Culture of mouse brain capillary endothelial cell lines that express factor VIII, γ-glutamyl transpeptidase, and form junctional complexes in vitro

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Abstract
The isolation and culture of cell lines from mouse brain capillary endothelium (MBE) is described. Cells migrating from collagenase-treated capillary fragments proliferated rapidly in the 1st wk of culture forming large epithelioid cobblestonelike colonies. The cells showed only marginal proliferation after 2 to 3 wk in culture, until peripheral cells migrated away from the colony which exhibited a marked degree of proliferation. These cells were trypsinized and subcultured to confluence. The cells can be maintained for well over 40 passages and seem to retain their endothelial morphology. The endothelial origin of these cells was demonstrated by positive immunoperoxidase reactivity with Factor VIII-related antigen, specific binding ofBandeiraea simplicifolia lectin and γ-glutamyl transpeptidase activity. Electron microscopic examination of the MBE cells showed junctional complexes including intermediate junctions, but no tight junctions. The overall ultrastructure indicates that a degree of dedifferentiation has occurred, the cells ultrastructurally resembling immature endothelium. An earlier investigation of cultured mouse brain endothelial cells reported a cell line that had lost many functional and structural characteristics. Our study demonstrates, as the previous one, that a certain degree of dedifferentiation needs to occur if MBE cells are to be maintained for long-term culture. However, the degree of dedifferentiation seems to be variable, depending in part on the culture conditions employed.