Adhesion and Growth of Brain Microvascular Endothelial Cells on Treated Glass

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Abstract
For most studies of cultured endothelium, cells are grown on tissue culture plastic. However, for microscopic studies involving the use of fluorescent dyes, the autofluorescence of the plastic can be a problem. Glass is a good alternative to plastic in this case, but standard methods for coating tissue culture plastic to provide an adhesive substrate do not work well on glass. We here report studies on the morphology and growth characteristics of rat brain microvascular endothelial cells growing on glass treated to improve adhesion by changing the collagen-to-glass binding properties and finally coated with collagen. Three methods were tested, using primary cultures of rat brain endothelial cells, and an immortalized rat brain endothelial cell line (RBE4). Cells grew poorly on an extracellular matrix laid down by prior culture of rat type I astrocytes. Cells grew equally well on glass coated with poly-L-lysine or its succinylated derivative. However, a further method gave an even more stable and confluent cell monolayer. This method involved the use of carbodiimide or silane compounds to create a covalent binding of collagen to glass. Rat tail collagen was better than a mixture of type I and IV collagen as the final coating substrate on the treated glass. These methods are likely to be useful for the culture of both endothelium and other cell types.