Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.

We report here that rings of rat aorta embedded in gels of fibrin or collagen and cultured in MCDB 131, an optimized growth medium for microvascular endothelial cells, generate branching microvessels in the absence of serum or other soluble protein supplements. The angiogenic response is self-limited and can be quantitated by counting the newly formed microvessels daily in the living cultures. The microvascular growth curves are characteristic for each gel. Growth of microvessels in collagen gel peaks at the end of the 1st week and is followed by a rapid regression in the 2nd week. Fibrin gels, as compared with collagen, stimulate angiogenesis by 170%, support growth during the 2nd week, and protect the newly formed microvessels from early regression. Angiogenesis is inhibited by adding hydrocortisone to the culture medium. Conversely, a 230% stimulation of angiogenesis is obtained when aortic rings are cultured in collagen gels floating in serum-free medium conditioned by sarcoma 180 cells. Our results demonstrate that: (a) angiogenesis can be obtained reproducibly in serum-free culture; (b) serum-free culture is a sensitive method for testing the inhibitory or stimulatory effects of soluble or matrix factors on angiogenesis; (c) the aortic ring model can be used as a quantitative assay for the study of angiogenesis under chemically defined culture conditions.