人脑微血管内皮细胞的分离培养及体外血脑屏障特性研究
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Diane Biegel a, Dennis D. Spencer b, Joel S. Pachter a,*
Department of Physiology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030, USA b Department of Surgery, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510, USA
184
D. Biegel et al. /Brain Research 692 wenku.baidu.com1995) 183-189
obtained, without sacrificing cell viability or the ability of the cells to express critical in vivo characteristics. These modifications allow as little as 300 mg of tissue to be used as starting material, an amount which can generate 12-15 confluent monolayers of BMEC, cultured in 48-well cluster dishes, within 1- week's time. Furthermore, due to its novel 3-dimensional nature and expression of in vivo barrier properties, this system is uniquely multi-purposive and ideally suited for evaluating a variety of physiologic and pathophysiologic processes that occur at the BBB.
alterations in BBB properties may contribute to disease pathogenesis or exacerbation [11,13,19]. It is of extreme clinical importance, therefore, that factors regulating BBB function in human brain be elaborated. While traditional histologic examination of brain tissue has contributed significantly to identifying molecular factors that contribute to a variety of neuropathologic conditions, this type of analysis is inherently limited in its ability to resolve complex processes occurring at the BBB - - such as leukocyte and tumor cell extravasation - which require the interaction of various cell types. Hence, it is desirable to have a BBB model system that can be resolved into its component parts, and would thus allow the specific contributions from particular cell types to be evaluated. To this end, we have recently described a 3-dimensional tissue culture model of the BBB that is derived from bovine BMEC, and that retains major phenotypic properties characteristic of the BBB in situ [3]. In this report, we now describe a similar BBB model derived from human BMEC. The use of human cells offers the distinct advantage of allowing for the study of pathologic processes that are restricted to human disease. This protocol is relatively quick, being able to be completed in less than 5 h. Moreover, specific adaptations have been made that allow for a relatively pure population of BMEC to be
Accepted 1 l April 1995
Abstract
A simplified protocol for isolating brain microvessel endothelial cells (BMEC) from human cortex and culturing them on a thick collagen plug is described. This method results in the establishment of monolayers of BMEC that retain numerous properties indicative of the blood-brain barrier (BBB) phenotype, such as elevated transendothelial electrical resistance, attenuated paracellular flux of sucrose, peripheral actin filament distribution and asymmetric localization of the efflux peptide, P-glycoprotein, to the apical (luminal) BMEC surface. The novel 3-dimensional nature of this model system renders it ideally suitable for assaying such varied aspects of BBB physiology as solute transport, pathogen penetrance, leukocyte infiltration and tumor metastasis into the brain. Moreover, the fact that the system is derived from human brain allows for the study of pathogenetic mechanisms that may only be operative in humans.
* Corresponding author. Fax: (1) (203) 679-1269. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 5 1 1 - 0
BRAIN RESEARCH
ELSEVIER
Brain Research 692 (I995) 183-189
Research report
Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro
Keywords: Blood-brain barrier; Endothelium; Microvessel; Tight junction; CNS inflammation
I. Introduction
In vertebrates, the cerebral microvessel endothelium is the major element of the the blood-brain barrier (BBB) and comprises the primary limitation to passage of substances, both soluble and cellular, from the blood into the brain. To accomplish this task, brain microvessel endothelial cells (BMEC) utilize unique features that distinguish themselves from those of peripheral endothelium. Most prominant among these are the following: (1) numerous intercellular 'tight junctions' that display high transendothelial electrical resistance and retard paracellular flux [7]; (2) absence of fenestrae and a reduced level of fluid-phase endocytosis [18] and (3) asymmetrically-localized enzymes and carrier-mediated transport systems that engender a truly 'polarized' phenotype [16,23]. Like peripheral endothelial cells, however, BMEC express, or can be induced to express, cell adhesion molecules on their surface that regulate the extravasation of leukocytes into the brain. Accumulating evidence indicates that, in a variety of neuroinfectious and neurodegenerative disorders,
2. Materials and methods
2.1. Isolation of human brain microvessels
Human brain microvessels were derived from samples of cortical tissue obtained from temporal lobe resections performed on patients with intractable seizure disorders at Yale New Haven Hospital. Typically, individual tissue samples ranged in size from approximately 1.0-1.8 g. Immediately after surgical removal, cortical samples were placed in ice-cold M199 medium (Gibco/BRL, Grand Island, NY), containing 50 mM Hepes, pH 7.4, and 3 X antibiotic-antimycotic (penecillin, streptomycin, amphotericin B; Gibco/BRL), and transferred to the laboratory in this same medium. Isolation of brain microvessels was performed by modifications of the method we previously used for the preparation of BMEC cultures from bovine brain [3,15]. As such, only significant changes in this protocol will be reported here. After the initial stages of tissue disruption and filtration, tissue fragments were subject to the first digestion with collagenase/dispase, in order to free the microvesels from the surrounding parenchymal tissue. Specifically, the tissue was resuspended in M199 medium containing 50 mM Hepes, pH 7.4, 1 × antibiotic-antimycotic, 20 U / m l DNase I (Type II, Sigma) and 0.147 /zg/ml of the protease inhibitor TLCK (tosyl-lysine-chloromethyl-ketone, Sigma). The inclusion of DNase I, which degrades the DNA liberated from broken cells, and TLCK, which inhibits the protease clostripain present in collagenase preparations, was for the purpose of fostering more efficient, though less destructive, digestion of microvessels away from the surrounding parenchyma [1]. A ratio of 0.5 ml of enzyme solution: gm of tissue sample was used, and the tissue uniformly dispersed by repeated pipetting through a cut-off 1.0 ml Eppendorf pipette tip. The enzyme/tissue mixture was then placed at 37°C in a shaking water bath for 1 h. After the first 30 min, the mixture was triturated with a tapered Pasteur pipette that had been coated with a 2% bovine serum albumin/phosphate buffered saline solution to minimize cell adherence [1]. After trituration, the tissue extract was placed back at 37°C for the remaining 30 min. Upon completion of the first enzymatic digestion, mylein debris was removed by centrifugation of the extract through
Department of Physiology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030, USA b Department of Surgery, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510, USA
184
D. Biegel et al. /Brain Research 692 wenku.baidu.com1995) 183-189
obtained, without sacrificing cell viability or the ability of the cells to express critical in vivo characteristics. These modifications allow as little as 300 mg of tissue to be used as starting material, an amount which can generate 12-15 confluent monolayers of BMEC, cultured in 48-well cluster dishes, within 1- week's time. Furthermore, due to its novel 3-dimensional nature and expression of in vivo barrier properties, this system is uniquely multi-purposive and ideally suited for evaluating a variety of physiologic and pathophysiologic processes that occur at the BBB.
alterations in BBB properties may contribute to disease pathogenesis or exacerbation [11,13,19]. It is of extreme clinical importance, therefore, that factors regulating BBB function in human brain be elaborated. While traditional histologic examination of brain tissue has contributed significantly to identifying molecular factors that contribute to a variety of neuropathologic conditions, this type of analysis is inherently limited in its ability to resolve complex processes occurring at the BBB - - such as leukocyte and tumor cell extravasation - which require the interaction of various cell types. Hence, it is desirable to have a BBB model system that can be resolved into its component parts, and would thus allow the specific contributions from particular cell types to be evaluated. To this end, we have recently described a 3-dimensional tissue culture model of the BBB that is derived from bovine BMEC, and that retains major phenotypic properties characteristic of the BBB in situ [3]. In this report, we now describe a similar BBB model derived from human BMEC. The use of human cells offers the distinct advantage of allowing for the study of pathologic processes that are restricted to human disease. This protocol is relatively quick, being able to be completed in less than 5 h. Moreover, specific adaptations have been made that allow for a relatively pure population of BMEC to be
Accepted 1 l April 1995
Abstract
A simplified protocol for isolating brain microvessel endothelial cells (BMEC) from human cortex and culturing them on a thick collagen plug is described. This method results in the establishment of monolayers of BMEC that retain numerous properties indicative of the blood-brain barrier (BBB) phenotype, such as elevated transendothelial electrical resistance, attenuated paracellular flux of sucrose, peripheral actin filament distribution and asymmetric localization of the efflux peptide, P-glycoprotein, to the apical (luminal) BMEC surface. The novel 3-dimensional nature of this model system renders it ideally suitable for assaying such varied aspects of BBB physiology as solute transport, pathogen penetrance, leukocyte infiltration and tumor metastasis into the brain. Moreover, the fact that the system is derived from human brain allows for the study of pathogenetic mechanisms that may only be operative in humans.
* Corresponding author. Fax: (1) (203) 679-1269. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 5 1 1 - 0
BRAIN RESEARCH
ELSEVIER
Brain Research 692 (I995) 183-189
Research report
Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro
Keywords: Blood-brain barrier; Endothelium; Microvessel; Tight junction; CNS inflammation
I. Introduction
In vertebrates, the cerebral microvessel endothelium is the major element of the the blood-brain barrier (BBB) and comprises the primary limitation to passage of substances, both soluble and cellular, from the blood into the brain. To accomplish this task, brain microvessel endothelial cells (BMEC) utilize unique features that distinguish themselves from those of peripheral endothelium. Most prominant among these are the following: (1) numerous intercellular 'tight junctions' that display high transendothelial electrical resistance and retard paracellular flux [7]; (2) absence of fenestrae and a reduced level of fluid-phase endocytosis [18] and (3) asymmetrically-localized enzymes and carrier-mediated transport systems that engender a truly 'polarized' phenotype [16,23]. Like peripheral endothelial cells, however, BMEC express, or can be induced to express, cell adhesion molecules on their surface that regulate the extravasation of leukocytes into the brain. Accumulating evidence indicates that, in a variety of neuroinfectious and neurodegenerative disorders,
2. Materials and methods
2.1. Isolation of human brain microvessels
Human brain microvessels were derived from samples of cortical tissue obtained from temporal lobe resections performed on patients with intractable seizure disorders at Yale New Haven Hospital. Typically, individual tissue samples ranged in size from approximately 1.0-1.8 g. Immediately after surgical removal, cortical samples were placed in ice-cold M199 medium (Gibco/BRL, Grand Island, NY), containing 50 mM Hepes, pH 7.4, and 3 X antibiotic-antimycotic (penecillin, streptomycin, amphotericin B; Gibco/BRL), and transferred to the laboratory in this same medium. Isolation of brain microvessels was performed by modifications of the method we previously used for the preparation of BMEC cultures from bovine brain [3,15]. As such, only significant changes in this protocol will be reported here. After the initial stages of tissue disruption and filtration, tissue fragments were subject to the first digestion with collagenase/dispase, in order to free the microvesels from the surrounding parenchymal tissue. Specifically, the tissue was resuspended in M199 medium containing 50 mM Hepes, pH 7.4, 1 × antibiotic-antimycotic, 20 U / m l DNase I (Type II, Sigma) and 0.147 /zg/ml of the protease inhibitor TLCK (tosyl-lysine-chloromethyl-ketone, Sigma). The inclusion of DNase I, which degrades the DNA liberated from broken cells, and TLCK, which inhibits the protease clostripain present in collagenase preparations, was for the purpose of fostering more efficient, though less destructive, digestion of microvessels away from the surrounding parenchyma [1]. A ratio of 0.5 ml of enzyme solution: gm of tissue sample was used, and the tissue uniformly dispersed by repeated pipetting through a cut-off 1.0 ml Eppendorf pipette tip. The enzyme/tissue mixture was then placed at 37°C in a shaking water bath for 1 h. After the first 30 min, the mixture was triturated with a tapered Pasteur pipette that had been coated with a 2% bovine serum albumin/phosphate buffered saline solution to minimize cell adherence [1]. After trituration, the tissue extract was placed back at 37°C for the remaining 30 min. Upon completion of the first enzymatic digestion, mylein debris was removed by centrifugation of the extract through