Isolation and culture of human neuromicrovascular endothelial cells for the study of angiogenesis in vitro.
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Isolation and culture of human neuromicrovascular endothelial cells for the study of angiogenesis in vitro. / Lamszus, K; Schmidt, Nils-Ole; Ergün, S; Westphal, M.
In: J NEUROSCI RES, Vol. 55, No. 3, 3, 1999, p. 370-381.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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T1 - Isolation and culture of human neuromicrovascular endothelial cells for the study of angiogenesis in vitro.
AU - Lamszus, K
AU - Schmidt, Nils-Ole
AU - Ergün, S
AU - Westphal, M
PY - 1999
Y1 - 1999
N2 - Neovascularization in the adult central nervous system occurs as a response to several pathophysiological conditions such as ischemia, wound repair, or neoplasia. Endothelial cells from different blood vessel types, different organs, and different species are heterogeneous; therefore, the appropriate cell type should be used to study specific aspects of vascular pathology. We have developed a method to isolate human cerebral microvascular endothelial cells (CMECs) from small, freshly obtained specimens of normal brain adherent to human arteriovenous malformations (AVMs). The isolation procedure involves enzymatic digestions and gradient centrifugations, yielding over 95% pure primary cultures. Alternative isolation methods using magnetic beads, panning, or cloning were not superior with regard to cell purity or yield. CMECs were identified by their immunoreactivity for vWF, CD34, EN4, binding of Ulex europeus lectin, and uptake of DiI-Ac-LDL. They displayed ultrastructural features characteristic of blood-brain barrier endothelial cells and expressed GLUT-1. CMECs were subcultured; however, prolonged culture led to reduced culture purity. Vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor/scatter factor stimulated the directional motility of CMECs, with dose-response profiles similar to human umbilical vein endothelial cells (HUVECs). In contrast, to stimulate proliferation, lower concentrations of growth factors tended to be necessary for CMECs than for the large vessel endothelial cells. CMECs formed capillary tube-like structures in an in vitro angiogenesis assay using matrigel. This study expands the spectrum of available tissue sources for the isolation of human neuromicrovascular endothelial cells, which are essential for the in vitro study of blood-brain barrier function and cerebral angiogenesis.
AB - Neovascularization in the adult central nervous system occurs as a response to several pathophysiological conditions such as ischemia, wound repair, or neoplasia. Endothelial cells from different blood vessel types, different organs, and different species are heterogeneous; therefore, the appropriate cell type should be used to study specific aspects of vascular pathology. We have developed a method to isolate human cerebral microvascular endothelial cells (CMECs) from small, freshly obtained specimens of normal brain adherent to human arteriovenous malformations (AVMs). The isolation procedure involves enzymatic digestions and gradient centrifugations, yielding over 95% pure primary cultures. Alternative isolation methods using magnetic beads, panning, or cloning were not superior with regard to cell purity or yield. CMECs were identified by their immunoreactivity for vWF, CD34, EN4, binding of Ulex europeus lectin, and uptake of DiI-Ac-LDL. They displayed ultrastructural features characteristic of blood-brain barrier endothelial cells and expressed GLUT-1. CMECs were subcultured; however, prolonged culture led to reduced culture purity. Vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor/scatter factor stimulated the directional motility of CMECs, with dose-response profiles similar to human umbilical vein endothelial cells (HUVECs). In contrast, to stimulate proliferation, lower concentrations of growth factors tended to be necessary for CMECs than for the large vessel endothelial cells. CMECs formed capillary tube-like structures in an in vitro angiogenesis assay using matrigel. This study expands the spectrum of available tissue sources for the isolation of human neuromicrovascular endothelial cells, which are essential for the in vitro study of blood-brain barrier function and cerebral angiogenesis.
M3 - SCORING: Zeitschriftenaufsatz
VL - 55
SP - 370
EP - 381
JO - J NEUROSCI RES
JF - J NEUROSCI RES
SN - 0360-4012
IS - 3
M1 - 3
ER -