This work was supported by the &#34;Else Kr&#246;ner-Fresenius-Stiftung&#34; (2018_A03 to TR), &#34;Innovative Medizinische Forschung (IMF) M&#252;nster&#34; (I-RU211811 to TR) and German Research Foundation (DFG, INST 2105/27-1, ME 3283/5-1, and ME 3283/6-1 to SGM). Illustrated images provided by Heike Blum.

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	<li>Rodrigues, S. F., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+cells+and+endothelial+barrier+function.">Blood cells and endothelial barrier function.</a> Tissue Barriers. 3 (1-2), e978720 (2015).</li><li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+functions.">Endothelial cell functions.</a> Journal of Cellular Physiology. 196 (3), 430-443 (2003).</li><li>Pierce, R. W., Giuliano, J. S., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+function+and+dysfunction+in+critically+ill+children.">Endothelial cell function and dysfunction in critically ill children.</a> Pediatrics. 140 (1), (2017).</li><li>Nasdala, I., Wolburg-Buchholz, K., Wolburg, H., 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+transmembrane+tight+junction+protein+selectively+expressed+on+endothelial+cells+and+platelets.">A transmembrane tight junction protein selectively expressed on endothelial cells and platelets.</a> Journal of Biological Chemistry. 277 (18), 16294-16303 (2002).</li><li>Sano, H., Sano, Y., Ishiguchi, E., 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=Establishment+of+a+new+conditionally+immortalized+human+skeletal+muscle+microvascular+endothelial+cell+line.">Establishment of a new conditionally immortalized human skeletal muscle microvascular endothelial cell line.</a> Journal of Cellular Physiology. 232 (12), 3286-3295 (2017).</li><li>Kappos, L., Bates, D., Edan, 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=Natalizumab+treatment+for+multiple+sclerosis:+Updated+recommendations+for+patient+selection+and+monitoring.">Natalizumab treatment for multiple sclerosis: Updated recommendations for patient selection and monitoring.</a> Lancet Neurology. 10 (8), 745-758 (2011).</li><li>Birbrair, A., Zhang, T., Wang, Z. 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=Skeletal+muscle+pericyte+subtypes+differ+in+their+differentiation+potential.">Skeletal muscle pericyte subtypes differ in their differentiation potential.</a> Stem Cell Research. 10 (1), 67-84 (2013).</li><li>Ruck, T., Bittner, S., Epping, L., Herrmann, A. M., Meuth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+murine+brain+microvascular+endothelial+cells.">Isolation of primary murine brain microvascular endothelial cells.</a> Journal of Visualized Experiments. (93), e52204 (2014).</li><li>Hwang, I., An, B. S., Yang, H., Kang, H. S., Jung, E. M., Jeung, 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=Tissue-specific+expression+of+occludin,+zona+occludens-1,+and+junction+adhesion+molecule+A+in+the+duodenum,+ileum,+colon,+kidney,+liver,+lung,+brain,+and+skeletal+muscle+of+C57BL+mice.">Tissue-specific expression of occludin, zona occludens-1, and junction adhesion molecule A in the duodenum, ileum, colon, kidney, liver, lung, brain, and skeletal muscle of C57BL mice.</a> Journal of Physiology and Pharmacology. 64 (1), 11-18 (2013).</li><li>Frye, C. A., Patrick, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+microvascular+endothelial+cells.">Isolation and culture of rat microvascular endothelial cells.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 38 (4), 208-212 (2002).</li><li>Le, G. Y., Essackjee, H. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+microvascular+endothelial+cells.">Isolation of microvascular endothelial cells.</a> Bio-protocol. 8 (12), (2018).</li><li>Ieronimakis, N., Balasundaram, G., Reyes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation,+culture+and+transplant+of+mouse+skeletal+muscle+derived+endothelial+cells+with+angiogenic+potential.">Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential.</a> PLoS One. 3 (3), e0001753 (2008).</li><li>Pham, M. T., Pollock, K. M., Rose, M. 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The authors declare no competing financial interests.

Microvascular endothelial cells provide barrier functions in all tissues and their dysfunction results in disease of the associated organs 3. Moreover, organ-specific studies of microvascular EC could pave the way for new therapeutic strategies. Therefore, a deeper understanding of microvascular EC function under physiological and pathophysiological conditions is of great scientific interest. Modulation of leukocyte/endothelium interaction is successfully used to treat multiple sclerosis patients ...

Via bloodstream, cells and organs are supplied with oxygen, substrates and other necessary molecules. This interchange takes place in capillaries, the smallest vessels. Capillaries are formed by an inner endothelial cell (EC) layer whose integrity remains a prerequisite to successful regulation of muscle homeostasis between the intravascular and interstitial space. To ensure a selective transition of soluble factors and cells, EC constitute a monolayer interconnected by tight and adherens junctions 1. Besides its role as barrier for nutrients or metabolic products, EC regulate the recruitment of leukocytes in inflammatory processes. Inflammation or tissue damage leads to an up-regulation of adhesion molecules on the EC surface and production of chemokines facilitating leukocyte attachment and transmigration into the target tissue 2. Consequently, EC are critically involved in the regulation of inflammatory processes such as the defense against pathogens or tissue repair.
A dysfunction of EC is directly associated with vascular diseases, chronic kidney failure, venous thrombosis severe pathogen infections. Furthermore, EC are virtually always involved in organ-specific autoimmunity such as diabetes mellitus or multiple sclerosis 3. The barrier function between blood stream and organs is therefore controlled by a concerted interplay of different cell types. In the skeletal muscle microvascular endothelial cells (MMEC) together with muscle cells, fibroblasts and pericytes form a functional unit, the &#34;myovascular unit&#34; (MVU). Therefore, a dysfunction of the MVU might play a critical role in the pathophysiology of myopathies. However, a deeper understanding of these regulatory mechanisms is still missing and currently precludes the identification of new, urgently needed, therapeutic targets in myopathies.
To investigate the complex physiological and pathophysiological mechanisms, animal models are commonly used. However, in vitro models offer the advantage to focus on the subject of interest by excluding a variety of confounding factors. To investigate processes in vitro it is necessary to isolate pure and viable primary cells. In contrast to cell lines, primary cells isolated from transgenic animals enable to investigate the consequences of genetic modifications in vitro.
Here, a method to isolate primary murine MMEC is described by using mechanical and enzymatic dissociation followed by magnetic activated cell sorting techniques (MCS) for purification. For this purpose, magnetic beads against specific surface markers are used. Platelet endothelial cell adhesion molecule-1 (PECAM1, CD31) is mainly expressed on EC and can be used to enrich this cell type. To warrant high cell purity, cells of hematopoietic origin are excluded by a negative selection for protein tyrosine phosphatase receptor type C (PTPRC, CD45). Further, quality controls, cultivation of primary murine MMEC, potential applications and limitations as well as special considerations are presented.

<table>
	<tbody>
		<tr>
			<td>0,25% Trypsin-EDTA</td>
			<td>Thermo Fisher</td>
			<td>25200-056</td>
			<td>ready to use</td>
		</tr>
		<tr>
			<td>ACK buffer</td>
			<td></td>
			<td></td>
			<td>150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in water at a pH of 7.3</td>
		</tr>
		<tr>
			<td>Anti-mouse CD31-FITC (clone MEC13.3)</td>
			<td>Biolegend</td>
			<td>102506</td>
			<td>Isotype control: FITC Rat IgG2a, &#954; Isotype Ctrl</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45-PE (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103106</td>
			<td>Isotype control: PE Rat IgG2b, &#954; Isotype Ctrl</td>
		</tr>
		<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>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 MicroBeads mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-418</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase-Dispase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td>Collagenase from V. alginolyticus, Dispase from B. polymyxa</td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated Multiple 6-Well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated anti-rat IgG antibody</td>
			<td>dianova</td>
			<td>712-166-153</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (ProLong Gold antifade reagent with DAPI)</td>
			<td>Thermo Fisher</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Desoxyribonuclease</td>
			<td>Sigma Aldrich</td>
			<td>D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas</td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonat treated water</td>
			<td>Thermo Fisher</td>
			<td>AM9916</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, containing Glutamin Supplement and pyruvate</td>
			<td>Thermo Fisher</td>
			<td>31966-021</td>
			<td>warm up to 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>dNTP Mix (10 mM)</td>
			<td>Thermo Fisher</td>
			<td>R0192</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Sarstedt</td>
			<td>551,579</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 70 &#956;m Cell Strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>FC buffer</td>
			<td></td>
			<td></td>
			<td>0.1% BSA, 0.2% NaN3, 2 mM EDTA</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Fetal calf serum</td>
		</tr>
		<tr>
			<td>Fixable Viability Dye eFluor780</td>
			<td>Thermo Fisher</td>
			<td>65-0865-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (serrated, straight, 12 cm)</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (serrated, straight, 12 cm)</td>
			<td>Fine Science Tools</td>
			<td>11009-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe 100 Solo 1ml (Omnifix)</td>
			<td>Braun</td>
			<td>9161708V</td>
			<td></td>
		</tr>
		<tr>
			<td>large magnetiv columns (LS columns)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>for CD45-MACS-step</td>
		</tr>
		<tr>
			<td>MCS buffer</td>
			<td></td>
			<td></td>
			<td>0.5% BSA, 2 mM EDTA in PBS at a pH of 7.2</td>
		</tr>
		<tr>
			<td>Medium magnetic column (MS column)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>for CD31-MACS-step</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Thermo Fisher</td>
			<td>R0581</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Phosphate buffered saline, ready to use</td>
		</tr>
		<tr>
			<td>PCR buffer (5x)</td>
			<td>Thermo Fisher</td>
			<td>EP0742</td>
			<td>in a kit with the reverse transcriptase</td>
		</tr>
		<tr>
			<td>Pecam1 rat &#945;-mouse</td>
			<td>SantaCruz</td>
			<td>Sc-52713</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>primary murine muscle cells</td>
			<td>celprogen</td>
			<td>66066-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer Cdh15 (M-Cadherin)</td>
			<td>Thermo Fisher</td>
			<td>Mm00483191_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Cldn5 (claudin-5)</td>
			<td>Thermo Fisher</td>
			<td>Mm00727012_s1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Ocln (occludin)</td>
			<td>Thermo Fisher</td>
			<td>Mm00500912_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Pax-7</td>
			<td>Thermo Fisher</td>
			<td>Mm01354484_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer Tjp-1 (Zonula occludens 1)</td>
			<td>Thermo Fisher</td>
			<td>Mm00493699_m1</td>
			<td>FAM labeled</td>
		</tr>
		<tr>
			<td>Primer 18s rRNA (Eukaryotic endogenous control)</td>
			<td>Thermo Fisher</td>
			<td>4310893E</td>
			<td>VIC labeled</td>
		</tr>
		<tr>
			<td>qPCR buffer (Maxima Probe/ROX qPCR Master Mix (2X)</td>
			<td>Thermo Fisher</td>
			<td>K0231</td>
			<td>2 x 1,25 mL; for 200 reactions each</td>
		</tr>
		<tr>
			<td>Random mixture of single-stranded primer</td>
			<td>Thermo Fisher</td>
			<td>SO142</td>
			<td>Random Hexamer Primer</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase (200 U/&#956;L) + PCR buffer (5x)</td>
			<td>Thermo Fisher</td>
			<td>EP0742</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase Inhibitor (40 U/&#956;L)</td>
			<td>Thermo Fisher</td>
			<td>EO0381</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor (cutting edge 23 mm, sharp/sharp) )</td>
			<td>Fine Science Tools</td>
			<td>14088-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissor (cutting edge 42 mm, sharp/blunt)</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Speed Coating solution</td>
			<td>PeloBiotech</td>
			<td>PB-LU-000-0002-00</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

One day after isolation, primary murine MMEC and residual other cells form conglomerates and adhere to the bottom of culture dishes (Figure 1A day 1). From day 7 on, flat and elongated cells can be observed. However, contamination of other, mostly spheroid cells, is still visible (Figure 1A day 7). Thus, another cycle of CD31 positive selection via MCS is required. Hereafter, primary murine MMEC proliferate to a density ...

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

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

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-primary-murine-skeletal-muscle-microvascular-endothelial

Research

JoVE Journal

Neuroscience

10.0K Views.  University of Muenster. 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 "myovascular unit".

Video: Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

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 Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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 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 ...

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 ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

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.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...