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.

<ol>
	<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. C., Ballard, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+adenosine+formation+and+release+by+freshly-isolated+vascular+endothelial+cells+from+rat+skeletal+muscle:+Effects+of+hypoxia+and/or+acidosis.">Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: Effects of hypoxia and/or acidosis.</a> Biochemical and Biophysical Research Communications. 450 (1), 93-98 (2014).</li><li>Cheung, K. C., Marelli-Berg, F. 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. D., 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=Generation+of+human+vascularized+brain+organoids.">Generation of human vascularized brain organoids.</a> Neuroreport. 29 (7), 588-593 (2018).</li></ol>

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 NH&lt;sub&gt;4&lt;/sub&gt;Cl, 10 mM KHCO&lt;sub&gt;3&lt;/sub&gt;, 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, &amp;kappa; 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, &amp;kappa; 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 &lt;em&gt;V. alginolyticus&lt;/em&gt;, Dispase from &lt;em&gt;B. polymyxa&lt;/em&gt;</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 &amp;deg;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 &amp;mu;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% NaN&lt;sub&gt;3&lt;/sub&gt;, 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 1 mL (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 &amp;alpha;-mouse</td><td>SantaCruz</td><td>Sc-52713</td><td>100 &amp;micro;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/&amp;mu;L) + PCR buffer (5x)</td><td>Thermo Fisher</td><td>EP0742</td><td></td></tr><tr><td>Rnase Inhibitor (40 U/&amp;mu;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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.1K 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

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Developmental Biology

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 of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biology

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Primary Mouse Lung Endothelial Cells

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

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

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

Immunology and Infection

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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 Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...