Microvascular endothelium and pericytes: high yield, low passage cultures

Cultured microvascular endothelial cells (MEC) have become a valuable model for studies of microvascular physiology and pathology. Most current techniques involve manual removal of undesirable cell types or cloning, require one to several months, and yield high population doubling level cultures derived from a relatively small sample of the original population. We have devised a technique to more rapidly produce larger numbers of MEC. This method provided primary cultures consisting predominantly of MEC within 1 wk. The technique involves selective aspiration of gray matter from the bovine cerebral cortex followed by homogenization, sieving, enzymatic dissociation, and then dense plating (10(4) to 10(5) vessel fragments/cm2) onto gelatin- or fibronectin-coated plastic. Typical yields were 0.1 to 0.5 X 10(6) fragments/g of aspirated gray matter. The optimal culture medium for these cells was 15% equine plasma derived serum, 20% conditioned medium, 2% retinal extract, 60% fresh medium, and 500 micrograms/ml heparin. Cells attached within 24 h, well-spread colonies were present within 1 to 2 d, and cultures approached confluence within 2 to 3 d. Alkaline phosphatase staining confirmed the microvascular origin of the material plated. Morphology, Factor VIII-related antigen staining and 1,1'-dioctacecyl-3,3,3'3,-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein uptake suggested that MEC predominated. Cultures could be passaged and additionally purified by sequential exposure to pancreatin and trypsin-EDTA. Pancreatin selectively removed MEC colonies leaving a relatively homogeneous pericyte population. The relative ease with which such cultures can be produced should facilitate the in vitro study of brain microvascular function and may also provide insights useful for growing MEC from other vascular beds.