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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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 with natalizumab, an antibody which prevents lymphocyte adhesion and thereby transmigration 6. However, various myopathies including the inflammatory subtypes still remain only poorly treatable to date. Therefore, elucidation of EC properties specifically in skeletal muscle endothelium could help to expand available treatments to myopathy or result in novel therapeutic approaches.
Immortalized endothelial cell lines (IECL) have been established and used for several in vitro models. These cell lines offer some advantages compared to primary microvascular EC due to immortalization and fast proliferation. Furthermore, primary cultured cells can undergo senescence and lose or change their morphology or physiological function after some time or passages. However, IECL only maintain some endothelial cell properties and cannot resemble the variety of different organ-borne primary microvascular EC. Of note, they are rarely derived from skeletal muscles. To date, only a single publication describes a human skeletal microvascular endothelial cell line named TSM15 5. In contrast, murine microvascular EC have not been described so far. Finally, primary cells from transgenic animals provide the opportunity to study genetic modifications in vitro. However, primary cell cultures also hold certain pitfalls. First, contamination with cells of no interest can interfere with the validity of experimental findings. Therefore, the accuracy of several selection steps and a high purity of isolated cells must be guaranteed. Cell suspension after dissociation of muscle tissue contains erythrocytes and different mononuclear cell types such as immune cells, satellite cells, pericytes, fibroblasts and endothelial cells 7. Therefore, cell fractions after the CD31 MCS step were tested for markers exclusively expressed by muscle satellite cells. As expected CD45- CD31- fraction and primary muscle cells showed Pax-7 and Cdh15 expression, whereas CD45- CD31+ and cultivated primary murine MMEC were negative for these markers after a second CD31 MCS step. Furthermore a quality control of isolated or cultured cells is inevitably to guarantee reliable and reproducible experiments. Several methods for quality control of primary murine MMEC are available: besides microscopy of morphological properties, flow cytometry, PCR and immunofluorescence stainings for characteristic EC markers (e.g. CD31, Sca-1) can be used. Purity assessment of freshly isolated primary murine MMEC was only about 60-70%, whereas a viability of about 70% could be detected. Microscopy of these cells partially shows cell conglomerates, which might consist of contaminating cells such as fibroblasts or pericytes attached to EC. After 7 days mainly flat and elongated cells and spheroid cells can be observed. However, two CD31 MCS passages resulted in high viability and purity. Alternative approaches to increase purity such as puromycin-containing media failed in primary murine MMEC while being effective in murine brain microvascular EC 8.
Since microvascular EC derived from capillaries express tight junction proteins, gene expression of Cld5, Ocln and Tjp1 in primary murine MMEC compared to differentiated muscle cells was examined. As expected all tight junction molecules were detected in primary murine MMEC. In comparison, muscle cells demonstrated only low expression of Tjp1, whereas no expression of Cld5 and Ocln could be determined. Similar expression patterns were previously demonstrated in whole murine skeletal muscle. Here, an expression on gene and protein level could be detected for Tjp1 but not for occludin 9. Further functional features such as transendothelial resistance could be measured.
The herein described protocol features only the isolation of microvascular EC from murine muscle tissue. However, it might be transferred to various other species as for example a similar protocol was published for rat microvascular EC derived from epididymal fat pads. Here, CD31 positive selection via MCS was preceded by density gradient centrifugation 10. Similar approaches were successful in macrovascular EC isolation from rat femoral arteries 11. A recently published method to isolate microvascular EC of heart and lung tissue uses CD31 and endoglin (CD105) as antigens for magnetic cell sorting 12. However, the purity of EC could not further be increased by using additional CD105 magnetic bead separation. Another possibility to isolate and purify primary murine MMEC is multicolor fluorescence activated cell sorting (FACS). A distinct population of Sca-1+, CD31+, CD34dim and CD45- cells from murine muscles could be isolated and was characterized as primary murine MMEC 13. An advantage of this method is a pure EC population which can be directly used or cultured for further experiments (Note that in our experience FACS leads to increased cell death due to higher cell stress). Further, dead cells or conglomerates of different cell types can significantly reduce the isolated cell numbers by FACS. Additionally, it is reported that primary murine MMEC isolated by FACS were unsuccessfully cultured at commonly used culture conditions of 21% O2 and 5% CO2. Here, the oxygen level had to be adjusted to 5% for a sufficient cultivation 13. Moreover, the FACS technique is more complex, time-consuming and the infrastructural prerequisites are expensive.
The musculus quadriceps femoris and the musculus triceps surae of male mice in the age of 4-12 weeks are most suitable for isolation of primary murine MEC as they are easily accessible and large enough for isolation of sufficient cell numbers (5 - 10 x 105 per gram muscle). Therefore, it must be ensured that conditions are comparable in independent experiments. As described above, primary cells can undergo senescence. Hence, use cells for respective experiments promptly in lower passages. After passage 8&#8211;10 primary murine MMEC change their morphology, demonstrate slow proliferation rates and lose their contact inhibition as signs for dedifferentiation and senescence.
Further there are some notes for troubleshooting for this protocol. Sterile working is essential to avoid contaminations. Quality controls are necessary to monitor purity and viability of isolated cells. Used materials in this protocol must be stored and applied according to manufacturer&#39;s instructions. Additional, age and sex of used animals, as well as different muscle groups might influence the quality and comparability of isolated cells in experiments.
The described protocol should be considered as platform method to isolate primary microvascular EC of skeletal muscles. Isolated cells can be used for several applications to gain further insights into blood-muscle-barrier function. Cells are suitable for both protein and RNA expression studies as well as functional assays (including transmigration and adhesion studies). However, this protocol was optimized for studies focusing on inflammatory processes and hence slight modifications might be necessary with respect to different research areas.
To model the complex spatial and functional cellular interactions within the MVU, primary murine MMEC can be cultivated in more complex 3D cell culture systems together with other cell types. Prospectively, it should also be possible to generate MVU organoids as it has been described for other tissues before 14. However, successful isolation of primary murine MMEC is inevitable for aiming at this next step.

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 of approximately 80&#8211;90%. Upon confluence they typically form a non-overlapping monolayer of longitudinally aligned cells (Figure 1A day 14). Proliferation stops upon confluence due to contact-inhibition. After 14 days about 5&#8211;10 x 105 cells can be used for further investigations.
Quality control via flow cytometry using fixable viability dye FVD780 (to stain for dead cells), PECAM1 (CD31) and PTPRC (CD45, as marker for cells of hematopoietic origin) showed values for both viability and purity ranging around 70% each for cells immediately after the isolation (Figure 2A). Cells cultivated after another CD31 positive selection via MCS, showed satisfying values for purity as well as for viability ranging up to 95% each (Figure 2B).
To evaluate the accuracy of the selection steps, obtained cells were further investigated for gene expression of the muscle satellite cell marker genes paired box protein 7 (Pax7) and M-cadherin (Cdh15) on mRNA level by quantitative PCR (qPCR). Primary murine MMEC (pmMMEC) and differentiated primary murine muscle cells (pmMC) were used as negative and positive control, respectively. Murine muscle cells were commercially purchased. As expected, only the CD45- CD31- fraction as well as the pmMC expressed Pax7 and Cdh15, whereas CD45- CD31+ and the primary murine MMEC were negative for these markers (Figure 2C).
EC derived from capillaries in the endomysium of skeletal muscle express tight junction proteins 4,5. By using qPCR, the expression of claudin-5 (Cld5), occludin (Ocln) and zonula occludens-1 (Tjp1 or ZO1) of confluent primary murine MMEC after the second CD31 MCS step was evaluated. PmMC were used as controls (Figure 2D). Primary murine MMEC expressed high levels of Cld5, Ocln and Tjp1, whereas pmMC only show low expression of Tjp1. Furthermore, immunofluorescence staining as quality control confirmed surface expression of the endothelium-specific marker PECAM1 in primary murine MMEC (Figure 2E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58901/58901fig1.jpg" /> 
Figure 1: Morphology of primary murine MMEC. (A) Representative image of cultured primary murine MMEC by phase contrast microscopy from days 1 to 14. d1 = Day 1, d7= Day 7, d14 = Day 14. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58901/58901fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58901/58901fig2v2.jpg" /> 
Figure 2: Quality control of primary murine MMEC. Gating strategy for flow cytometric analysis of primary murine MMEC immediately after isolation (A) and on day 15 (B) using (I) FSC/SSC, (II) FVD780 and (IIIa) CD45, (IIIb) CD31 (dashed lines = isotype control). (C) Expression level of Pax7 and M-Cadherin (Cdh15) in CD45- CD31-, CD45- CD31+ fractions after MCS isolation as well as in primary murine MMEC (pmMMEC) and primary murine muscle cells (pmMC). Expression levels are shown as &#916;Ct values (sample - 18S rRNA), n.d. = not determined. (D) Expression level of tight junction proteins claudin-5 (Cldn5), occludin (Ocln) or zonula occludens-1 (Tjp1 or ZO-1) of pmMMEC and pmMC, shown as &#916;Ct values. (E) Immunofluorescence staining for PECAM1 (red) in cultivated primary murine MMEC 24 h after second CD31 positive selection. Nuclear staining with DAPI-containing (blue) mounting medium; left = anti-PECAM1/DAPI, right: negative control/DAPI. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58901/58901fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Primary Murine 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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Nanyang Technological University

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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 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 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 ...

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 ...