D culture for 4 weeks; high oxygen tension alone does not appear to stimulate cytochrome c oxidase activity.
OBJECTIVE: Here we investigate whether monolayer culture or culture at 21% oxygen influences activity of cytochrome c oxidase, the terminal enzyme in the respiratory chain whose activity is essential for oxidative metabolism and whether return to three dimensional (3-D) culture restores cytochrome c oxidase activity to original levels. METHODS: Primary bovine articular chondrocytes were cultured in alginate beads (3-D) for 4 weeks or in monolayer under 1% and 21% oxygen for up to 9 days and then returned to 3-D culture for up to 4 weeks. Cells were stained to localise cytochrome c oxidase within the cells. Mitochondrial protein content and cytochrome c oxidase enzymatic activity were determined. Expression of cytochrome c oxidase subunits, COXI and COXIV, was assessed by qRT-PCR. RESULTS:Cytochrome c oxidase staining remained minimal in chondrocytes cultured in alginate for 4 weeks under 21% oxygen. Mitochondrial protein content and cytochrome c oxidase activity increased significantly during 9 days of chondrocyte expansion in monolayer, accompanied by up-regulation of the COXI mitochondrial gene but not the COXIV nuclear-encoded gene. Cytochrome c oxidase staining increased from day 5 of monolayer culture and remained high even after the cells were returned to 3-D culture for 4 weeks. CONCLUSIONS: Culture of chondrocytes in monolayer leads to a rapid increase in mitochondrial protein content and cytochrome c oxidase activity. The increase in cytochrome c oxidase activity is not reversed even after chondrocytes are returned to 3-D culture for 4 weeks; high oxygen tension alone does not appear to stimulate cytochrome c oxidase activity.