L. E. DeBault

Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization.

SummaryMicrovessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 μm to 25 μm and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present.

Cerebral microvessels and derived cells in tissue culture: II. Establishment, identification, and preliminary characterization of an endothelial cell line.

SummaryAn endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C6 glioma-conditioned medium.An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium.The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence).

Morphologic effects of antibody to mouse brain endothelium in vivo.

The purpose of this study was to assess the morphologic effects of antiendothelial antibodies (EAB) on mouse brain endothelium in vivo. The antigen utilized was the plasma membranes fraction of cultured mouse brain endothelial cells, which was injected into rabbits with complete Freunds adjuvant. The resultant antibody-containing serum was injected back into mice via tail veins in varying time courses and dosages, followed by perfusion of the animals. The antibody was primarily IgG, and was visualized on the brain endothelium by electron microscopy, using the peroxidase antiperoxidase (PAP) labeling technique. Normal rabbit sera and saline were used as controls. Results showed a significantly greater number of micropinocytotic vesicles in the endothelium of the test animals compared to controls. The number of multivesicular bodies and the thickness of the endothelium were also greater in the test animals. At no time was antibody visualized internal to the endothelial luminal membrane, and no lesions such as inflammation or necrosis were observed. This study shows that serum-containing antiendothelial antibodies has a direct, but apparently limited, effect on endothelium.

Platelet factor and cerebral vascular endothelium: platelet-induced mitogenesis.

Abstract Intact bovine platelets or their products were added to pre-confluent or contact-inhibited monolayers of endothelial cells isolated from mouse cerebral microvessels. Mitogenic responses of these cerebral microvascular endothelial cells were determined by tritiated thymidine incorporation. In pre-confluent cultures the incorporation produced by whole platelets and platelet-rich plasma serum approached that of whole blood serum controls. Serial platelet dilutions induced a graded response, with a discrete threshold. Whole blood plasma serum, platelet-rich plasma serum, and thrombin-activated platelet supernatant augmented the low incorporation produced by platelet-poor plasma serum. Contact-inhibited, complete monolayers did not respond. These results were reproducible, even after sera were frozen and retested at varied intervals.