This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) M&#252;nster (SEED 03/12, SB), by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC 1003 &#8211; CiM), University of Muenster (to SB and SGM) and by the Else Kr&#246;ner-Fresenius-Stiftung (2012_A88 to SB and SGM). We thank Heike Blum for the excellent illustrations.

All animal experiments were approved by the local authorities (&#8220;Landesamtf&#252;rNatur, Umwelt und Verbraucherschutz NRW; Fachbereich 87, Tiergesundheit, Tierschutz; Recklinghausen, Germany&#8221;) and conducted according to the German law of animal protection.
1. General Comments for Mouse Experiments
<ol>
	<li>Perform all experiments using mice in accordance with the guidelines of the respective Institutional Animal Care and Use Committee.</li>
	<li>Keep the mice under pathogen-free conditions and enable them to have access to food and water ad libitum.</li>
	<li>Use age- and sex-matched mice in experimental groups because biological properties can vary with age and gender.</li>
	<li>Whenever possible try to reduce, refine or replace animal experiments.</li>
</ol>
2. Preparation of the Percoll Gradient
<ol>
	<li>Mix 19 ml of 1x PBS with 1 ml 10x PBS, 1 ml FCS and 10 ml Percoll.</li>
	<li>Perform sterile filtration in a glass tube (diameter of filter pores: 0.2 &#181;m). Afterwards store the solution at 4 &#176;C.</li>
</ol>
3. Preparation of Endothelium Cell Medium and Coating of Cell Culture Plates
<ol>
	<li>Endothelial cell medium:
	<ol>
		<li>Preparation of 500 ml medium: Mix 400 ml DMEM medium (approx. 80%) with 100 ml PDS (approx. 20%), 0.25 ml bFgF (20 &#181;g/ml; approx. 0.05%), 0.5 ml heparin (100 &#181;l/ml; approx. 0.1%) and 0.5 ml pyrumycin (4 mg/ml; approx. 0.1%). Add pyrumycin only for cell culture medium used in the first 2 days of culture.</li>
		<li>Perform sterile filtration in a glass tube (diameter of filter pores: 0.2 &#181;m). Afterwards store the solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Coating of cell culture plates:
	<ol>
		<li>For 1 ml of coating solution, prepare a solution of 500 &#181;l ddH2O, 400 &#181;l collagen IV (0.4 mg/ml) and 100 &#181;l fibronectin (0.1 mg/ml).</li>
		<li>Cover the whole surface of each well of the cell culture plate with the coating solution. Store the plate at 4 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
4. Isolation of Murine Brain Microvascular endothelial cells (MBMEC)
<ol>
	<li>Sacrifice 10 adult mice (8 - 12 weeks old, C57BL/6) by cervical dislocation. Afterwards isolate the mouse-brains and store in 5 ml of PBS (Caveat: Work sterile!).</li>
	<li>Remove brain stems, cerebella and thalami with a forceps. Detach meninges by carefully rolling the brains on a sterile blotting paper. Collect the resulting meninges-free forebrains.</li>
	<li>Transfer the brains to a 50 ml Falcon tube filled with 13.5 ml of DMEM.</li>
	<li>Mince the tissue, first with a 25 ml pipette, then with a 10 ml pipette until the media becomes milky.</li>
	<li>Then digest the tissue with a mixture of 0.6 ml collagenase CLS2 (10 mg/ml in DMEM) and 0.2 ml DNAse (1 mg/ml in PBS) in Dulbecco&#8217;s Modified Eagle Medium (DMEM) for 1 hr at 37 &#176;C on an orbital shaker at 180 rpm.</li>
	<li>Add 10 ml of DMEM added to the tissue suspension and centrifuge the cells at 1,000 x g for 10 min at 4 &#176;C.</li>
	<li>Discard supernatant (Caveat: PELLET IS EASY TO LOSE!)</li>
	<li>To remove the myelin, resuspend the pellet with a 25 ml pipette in 25 ml of BSA-DMEM (20% w/v) (approx. 25 times), and centrifuge at 1,000 x g for 20 min at 4 &#176;C.</li>
	<li>Discard the upper myelin layer (milky) with a broken glass Pasteur pipette with larger diameter, and then discard the BSA layer with a normal Pasteur pipette.</li>
	<li>Resuspend the pellet in 9 ml DMEM and add 1 ml collagenase/dispase (final concentration is 1 mg/ml) and 0.1 ml DNAse. Digest the solution for 1 hr at 37 &#176;C on an orbital shaker at 180 rpm (caveat: do not use the pipette to resuspend &#8211; cells can be lost).</li>
	<li>During the digestion, centrifuge the Percoll solution to set up the density gradient using an ultracentrifuge at 3,000 x g for 1 hr at 4 &#176;C, with acceleration, without brake.</li>
	<li>Add 10 ml of DMEM to the digested cell suspension. Centrifuge the suspension at 1,000 x g for 10 min at 4 &#176;C. Then discard the supernatant and resuspend the pellet in 2 ml of DMEM.</li>
	<li>Add the resuspended cells carefully on top of the Percoll gradient and centrifuge at 700 x g for 10 min at 4 &#176;C without acceleration and brake. Note: The cells are visible as a cloud in the interphase.</li>
	<li>Take approx. 12 ml of the interphase with cells with a long sterile needle and place it in a new 50 ml Falcon with 5 ml of DMEM.</li>
	<li>Centrifuge the cells again at 1,000 x g for 10 min at 4 &#176;C. Afterwards resuspend the pellet in endothelial cell medium (use approx. 0.2 ml of medium per 1 cm2 surface of cell culture plate).</li>
	<li>Remove coating solution from coated cell culture plates (see step 3.2) and rinse every well with sterile PBS in two consecutive washing steps.</li>
	<li>Maintain cell cultures in endothelial cell medium at 37 &#176;C and 5% CO2 in a sterile incubator. Change the medium every two to three days. Split cells at 90% confluence.</li>
</ol>

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<li>Zonta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuron-to-astrocyte+signaling+is+central+to+the+dynamic+control+of+brain+microcirculation%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.</a> Nature Neuroscience. 6, 43-50 (20003).</li>
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<li>Gerhardt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VEGF+guides+angiogenic+sprouting+utilizing+endothelial+tip+cell+filopodia%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.</a> Journal of Cell Biology. 161, 1163-1177 (2003).</li>
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</ol>

The authors declare that they have no competing financial interests.

Blood-brain-barrier dysfunction is a hallmark of various deleterious CNS diseases, e.g. a breakdown of the BBB in multiple sclerosis extensively facilitates the CNS invasion by autoreactive immune cells and enables the encounter and destruction of oligodendrocytes. In ischemic stroke the disruption of the BBB and the subsequent formation of brain edema are critical factors that influence secondary infarct growth and the overall survival of patients6. Furthermore, by alterations of the BBB in remote areas focal ischemic lesions are able to induce widespread functional alterations of the brain. However, the underlying molecular mechanisms are largely unknown5. In contrast, the high density and diversity of efflux transporters in functional BMECs reduces the concentration of beneficial drugs such as chemotherapeutics for brain malignancies or antibiotics for infectious diseases. Therefore, a deeper understanding of the blood-brain-barrier function under physiological and pathophysiological conditions is required to develop pharmacological agents that are able to specifically regulate the barrier function in the favored direction21. Reliable in vitro models of the BBB are indispensable tools to study the regulatory mechanisms at the BBB.
Many immortalized brain endothelial cell lines have been established and employed as in vitro BBB models22. These cell lines offer certain advantages over primary BMECs as they grow faster and keep certain BBB characteristics over several passages. However, endothelial cell lines cannot fully substitute for primary cells as important properties are changed that intermingle with the transferability of experimental findings to the in vivo situation. For instance, the murine brain endothelial cell line bEnd.5 expresses only low levels of discontinuously distributed tight junction proteins leading to high paracellular permeability23. BEnd.3, another frequently used murine brain endothelial cell line, demonstrates dense and continuous distributed tight junctions, a high transendothelial resistance, several efflux transporters and low paracellular permeability. However, bEND.3 cell layers compared to primary BMECs lack a real discrimination with respect to the permeation of certain transcellular and paracellular substrates24.
In preparations of primary cell cultures, contaminating cells can significantly interfere with the validity of experimental findings. In the described protocol, pyrumicin is used as a selective agent for endothelial cells to achieve high purity. Nevertheless, a regular quality control of the cell culture is inevitably needed to guarantee for reliable experiments. There are several methods for quality control, besides typical morphological properties of endothelial cells (see Figure 1A), the transendothelial resistance might be measured or the expression of endothelial cell marker such as CD31 (see Figure 1B) or von Willebrand factor (vWF) might be evaluated.
The number of brain endothelial cells isolated from one mouse brain is relatively low and to set up a proper cell culture for multiple experiments, the brains of several mice have to be pooled. In our experience, at least 10 mice need to be used for one experiment which might be a limiting factor depending on the resources of the respective animal facility. In order to avoid bias or confounding factors, only mice with an identical genetic and environmental background should be pooled. In contrast, bovine and porcine brain endothelial cell preparations provide a large number of brain endothelial cells available in one brain. However, besides the uncomplicated breeding and housing, the broad and ever increasing repertoire of available transgenic mice renders primary murine BMECs as an ideal model to study blood-brain-barrier function.
Several key findings concerning the functions of the BBB and the underlying molecular mechanisms have come from studies in 2D tissue culture systems. Recently, 3D culture systems have been developed25, where endothelial cells are placed within a collagen matrix enabling them to form lumen and tubular networks. These new cell culture systems allow for an even more accurate reproduction of vascular processes in the intact organism in vivo. The involvement of further cells of the NVU in 3D cell culture systems, such as pericytes and astrocytes might revolutionize the in vitro studying of blood-brain-barrier function; first models have recently been developed26.
There are some recommendations for troubleshooting. First of all sterile working is essential for the quality of the endothelial cell culture. Second, pyrumycin should only be added for cell culture medium used in the first 2 days of culture, longer application might lead to increased MBMEC cell death and low quality barrier function. Third, the enzymes applied in this protocol should be used and stored according to the manufacturer&#8217;s instructions; otherwise their proper function might be compromised. Furthermore, the coating of cell culture plates might be amended if endothelial cells do not attach. Concentrations of fibronectin and collagen might be changed or other matrix proteins might be added to the coating solution. Lastly, age, gender, season of the year and environmental conditions within the animal facility are important factors that might influence the quality of MBMEC isolation and comparability of experiments. It should be ensured that conditions are comparable between independent experiments.
The described protocol should be considered as a basic neuroimmunological method to isolate murine BMECs. The isolated cells might be used for a wide variety of applications to gain further insight into BBB function, such as migration assays, gene and protein expression studies, electrophysiological evaluations or permeability experiments. As mentioned above, 3D cell culture systems of BMECs might provide new opportunities to study the complex mechanisms involved in the regulation of the BBB in the context of the NVU. Therefore, the isolation of primary endothelial cells will remain an invaluable technique in the field of BBB research in the future.

Brain microvascular endothelial cells (BMEC) form monolayers that are an integral part of the highly specialized blood-brain-barrier (BBB). BMECs are interconnected by junctional proteins attached to a basement membrane. Together with pericytes, smooth muscle cells, astrocytic end feet and circulating blood cells they build up the so-called neurovascular unit (NVU)1. Against the previous notion of an impermeable barrier between blood and the central-nervous-system (CNS), the NVU is a dynamic, highly specific and regulated interface that controls the transition of fluids, molecules and cells between cerebral blood vessels and the CNS2. A dysfunction or dysregulation of the NVU may initiate and/or contribute to a variety of neurovascular, infectious, inflammatory or degenerative diseases of the CNS, such as ischemic stroke, HIV-encephalopathy, multiple sclerosis, Alzheimer&#8217;s or Parkinson&#8217;s disease3-6.
The BMEC monolayer is tightly sealed by a junctional complex constituted of tight (TJ) and adherens junctions (AJ)7. The high electrical resistance and the low paracellular permeability of the BBB are mainly based on TJ proteins8. The TJs are complexes formed by the transmembrane proteins of the claudin and occludin family, which are linked to the cytoskeleton of the endothelial cells by adapter molecules, such as the zonulaoccludens (ZO) proteins ZO1-34. Adherens junctions are mainly assembled by the integral membrane protein vascular endothelial (VE)-cadherin, which is linked to the cytoskeleton via catenins8.
The tight sealing of the BBB prevents the free exchange of substrates and cells between blood and CSF. Exceptions to this rule are lipophilic, small molecules with a molecular weight &#60;400 Da, which are able to cross the BBB by lipid-mediated diffusion9. The passage of larger and/or hydrophilic molecules, such as glucose, amino acids, peptides, proteins and many drugs is restricted to highly controlled transcellular transport systems10, which can be classified into five main categories: carrier-mediated transport, ion transport, active efflux transport, receptor-mediated transport, and caveolae-mediated transport4. These transporter systems help maintaining the homeostasis of the CNS, which is required for an accurate signal generation, transduction and integration. Moreover, BMECs are able to actively control the transition of distinct molecules by the expression of a variety of ectoenzymes. These enzymes are localized on the cell surface and modify specific endogenous and exogenous substrates hindering or allowing the transition of the BBB11.
Whereas the CNS was considered as an immune-privileged organ for a long-time, recent findings suggest a rather dynamic and tightly regulated system of immune surveillance of the CNS. The BMECs are critically involved in the regulation of immune cell transmigration. By the expression of selectins on their surface lymphocytes are selectively induced to loosely attach to the endothelium. The secretion of chemokines that encounter with specific receptors on leukocytes leads to the expression or conformational changes of leukocyte integrins, such as LFA-1 (lymphocyte function associated antigen-1) and VLA-4 (very late antigen-4). The integrins mediate a firm adhesion by binding their endothelial counterreceptors, e.g. VCAM-I (vascular cell adhesion molecule), ICAM-I (intercellular adhesion molecule) enabling the transmigration into the brain parenchyma between or through BMECs of the BBB12-14. These and other findings underline the active role of the endothelium itself in regulating immune cell migration.
Furthermore, BMECs as part of the NVU are involved in the regulation of the cerebral blood flow linked to local neuronal metabolic demands. Upon astrocytic stimulation endothelial cells produce vasoactive substances such as nitric oxide leading to relaxation of vascular smooth muscle cells15.
Angiogenesis and neurogenesis in the developing as well as in the adult brain show parallel patterning and development and share many properties of regulation1,4. Endothelial cells are critically involved in these processes16,17.
In summary, BMECs provide essential features to warrant a proper development and functioning of the CNS. BBB dysfunction is linked to many severe neurologic disorders. However, only very few targets have been identified at the brain-vasculature interface for a specific and efficient treatment18. Simplified in vitro models have been used to understand the mechanisms involved in function and regulation of complex physiologic systems for a long time. The isolation as described by this manuscript and the in vitro study of murine BMECs, given the wide variety of specific mouse knockout-strains, might provide a further understanding of BBB function and regulation under physiological and pathophysiological conditions opening up new therapeutic avenues.

<table><tbody><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>Basic fibroblast growth factor</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A9418</td><td>Bovine serum albumin</td></tr><tr><td>Collagenase CLS2</td><td>Worthington</td><td>LS004176</td><td>High relative level of protease activity</td></tr><tr><td>Collagenase-dispase</td><td>Roche</td><td>10269638001</td><td>Collagenase from &lt;em&gt;V. alginolyticus&lt;/em&gt;, Dispase from &lt;em&gt;B. polymyxa&lt;/em&gt;</td></tr><tr><td>Collagen IV</td><td>Sigma Aldrich</td><td>C0543</td><td>From Engelbreth-Holm-Swarm murine sarcoma basement membrane</td></tr><tr><td>DMEM</td><td>Sigma Aldrich</td><td>D5030</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>DNAse</td><td>Sigma Aldrich</td><td>D4513</td><td>Deoxyribonuclease I from bovine pancreas</td></tr><tr><td>FCS</td><td>Sigma Aldrich</td><td>F6178</td><td>Fetal calf serum (also named fetal bovine serum)</td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H3393</td><td>Anticoagulant</td></tr><tr><td>PDS</td><td>First Link UK Ltd.</td><td>60-00-850</td><td>Plasma-derived serum</td></tr><tr><td>Percoll</td><td>Sigma Aldrich</td><td>P1644</td><td>Warm to room temperature before use</td></tr><tr><td>Pyrumycin</td><td>Sigma Aldrich</td><td>P8833</td><td>Should only be used in first 2 days of culture</td></tr></tbody></table>

isolation of primary murine brain microvascular endothelial cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Immediately after isolation, murine BMECs and contaminating cells form conglomerates floating in the cell culture medium (Figure 1A, day 0). The treatment with pyrumycin leads to a selection of murine BMECs since they express high levels of efflux transporters such as P-glycoprotein leading to a relative resistance to toxins. Contaminating cells are much more susceptible to pyrumycin mediated cytotoxicity19. During the process of pyrumicin selection, contaminating cells enter apoptotic or necrotic cell death, whereas MBMECs begin to attach to the collagen IV/fibronectin coated cell culture plates (Figure 1A, day 1 and day 2). Until day 5, the MBMECs proliferate to a density of approx. 80 - 90%. They are characterized by the typical spindle-shaped appearance. At confluence, they form a monolayer of tightly packed, longitudinally aligned and non-overlapping contact-inhibited cells (Figure 1A, day 5). By pyrumicin selection, a high purity of up to 99% MBMEC is reached as demonstrated by the endothelial cell marker CD31 (PECAM1; Figure 1B). The MBMECs form tightly sealed monolayers interconnected by tight junction proteins. After 7 - 8 days of culture, the endothelial cell layers build up stable values for electrical resistance (reaching typical values between 20 - 30 &#937; x cm2) and capacitance (Figure 1C). At this time-point functional assays such as cell-migration experiments3,20,21 and permeability tests, e.g. with Evans blue or Dextran, should be performed.
<img alt="Figure 1" src="/files/ftp_upload/52204/52204fig1highres.jpg" width="500" /> 
Figure 1. Morphological and Functional Properties of Murine BMECs. (A) Representative time course of cultured BMECs viewed by transmission light microscopy over 5 days. Scale bar applies for all images. (B) Immunofluorescence staining for the endothelial cell marker CD31 at day 5. CD31 (green) and DAPI (blue). (C) Cells were precultured for 5 days and then transferred to an automated system for the analysis of biophysical properties (resistance and capacitance). <a href="https://www.jove.com/files/ftp_upload/52204/52204fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Primary Murine Brain Microvascular Endothelial Cells at JoVE.com

Isolation of Primary Murine Brain Microvascular Endothelial Cells

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

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Research

JoVE Journal

Neuroscience

32.5K Views.  University of Münster. Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB. 

Video: Isolation of Primary Murine Brain Microvascular Endothelial Cells

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Medicine

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Biology

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Source: H&#248;rlyck, S. et al. <a href="https://www.jove.com/video/61253" target="_blank">Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.</a>&#160;J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

Source: H&#248;rlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.&#160;J. Vis. Exp. ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

Summary

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