This work was supported by a National Alliance for Research on Schizophrenia and Depression (NARSAD) Young Investigator Award and National Institutes of Health grant R01NS073635 to AV.

1. Preparation of Reagents and Solutions
<ol>
	<li>Coating of 35 mm culture dish: Collagen Type 1 solution is supplied as an aqueous solution in 20 mM acetic acid (~100 mg protein/vial). Dilute an appropriate volume of collagen solution to a working concentration of 0.01% using sterile tissue culture grade water. Coat dishes with 1 ml of collagen solution for 3-4 hr at room temperature (RT) or 37 &#176;C, or overnight at 2-8 &#176;C. Remove excess solution from the coated dish and allow it to dry overnight. Rinse the dish with tissue culture grade water or PBS before adding media. Coated dishes can be stored at 4 &#176;C for one month.</li>
	<li>Prepare complete Dulbecco&#39;s Modified Eagle Medium (DMEM). Supplement the DMEM (500 ml) with 10% FBS and antibiotic-antimycotic solution (1x concentration; 1 ml/100 ml). Complete DMEM can be stored at 4 &#176;C for two months.</li>
	<li>Prepare an aliquot (50 ml) of DMEM with DNase I (0.001 mg/ml), (referred to as DMEM-DNase I).</li>
	<li>Prepare another aliquot (10 ml) of DMEM with collagenase and dispase (1 mg/ml) (referred to as DMEM-collagenase/dispase).</li>
	<li>Prepare Endothelial Cell Culture Medium (ECCM, 500 ml), supplemented with EGF (5 &#956;g), ECGS (100 mg) and 5 &#956;l of antibiotic-antimycotic solution. ECCM can be stored at 4 &#176;C for two months.</li>
	<li>Prewarm all media before use.</li>
	<li>Prepare Purification buffer: PBS, pH 7.2 with 0.5% BSA and 2 mM EDTA. Keep buffer cold (2-8 &#176;C).</li>
</ol>
2. Removal of Embryos and Dissection of Telencephalon
All experiments using laboratory animals are approved by the animal care and use committees of McLean Hospital and conform to NIH guidelines for the care and use of laboratory animals.
<ol>
	<li>Use timed pregnant CD1 mice (embryonic day 15). The day of vaginal plug discovery is considered embryonic day 0 (E0). CD1 dams generally produce large litters, so 10-12 embryos are expected from a pregnant dam.</li>
	<li>For hysterotomy, anesthetize pregnant mice with an intraperitoneal injection of Ketamine (100 mg/kg)/Xylazine (5 mg/kg). For mice approximately 20-30 g, deliver 0.3 ml of a 10 ml/kg Ketamine/Xylazine anesthetic solution and ensure that pain and distress is minimal during intraperitoneal injections of the anesthetic. Use hand restraint for the injections.</li>
	<li>Check deep anesthesia by toe pinch. Remove embryos from deeply anesthetized mice. Decapitate each embryo immediately upon removal from the mother. Place the embryonic heads in sterile Petri dishes in ice cold PBS.</li>
	<li>Follow hysterectomy by immediate euthanasia with anesthetic overdose (130 mg/kg pentobarbital, i.p.), a method for euthanasia, which is consistent with the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.</li>
	<li>Under a stereo microscope, remove the brain from the head with fine microtip scissors and fine forceps. Note: A high quality dissection is essential to successfully remove the pial membrane from the embryonic brain. This is achieved by practice. Fine forceps and microtip scissors are also key tools for getting a good dissection.</li>
	<li>Use the forceps to get a firm grip on the brain and the microtip scissors to peel away the pial membrane. Remove the mesencephalon and metencephalon and isolate the telencephalon. Transfer pial-free telencephalon from all embryos to a 35 mm culture dish with 2 ml PBS in ice.</li>
</ol>
3. Cell Isolation
<ol>
	<li>Mince the telencephalon into 1-2 mm fragments with a scalpel blade and collect tissue in a 15 ml Falcon tube containing 2 ml of complete DMEM.</li>
	<li>Dissociate tissue gently but thoroughly with a 1 ml pipette until clumps disappear and a milky suspension is achieved.</li>
	<li>Centrifuge dissociated cells at 800-1,000 x g for 5 min at RT.</li>
	<li>Carefully remove the supernatant and resuspend pellet in DMEM-DNase I. Gently dissociate the cells again using 1 ml pipette and centrifuge at 800-1,000 x g for 5 min at RT.</li>
	<li>Resuspend the pellet in warm complete DMEM. Filter cells through a sterile 70 &#956;m Nylon mesh. Collect filtered cells and keep in ice.</li>
	<li>Collect the cell-portions retained on the mesh and digest further with DMEM-collagenase/dispase (2 ml) for 1-2 min at RT. Wash cells 3x in DMEM-DNase I through centrifugation at 800-1,000 x g for 5 min at RT. Pool these cells with the filtered cells collected in step 3.5. Wash total cells once with complete DMEM. Proceed to determine cell number and magnetic labeling steps. Note: It is important to obtain a single-cell suspension before magnetic labeling.</li>
</ol>
4. Magnetic Labeling
<ol>
	<li>This protocol shows the magnetic labeling of 107 cells. For cell numbers lower than 107, use the same reagent volume and for cell numbers greater than 107, scale up all reagent volumes and total volumes accordingly (e.g. for 2 x 107 total cells, use twice the volume of all indicated reagents). Keep all the reagents and cells in ice.</li>
	<li>Determine cell number with a hemocytometer.</li>
	<li>Centrifuge cell suspension at 300 x g for 10 min at RT. Aspirate supernatant completely.</li>
	<li>Add 90 &#956;l/107 cells of purification buffer to the cell pellet followed by 10 &#956;l of CD31 MicroBeads. The Microbeads provided by manufacturer are conjugated to monoclonal anti-mouse CD31 antibody.</li>
	<li>Mix well and incubate for 15 min in the refrigerator. Note: Higher temperatures and/or longer incubation time during magnetic labeling may result in nonspecific binding.</li>
	<li>Wash cells by adding 1-2 ml/107 cells of purification buffer and centrifuge at 300 x g for 10 min. Remove supernatant and resuspend cells in 500 &#956;l of purification buffer. Proceed to magnetic separation or purification step.</li>
</ol>
5. Purification
<ol>
	<li>Place MS column in the magnetic field of MACS Separator.</li>
	<li>Rinse column with 500 &#956;l of purification buffer.</li>
	<li>Apply cell suspension onto the column. Load appropriate number of cells to MS column as higher cell number may clog the column. Caution: Each MS column can accommodate a maximum number of 2 x 107 cells, for a higher cell number, use more columns.</li>
	<li>Collect and discard flow-through containing unlabeled cells.</li>
	<li>Wash column with 500 &#956;l of purification buffer three times. During wash steps, make sure that the column reservoir is empty before adding subsequent purification buffer aliquots.</li>
	<li>Remove column from the MACS separator and place it on a 15 ml Falcon tube.</li>
	<li>A plunger is supplied along with the MS column. Pipette 1 ml of purification buffer onto the MS column and immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.</li>
	<li>Plate cells on a 35 mm culture dish precoated with collagen Type 1 and grow cells in ECCM in an incubator set to 37 &#176;C with 95% O2 and 5% CO2.</li>
	<li>Change the medium every four days. Use PVECs for experiments or subculture after 10-12 days when the dish is confluent.</li>
</ol>
6. Subculture of PV ECS
<ol>
	<li>Remove the ECCM and rinse the monolayer of PVECS with 1-2 ml of sterile PBS.</li>
	<li>Gently add 1.5 ml of 0.25% Trypsin-EDTA solution to cover the cell monolayer.</li>
	<li>Return the dish to the incubator. Within 5-7 min, cells will detach from the surface of the dish.</li>
	<li>After the cells have detached, immediately add an equal volume of trypsin inhibitor and triturate several times.</li>
	<li>Transfer the cells to a sterile 15 ml Falcon tube. Centrifuge the tube at 500 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 ml of prewarmed ECCM.</li>
	<li>Seed the cells for expanding the culture, imaging or other assays.</li>
</ol>

<ol>
	<li>Shen, Q., S, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+stimulate+self-renewal+and+expand+neurogenesis+of+neural+stem+cells.">Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.</a> Science. 304, 1338-1340 (2004).</li><li>Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+three-dimensional+system+to+study+interactions+between+endothelial+cells+and+neural+cells+of+the+developing+central+nervous+system.">A novel three-dimensional system to study interactions between endothelial cells and neural cells of the developing central nervous system.</a> BMC Neurosci. 8, 3 (2007).</li><li>Rauch, M. F., Michaud, M., Xu, H., Madri, J. A., Lavik, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+primary+neural+progenitor+and+endothelial+cells+in+a+macroporous+gel+promotes+stable+vascular+networks+in+vivo.">Co-culture of primary neural progenitor and endothelial cells in a macroporous gel promotes stable vascular networks in vivo.</a> 19, 1469-1485 (2008).</li><li>Vasudevan, A., Long, J. E., Crandall, J. E., Rubenstein, J. L., Bhide, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartment-specific+transcription+factors+orchestrate+angiogenesis+gradients+in+the+embryonic+brain.">Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain.</a> Nat. Neurosci. 11, 429-439 (2008).</li><li>Vasudevan, A., Bhide, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+embryonic+CNS:+a+new+twist+on+an+old+tale.">Angiogenesis in the embryonic CNS: a new twist on an old tale.</a> Cell Adh. Migr. 2, 167-169 (2008).</li><li>Won, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+vascular+networks+synchronize+GABA+neuron+migration+in+the+embryonic+forebrain.">Autonomous vascular networks synchronize GABA neuron migration in the embryonic forebrain.</a> Nat. Commun. 4, 2149-2162 (2013).</li><li>Dong, Q. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arterioscler. Thromb. Vasc. Biol. 17, 1599-1604 (1997).</li><li>Erblich, B., Zhu, L., Etgen, A. M., Dobrenis, K., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+colony+stimulation+factor-1+receptor+results+in+loss+of+microglia,+disrupted+brain+development+and+olfactory+deficits.">Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.</a> PLoS One. 6, (2011).</li><li>Wichterle, H., Garcia-Verdugo, J. M., Herrera, D. G., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Young+neurons+from+medial+ganglionic+eminence+disperse+in+adult+and+embryonic+brain.">Young neurons from medial ganglionic eminence disperse in adult and embryonic brain.</a> 2, 461-466 (1999).</li><li>Snapyan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculature+guides+migrating+neuronal+precursors+in+the+adult+mammalian+forebrain+via+brain-derived+neurotrophic+factor+signaling.">Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brain-derived neurotrophic factor signaling.</a> J. Neurosci. 29, 4172-4188 (2009).</li><li>Whitman, M. C., Fan, W., Rela, L., Rodriguez-Gil, D. J., Greer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+form+a+migratory+scaffold+in+the+rostral+migratory+stream.">Blood vessels form a migratory scaffold in the rostral migratory stream.</a> J. Comp. Neurol. 516, 94-104 (2009).</li></ol>

No conflicts of interest declared.

PVEC&#39;s are more physiologically relevant than adult brain endothelial cells and ECs from other tissue sources for studies focusing on neurovascular interactions and also have therapeutic potential. For PVEC preparation, it is critical beginning with dissection to work fast to achieve a good viability since dead cells may bind nonspecifically to CD31 MicroBeads. In addition, if single-cell suspension is not achieved prior to the magnetic labeling step, this will result in troubleshooting since cell clumps will clog th...

Angiogenesis, neurogenesis and neuronal migration are critical events in central nervous system (CNS) development, repair and regeneration. Several elegant studies have shown that endothelial cells stimulate neuronal proliferation and vice versa through release of soluble factors and by direct contact. We found it curious that in majority of these studies1-3, while neuronal progenitors/neural stem cells are isolated from the embryonic brain, they are cocultured with endothelial cells from the adult brain, other adult tissue sources, or with endothelial cell lines. This might in part be due to the technical difficulties associated with isolating and culturing pure populations of endothelial cells from the embryonic brain. However, angiogenesis, neurogenesis, and neuronal migration are concurrent events occurring in orders of magnitude more robust in the embryonic brain than in the normal adult brain. The periventricular vascular network of the embryonic forebrain (telencephalon) originates from a vessel located within the basal ganglia primordium and develops in the form of an orderly gradient from ventral to dorsal telencephalon by embryonic day 11 (E11)4,5. This plexus of vessels of the periventricular vascular network are distinct from pial vessels based on origins, anatomical location, growth patterns, and developmental regulation4,5. The direction of propagation of the periventricular angiogenesis gradient matches the telencephalic transverse neurogenetic gradient. Within the telencephalon, the periventricular angiogenesis gradient and the gradient of GABA neurons migrating tangentially overlaps spatially as well6. With respect to timing, the angiogenesis gradient is in advance of the neurogenetic gradient and GABA neuron gradient by about a day. Thus, periventricular endothelial cells are spatially and temporally well positioned to provide critical cues to support telencephalic neurogenesis and neuronal migration4,6. Therefore, use of embryonic periventricular endothelial cells in coculture experiments with neuronal progenitors and/or neurons would provide a more favorable model for studying neurovascular interactions and developing novel avenues for treatment of neurodegenerative disease or ischemic/traumatic brain injury.
We emphasize the importance of removing the pial membrane, not only to limit epithelial cell contamination but also to separate pial endothelial cells which are molecularly and functionally distinct from endothelial cells of the periventricular vascular network4,6 (termed PVECs to distinguish from pial ECs). Here, we describe the method that we routinely use in our laboratory to obtain a rich and pure yield of PVECs. These endothelial cells are prepared from embryonic forebrains isolated from a single timed-pregnant mouse. They can be expanded, subcultured, and frozen down successfully for future use.

<table><tbody><tr><td>DNase I</td><td>Sigma</td><td>D-4527</td><td>&amp;nbsp;</td></tr><tr><td>Collagen, Type 1 solution from rat tail&amp;nbsp;</td><td>Sigma</td><td>C3867</td><td>&amp;nbsp;</td></tr><tr><td>DPBS</td><td>Quality Biologicals</td><td>114057-131</td><td>&amp;nbsp;</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>M4055</td><td>&amp;nbsp;</td></tr><tr><td>BSA</td><td>Sigma</td><td>A2058</td><td>&amp;nbsp;</td></tr><tr><td>MS column</td><td>Miltenyi Biotech</td><td>130-042-201</td><td>&amp;nbsp;</td></tr><tr><td>CD31 microbeads</td><td>Miltenyi Biotech</td><td>130-097-418</td><td>&amp;nbsp;</td></tr><tr><td>MACS separator</td><td>Miltenyi Biotech</td><td>130-042-102</td><td>&amp;nbsp;</td></tr><tr><td>MACS multi stand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td>&amp;nbsp;</td></tr><tr><td>Cell strainer 70 &amp;micro;m</td><td>BD Bioscience</td><td>352350</td><td>&amp;nbsp;</td></tr><tr><td>Antibiotic and antimycotic solution</td><td>Sigma</td><td>A5955</td><td>&amp;nbsp;</td></tr><tr><td>FBS</td><td>Sigma</td><td>F4135</td><td>&amp;nbsp;</td></tr><tr><td>Collagenase/Dispase</td><td>Roche</td><td>10269638001</td><td>&amp;nbsp;</td></tr><tr><td>DMEM&amp;nbsp;</td><td>Lonza</td><td>12-604F</td><td>&amp;nbsp;</td></tr><tr><td>35 mm Culture dish</td><td>BD Bioscience</td><td>353001</td><td>&amp;nbsp;</td></tr><tr><td>15 ml Falcon tube&amp;nbsp;</td><td>BD Bioscience</td><td>352097</td><td>&amp;nbsp;</td></tr><tr><td>50 ml Falcon tube</td><td>BD Bioscience</td><td>352098</td><td>&amp;nbsp;</td></tr><tr><td>ECCM kit</td><td>BD Bioscience</td><td>355054</td><td>Kit Includes Endothelial cell growth supplement, EGF, and Soybean Trypsin Inhibitor</td></tr><tr><td>Endothelial Cell Growth Supplement (ECGS)</td><td>BD Bioscience</td><td>354006</td><td>&amp;nbsp;</td></tr><tr><td>RBECGM</td><td>Cell Applications</td><td>R819-500</td><td>&amp;nbsp;</td></tr><tr><td>DMEM F12&amp;nbsp;</td><td>Life Technologies</td><td>10565-018</td><td>&amp;nbsp;</td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>305050-061</td><td>&amp;nbsp;</td></tr><tr><td>Tissue culture grade water</td><td>Life Technologies</td><td>15230162</td><td>&amp;nbsp;</td></tr><tr><td>0.25% Trypsin</td><td>Life Technologies</td><td>15050</td><td>&amp;nbsp;</td></tr><tr><td>Soybean trypsin inhibitor</td><td>BD Bioscience</td><td>5425</td><td>&amp;nbsp;</td></tr><tr><td>Matrigel</td><td>BD Bioscience</td><td>354234</td><td>&amp;nbsp;</td></tr><tr><td>Qtracker 655 Cell Labeling Kit</td><td>Life Technologies</td><td>Q25021</td><td>&amp;nbsp;</td></tr><tr><td>CellLight Plasma Membrane-RFP, BacMam 2.0</td><td>Life Technologies</td><td>C10608</td><td>&amp;nbsp;</td></tr><tr><td>Biotinylated Isolectin B4 antibody</td><td>Sigma</td><td>L2140</td><td>&amp;nbsp;</td></tr><tr><td>Anti-Von Willebrand factor</td><td>Sigma</td><td>F3520</td><td>&amp;nbsp;</td></tr><tr><td>Anti-CD31/PECAM-1</td><td>BD Pharmingen</td><td>550274</td><td>&amp;nbsp;</td></tr><tr><td>VECTASHIELD Hardset Mounting Media with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td>&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Butler Schein Animal Health Supply</td><td>44028</td><td>&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>1009</td><td>&amp;nbsp;</td></tr><tr><td>Stereomicroscope</td><td>Motic</td><td>SMZ-168</td><td>&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>267110</td><td>&amp;nbsp;</td></tr><tr><td>Inverted microscope</td><td>Olympus CK-40</td><td>CK-40</td><td>&amp;nbsp;</td></tr><tr><td>Fluorescent microscope</td><td>Olympus FSX-100</td><td>FSX-100</td><td>&amp;nbsp;</td></tr><tr><td>Fine forceps</td><td>Roboz Surgical Instrument</td><td>7 inox</td><td>&amp;nbsp;</td></tr><tr><td>Fine microtip scissors</td><td>Roboz Surgical Instrument</td><td>RS5611</td><td>&amp;nbsp;</td></tr></tbody></table>

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.

The phenotypic characterization of PVECs from day 1-12 is shown by phase-contrast light microscopy (Figure 2). The cells attached to the dish on day 1 show morphology characteristic of cell division (Figure 2A). Between 5-8 days, PVECs transition from cobble stone to spindle shaped morphology typical for endothelial cells and more akin to its in vivo state (Figures 2B and 2C). By day 12 the PVEC culture achieves full confluence (Figure 2...

Watch this Scientific Journal Video about Isolation and Culture of Endothelial Cells from the Embryonic Forebrain at JoVE.com

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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 ...

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Neuroscience

15.3K Views.  Harvard Medical School. 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.

Video: Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

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 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.

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 and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Biology

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

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 ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

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 ...

Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

Source: Molino, Y. et al. <a href="https://www.jove.com/v/51278/setting-up-an-vitro-model-rat-blood-brain-barrier-bbb-focus-on-bbb">Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport.</a> J. Vis. Exp.&#160;(2014)
This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

Hodowla komórek śródbłonka z mikronaczyń mózgu szczura: procedura izolacji i hodowli komórek śródbłonka z mikronaczyń mózgu szczura

Source: Molino, Y. et al. Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

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

Isolation of Primary Murine Brain Microvascular Endothelial Cells

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Embryonic Ventricular Endothelial Cells

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Mouse Coronary Endothelial Cells

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Hodowla komórek śródbłonka z mikronaczyń mózgu szczura: procedura izolacji i hodowli komórek śródbłonka z mikronaczyń mózgu szczura