Cerebral microvessels and derived cells in tissue culture: Isolation and preliminary characterization

Summary Microvessels 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. This work was supported in part by an Arteriosclerosis Specialized center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health (HL-14230) and Grant 584-127703 from the Veterans Administration.     

Phenotypic diversity in cultured cerebral microvascular endothelial cells

Summary Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences in colonies of cells that grew in primary cultures. The morphologies ranged from a cobblestone phenotype considered typical of EC in culture to elongated and stellate cell appearances. Serially passaged cell lines were established based on two parameters: initially by growth and, second, on differences in primary colony morphology using selective weeding techniques. Each culture was examined for the presence of EC-characteristic markers which include Factor-VIII-related antigen, angiotensin-I-converting enzyme activity, collagen type IV synthesis, and PGI₂ production. Variable expression of each of these characteristics among the established EC lines was observed. Growth curves established for each of the EC cultures demonstrated differences in both population doubling rates and cell densities at confluence. The endocytic capacity of each EC line was also evaluated. Our ability to isolate and establish a number of morphologically distinct EC cultures indicates that diversity exists within the EC that comprise the cerebral microvasculature. Diversity in the established cell lines suggests either the EC that line the brain microvasculature exist as a mosaic or that morphologically distinct cultures may originate from different microanatomical origins (arteriolar, true capillary, or venular) or may have resulted from cells at different points in their in vitro life spans at the time of isolation. This research was supported by grants HLO3227 and HLO1514 from the National Institutes of Health, Bethesda, MD.     

Isolation of adult canine venous endothelium for tissue culture

Summary In order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact. This research was supported by National Heart, Lung and Blood Institute Contract NIH-NO1-HV-2054 and Grant HL23345.     

Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

Cerebral microvascular smooth muscle in tissue culture

Summary Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco’s modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a “hill and valley” pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-³⁵]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, α-actin. The identity of α-actin is confirmed by analysis of NH₂-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of α-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation. This work was supported by Veteran’s Administration Research Funds, National Institutes of Health Grant HL 14230 (Arteriosclerosis Specialized Center of Research, sponsored by the National Heart, Lung, and Blood Institute), and Cardiovascular Research Program Project Grant from the National Institutes of Health to the University of Iowa (HL-14388). A. R. S. is a postdoctoral fellow of the National Institute of General Medical Sciences (GM-09020). P. A. R. is an established investigator of the American Heart Association.     

Expression of fetal antigens in fetal and adult cells during long-term culture

Summary Expression of fetal antigens in early and late passages of tissue culture cells derived from C3H/HeN and C57BL/KaLw mouse fetal cells and from lung tissue of young C57BL/6N mice was investigated by the isotopic antiglobulin technique. The late passage lines of fetal cells had undergone “spontaneous” neoplastic transformation in culture. The antisera were produced by syngeneic immunization with 5000 R x-irradiated tissues from C3H/HeN and C57BL/6N fetuses of 1 to 2 weeks gestation. Fetal antigens were found to be retained even after 5 years in cell lines derived from fetal tissues In these lines no consistent change in fetal antigen expression could be correlated with neoplastic transformation. In contrast, the early passages of adult cells did not have detectable amounts of fetal antigens. However, fetal antigen(s) was demonstrated in cells of the late passages, and cells of both lines grew as sarcomas when next assayed 55 days later. In addition, fetal antigens were also present in established tumor lines in culture.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Tissue culture of human and canine thoracic duct endothelium

Summary Endothelial cells from the canine or human thoracic duct were harvested using 0.2% collagenase digestion and grown in Media 199, supplemented with fetal bovine serum. The canine endothelial cells grew to confluence (4.4 to 12×10⁴ cells/cm²) in 6 to 10 d; doubling times ranged from 1.5 to 2.8 d. There was a minimum critical density for cell growth between 500 and 10 000 cells/cm². The canine endothelial cells have been maintained in culture for periods up to 11 mo. The human thoracic duct endothelial cells are more difficult to grow and maintain. Endothelial cells were isolated from 5 out of 35 human thoracic ducts and grew for periods of up to 2 wk before degenerating. Both human and canine endothelial cells were Factor VIII positive. It has thus been demonstrated that it is possible to grow canine and, less easily, human thoracic duct endothelium in tissue culture.     

Efficient stem cell isolation from under vacuum preserved tissue samples

Different approaches for the isolation of stem/progenitor cells have been reported, including stem cell selection in stringent culture conditions. We evaluated the possibility of isolating human progenitor cells from surgical specimens preserved by under vacuum sealing and cooling, a clinical practice approached by several hospitals as alternative to formalin. Renal tissue samples (n = 20) maintained under vacuum from 6 to 48 h at 4°C were used to isolate human renal CD133⁺ progenitor cells. We obtained CD133⁺ progenitors from unsorted cells derived from disaggregated tissues from each sample. Phenotypic characterization as well as in vitro and in vivo differentiation of the obtained CD133⁺ lines showed results comparable with sorted CD133⁺ cells obtained from fresh tissue. These results indicate that the process of sealing under vacuum and cooling appears as a suitable tissue treatment to isolate hypoxia resistant cells, such as human stem/progenitor cells, and that this procedure can be exploited to render the extraction of stem cells from human samples more practical and feasible.     

Efficient stem cell isolation from under vacuum preserved tissue samples

Different approaches for the isolation of stem/progenitor cells have been reported, including stem cell selection in stringent culture conditions. We evaluated the possibility of isolating human progenitor cells from surgical specimens preserved by under vacuum sealing and cooling, a clinical practice approached by several hospitals as alternative to formalin. Renal tissue samples (n = 20) maintained under vacuum from 6 to 48 h at 4°C were used to isolate human renal CD133⁺ progenitor cells. We obtained CD133⁺ progenitors from unsorted cells derived from disaggregated tissues from each sample. Phenotypic characterization as well as in vitro and in vivo differentiation of the obtained CD133⁺ lines showed results comparable with sorted CD133⁺ cells obtained from fresh tissue. These results indicate that the process of sealing under vacuum and cooling appears as a suitable tissue treatment to isolate hypoxia resistant cells, such as human stem/progenitor cells, and that this procedure can be exploited to render the extraction of stem cells from human samples more practical and feasible.     

In situ fluorescence labeling of sheep lung microvascular endothelium

Summary Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.     

Angiogenesis and the formation of lymphaticlike channels in cultures of thoracic duct

Summary Segments of rat thoracic duct cultured in plasma clot or in collagen gel produced microvascular and fibroblastic outgrowths. Lymphaticlike channels (LLC) with a highly attenuated endothelium, which was barely visible by light microscopy, were found in 8 out of 25 cultures (32%). Serial histologic sections revealed that the endothelium of the LLC was continuous with the intimal endothelium of the throacic duct and was therefore of lymphatic origin. In addition to the LLC, vascular channels lined by a thick endothelium with hump-shaped, cross-sectional profiles were found in 10 cultures (40%). These channels were indistinguishable from the microvessels of blood vascular origin that formed in parallel cultures of rat aorta or periductal adipose tissue and were termed hematiclike channels (HLC). Contrary to the LLC, the HLC did not originate from the lymphatic endothelium of the thoracic duct. The frequent association of the HLC with the adventitia of the thoracic duct and with the surrounding adipose tissue suggested that they probably developed from the hematic microvessels of the periductal soft tissues. This research was supported by grants from the National Cancer Institute, NIH, National Bladder Cancer Project (CA14137), and the W. W. Smith Charitable Trust.     

Macromolecular selectivity of chick chorioallantoic membrane microvessels during normal angiogenesis and endothelial differentiation

Progressive angiogenesis and endothelial differentiation in the chick chorioallantoic membrane (CAM) serve to accommodate oxygen demands of the growing embryo. The present study evaluated CAM microvascular endothelial permselectivity during the most rapid phase of angiogenesis (day 10) and after initiation of endothelial cytodifferentiation (day 14). Chick embryos were incubated using established shell-less culture techniques tor intravital and ultrastructural observations. Systemic microinjections of FITC-dextrans (40, 70 and 150 KDa) provided an index of endothelial permselectivity after 2.5 min and 10 min perfusions. Ultrastructural examinations of the same dextran probes served to detect small intermittent foci within the perivascular interstitium. Although minor variations of dextran particle distributions around specific segments of the microcirculation were observed ultrastructurally, perivascular accumulation was not sufficient to elicit a detectable fluorescent signal. Thus, substantial accumulation of the graded-dextran series in the perivascular intcrstitium was not detected. Morphometric analyses of the precapillary, capillary, and postcapillary microvascular segments served to demonstrate a continuous endothelium which displayed cytoplasmic attenuation at day 14. Plasmalemmal vesicles were few and uniform within the microvascular units at day 10. A three-fold increase in vesicle densities characterized the precapillary endothelia at day 14. Average widths of the endothelial junctional clefts were homogeneous within the segmental microvascular endothelia at both days 10 and 14. Junctional cleft lengths were also homogeneous, except the significantly longer capillary endothelial clefts observed at day 10. These results are consistent with the concept that, despite certain differences in segmental vesicle densities and junctional cleft lengths, neovascularization of the CAM is achieved without excessive macromolecular efflux across the microvascular endothelia.     

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

Summary 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≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈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 γ-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. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).     

Characterization of a strain of cerebral endothelial cells derived from goat brain which retain their differentiated traits after long-term passage

Summary A strain of cerebral endothelial cells was established from isolated cortical microvessels of caprine brain. These cells, which are referred to as ECl cells, can be routinely subcultured to 32 passages without the loss of differentiated morphologic and immunologic traits. The ability to routinely subculture ECl cells is an important asset, given that isolated cerebral endothelial cells in mammals generally lose their differentiated traits after only 2 to 3 passages. ECl cells were shown to contain Factor VIII-related antigen, which is a specific marker for cells of endothelial origin. ECl cells morphologically demonstrated a scarcity of pinocytotic vesicles on their apical surfaces, a lack of trans-cytoplasmic vesicles, and the ability to form in culture confluent monolayers with tight junctional complexes. Therefore, ECl cells possess specific antigenic and ultrastructural features which classify them as being small vessel endothelial cells of the blood-brain barrier type. Cytogenetic evaluation of ECl cells demonstrated a normal female goat 60,XX karyotype and confirmed the apparent non-transformed nature of ECl cells due to the lack of chromosome abnormalities or rearrangements. Using scanning electron microscopy, ECl cells were also shown to form confluent monolayers on mixed nitrocellulose filters, a feature that will enable the development of an in vitro system to study trans-endothelial transport. Given that ECl cells are readily subcultured and grow well on nitrocellulose filters, and that they resemble cerebral endothelium in vivo, it seems evident that ECl cells can be used as a versatile model for the study of blood-brain barrier function, regulation, and pathology.     

Brain Microvessels Produce 12-Hydroxyeicosatetraenoic Acid

Cerebral microvessels isolated from perfused, adult murine brain produce a compound with the chromatographic properties of a monohydroxyeicosatetraenoic acid when incubated with arachidonic acid or stimulated with calcium ionophore A23187. The formation of this arachidonic acid metabolite is not reduced in the presence of the cyclooxygenase inhibitor ibuprofen, but it is abolished by the lipoxygenase inhibitor nordihydroguaiaretic acid. Analysis by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives of the metabolite, indicate that the compound is 12-hydroxyeicosatetraenoic acid. Fractions of isolated microvessels enriched with capillaries produce 2.1 times more 12-hydroxyeicosatetraenoic acid per microgram of protein than do fractions of microvessels enriched with arterioles. These studies confirm that brain microvessels can produce 12-hydroxyeicosatetraenoic acid and strongly suggest that cerebral endothelia are the primary source of microvessel-derived 12-hydroxyeicosatetraenoic acid. They further suggest that in brain injury, the liberation and accumulation of arachidonic acid in cerebral tissues may lead to the production of 12-hydroxyeicosatetraenoic acid within microvessels. The 12-hydroxyeicosatetraenoic acid formed in this way may mediate some of the blood-brain barrier and cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.     

Culture and characterization of bovine mesenteric lymphatic endothelium

Summary Lymphatic endothelial cells isolated from bovine mesenteric lymphatic vessels were cultured and characterized. Lymphatic endothelial cells grew as a monolayer displaying an elongated morphology in preconfluent primary cultures. When confluent, the cells exhibited a polygonal morphology to form a “cobblestone” pattern previously described for cultured vascular endothelium. All culture lymphatic endothelial cells expressed Factor VIII-related antigen and boundUlex europaeus I lectin. Ultrastructurally, cultured lymphatic endothelium was characterized by the presence of Weibel-Palade bodies as well as the usual cytoplasmic organelles.     

Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

, , , , , and 1992. Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting. International Journal for Parasitology 22: 1083–1088. Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75,48, 30, 24 and 22 kDa).