Name: isolation of primary murine brain microvascular endothelial cells
Uploaded: 2014-11-14T15:00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Isolation of Primary Murine Brain Microvascular Endothelial Cells at JoVE.com

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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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.

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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">bFGF</td>
			<td style="width:133px;">Peprotech</td>
			<td style="width:123px;">100-18B</td>
			<td style="width:331px;">Basic fibroblast growth factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BSA</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">A9418</td>
			<td>Bovine serum albumine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Collagenase CLS2</td>
			<td style="width:133px;">Worthington</td>
			<td style="width:123px;">LS004176</td>
			<td>High relative level of protease activity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Collagenase-dispase</td>
			<td style="width:133px;">Roche</td>
			<td align="right" style="width:123px;">10269638001</td>
			<td style="width:331px;">Collagenase from V. alginolyticus, Dispase from B. polymyxa</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Collagen IV</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">C0543</td>
			<td style="width:331px;">From Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMEM</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">D5030</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DNAse</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCS</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">F6178</td>
			<td>Fetal calf serum (also named fetal bovine serum)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Heparin</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">H3393</td>
			<td>Anticoagulant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">PDS</td>
			<td style="width:133px;">First Link UK Ltd.</td>
			<td style="width:123px;">60-00-850</td>
			<td>Plasma-derived serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Percoll</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">P1644</td>
			<td>Warm to room temperature before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pyrumycin</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">P8833</td>
			<td>Should only be used in first 2 d 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 ...

isolation-of-primary-murine-brain-microvascular-endothelial-cells

Research

JoVE Journal

Neuroscience

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 ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.