An in vitro Three-Dimensional Coculture Model of Cerebral Microvascular Angiogenesis and Differentiation

• Published in: In vitro cellular & developmental biology. Animal 1997-10, Vol.33 (9), p.684-691
• Main Authors: Ment, Laura R., Stewart, William B., Dominick Scaramuzzino, Madri, Joseph A.
• Format: Article
• Language: English
• Subjects: Angiogenesis; Animals; Animals, Newborn; Antibodies; Astrocytes; Astrocytes - cytology; Brain - blood supply; Brain - cytology; Cell Differentiation; Cells; Cellular Models; Coculture techniques; Coculture Techniques - methods; Collagens; Cultured cells; Dogs; Endothelial cells; Endothelium, Vascular - cytology; Enzyme Activation; Fibronectins - genetics; Gels; Hemorrhage; Immunohistochemistry; Laminin - genetics; Microcirculation - cytology; Microscopy, Confocal; Microscopy, Fluorescence; Models, Biological; Neovascularization, Physiologic; Rats; RNA, Messenger - metabolism; Urokinase-Type Plasminogen Activator - metabolism
• ISSN: 1071-2690; 1543-706X
• DOI: 10.1007/s11626-997-0126-y

The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral microvasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.