In vitro cultivation and behavior of aortic endothelium cells in a low serum culture medium

Endothelial cells isolated from calf aorta were used in the subculture No. 4 to 10 for experiments to establish standardized and well reproducible conditions of cultivation. The cells can be cultivated in the commercial medium Eagle-MEM with following supplements: 0.1 g L-glutamine, 5.0 g peptone, 0.5 g serum albumin, and 10 ml (= 1%) bovine serum per 1000 ml medium (MEMPAS). With the aid of immunofluorescence technique the cell type specific marker Factor VIII antigen was shown to be localized especially in the perinuclear region of the cells. The cells were characterized with regard to their growth behaviour. Both, the MEMPAS and the Eagle-MEM with 10 per cent serum increases the cell number in the first 4 days of the exponential growth to the same values. The use of MEMPAS in connection with a strict cultivation regime from the deep frozen cell suspension to culture in scintillation vials guaranteed well reproducible conditions of cultivation. In 14 non-selected experiments distributed over a longer period of time it was found that with regard to the values of the cell number on the respective day the cultures can be divided into two groups, which differ with statistical significance. In further experiments it was possible to confirm this result. Medium, conditioned by endothelial cells (K-MEMPAS) increases the cell number and the growth rate. From these results it was concluded that endothelial cells of vessels are able to produce growth factors with self-stimulating effects. At this time the endothelial cell line is stored in deep frozen state up to the 25th subculture. The endothelial cells cultivated in the described standardized conditions are useful for screening of cell type specific factors with angiogenic activity.