The effect of passage number on the stimulation by hypoxia of growth and sprouting activity in cultured aortic endothelium from the rat.

Primary cultures of endothelium from aortas of rats were harvested at different times to obtain a series of cell lines which had been through a varying number of passages ranging from 3 to 30. These were plated into tissue culture flasks half of which were gassed with 5% CO2 and air to act as controls. The other half were grown in a hypoxic environment employing a gas mixture containing 5.3% O2 and 6% CO2. Cells in their 36th passage were also used to derive growth curves of hypoxic and control cells. The degree of endothelial sprouting of all cultures after 9 days growth was assessed by point counting. At all passage numbers sprouting was more extensive in hypoxic than control cultures but this difference was most significant in early passages. The degree of sprouting stimulated by hypoxia remained constant for passages 3-5 whereas the sprouting activity of equivalent control cultures declined steadily. In late passages the degree of spontaneous sprouting fell sharply to a constant low level, as did that in hypoxic cultures, but not to such a low value. Hypoxia did not induce any marked increase in growth of endothelium in its 36th passage, unlike its previously reported effect on early passages. It is concluded that the stimulatory influence of hypoxia on the growth and sprouting activity of cultured endothelium falls with repeated passage and this decline is probably dependent upon the number of population doublings through which the cells have passed.