A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Abstract

A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or "weeding." The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin approximately laminin much greater than gelatin greater than poly-D-lysine approximately Matrigel greater than hyaluronic acid approximately plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-D-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7 +/- 2.6% (n = 6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of gamma-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation.

Dexamethasone increases expression and activity of multidrug resistance transporters at the rat blood-brain barrier

“…Rats used for isolation of primary endothelial cells were euthanized in accordance with the procedures approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center and are in compliance with the guidelines of the American Association for Laboratory Animal Science. Primary brain microvascular endothelial cells were prepared as described previously (17). Briefly, brains of 3-wk old male rats (30 -40 g, Sprague-Dawley) were mechanically homogenized in dissecting medium (DM).…”

Human astrocytes/astrocyte-conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

“…The structure and physiological properties of the BBB have been extensively studied in isolated microvessels and in primary cultures of brain microvascular endothelial cells derived from non-human origin such as rodents and cows [30,39,45,75]. We have previously developed the in vitro BBB model with HBMEC [25,53,72].…”

Expression of receptors for native and chemically modified low‐density lipoproteins in brain microvessels

“…Fax: (39) (6) 49766606. E-mail: luc@dbu.uniromal.it Abbreviations: AcLDL, acetylated low-density lipoprotein; BBB, blood-brain barrier; CNS, central nervous system; Ecs, endothelial cells; LDL, low-density lipoprotein; LDL-R, low-density lipoprotein receptor; OxLDL, oxidized low-density lipoprotein; RT, reverse transcriptase; SCAV-R, scavenger receptor; SMCs, smooth muscle cells blotting and/or by 125 I-labeled or fluorescent modified LDL uptake, to be present in macrophages [5][6][7], in various kinds of vascular endothelial cells (Ecs) [5,6,[8][9][10], in fibroblasts and in smooth muscle cells (SMCs) [11,12]. Their presence has been reported in brain by some authors [13] but not by others [5].…”

“…In particular, although cultured cells from major cerebral arteries have been shown to be able to take up AcLDL [17], only a few authors [10,18,19] have demonstrated AcLDL uptake by microvascular Ecs isolated from bovine or rat brain, in contrast with others who failed to demonstrate such an activity on AcLDL and even on unmodified LDL [9]. Even those authors who have reported specific uptake of AcLDL by bovine cerebral Ecs in vitro have omitted to describe any sub-type distinction or data concerning the preferential transcription of these receptors.…”