Fermentation of bovine endothelial cells for preparation of endothelial cell-surface heparan sulphate

The polysaccharide chains of a proteoheparan sulphate located on the endothelial cell surface are responsible for athrombogenicity of blood vessel walls. Mass cultivation of endothelial cells is the only way to isolate adequate amounts of this proteoheparan sulphate. In order to establish a method for fermentation of bovine endothelial cells, colonization of microcarriers, growth phase and cultivation of confluent carriers were optimized. The colonization process was varied relative to the number of beads, number of cells, total volume and kind of vessel. Two basal media were tested at different serum contents by growth assays. The same basal media without serum were supplemented with mitogen, bovine lipoprotein, insulin and transferrin and tested by activity assays on confluent cultures. The best method yields more than 80% of the cells on microcarriers. During the fermentation glucose and lactate concentrations were measured at constant perfusion rate and glucose consumption and lactate production were determined. Under optimized conditions we achieved a final cell titre of 4 x 10(9) cells/l and a calculated cell density of 7-9 x 10(4) cells/cm2 offered substrate surface. The minimal doubling time of the cell culture was about 18 h under optimized fermentation conditions. Removal of the core-protein by enzymatic digestion or beta-elimination releases the endothelial cell surface heparan sulphate.