Krankenhaus Amato Lusitano, Castelo Branco Portugal. Krankenhaus Sousa Martins, Guarda Portugal.

<ol><li> Arterien sind von UC Stücke von 3-7 cm erreicht. </li><li> Wharton Sulze, dass die Arterien umgibt wurde sorgfältig durch Schneiden mit der Schere entfernt. </li><li> A blunt-end Nadel aus einem äußeren Durchmesser von 0,6-1mm war ca. 7mm von einem Ende des seziert Arterien eingesetzt. </li><li> So entfernen Sie das verbleibende Blut in den Gefäßen sind die Arterien senkrecht gehalten und perfundierten mit etwa 20 bis 40 mL, physiologischer Kochsalzlösung (PSS), mit einem 20-ml-Spritze verbunden mit einer Nadel. Wenn nötig, wurde dieser Waschschritt für mehrere Male wiederholt. </li><li> Das gleiche Verfahren wurde wiederholt mit RPMI 1640 mit 10% fötalem Rinderserum (FBS) ergänzt. </li><li> Ein Ende des Blutgefäßes geschlossen wurde. Ein 10-ml-Spritze wurde mit der Nadel verbunden und perfundierten mit 3 ml Kollagenase Typ I (800 Einheiten / ml in gepufferten Salzlösung Hanks Solution (HBSS)). Dann werden die anderen extremityof das Schiff war ebenfalls geschlossen. </li><li> Das Schiff wurde inkubiert mit Phosphat-gepufferter Kochsalzlösung (PBS) für 15 min bei 37 ° C unter Rühren. </li><li> Nach dem Ende des Schiffes wurden geschnitten, um den freien SMCs sammeln. Die Arterien wurden perfuse mit etwa 20 bis 40 ml DMEM-F12 mit 5% FBS und Antibiotika, um eine 50-ml-Röhrchen ergänzt. </li><li> Das Rohr wurde bei Raumtemperatur (250 g, 8 min) zentrifugiert und Zellpellet wurde in 5 ml DMEM-F12 mit 5% FBS resuspendiert. </li><li> Die Zellsuspension wurde zu einer Kollagen-beschichteten T-Kolben überführt und bei 37 ° C unter einer 5% CO 2 befeuchteten Atmosphäre. </li><li> Nach 5 16 h, die nicht haftenden Zellen und zelluläre drebis wurden entfernt, 5 ml Kulturmedium zugegeben und die Zellen wurden bei 37 ° C unter einer 5% CO 2 befeuchteten Atmosphäre. </li><li> Die Zellkulturmedien wurde alle 2 3 Tage, bis SMCs Konfluenz erreichten. </li><li> Nachdem die Zellen erreicht Zusammenfluss wurden sie in zwei oder drei 25 cm 2 T-Flaschen ausgesät. </li></ol> Cleanup <ol><li> Entsorgen von Nadeln in Sharps Container. </li><li> Entsorgen von Spritzen in großen Behälter für biologischen. </li><li> Legen Sie alle Gewebe in einen kleinen Beutel für biologisches Risikomaterial. Schließen Sie die Tasche und bei -20 ° Celsius bis Verbrennung. </li><li> Weichen Sie alle Instrumente in viruzid für mindestens 10 min. </li><li> Nicht verwendete Medien Kühlschrank. </li><li> Entsorgen von Handschuhen in biohazard Abfall. </li></ol>

<ol>
	<li>Van Rijen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+in+human+umbilical+cord+endothelial+cells+contain+multiple+connexins.">Gap junctions in human umbilical cord endothelial cells contain multiple connexins.</a> The American journal of physiology. 272 (1 PT 1), C117-C117 (1997).</li><li>Mart&#237;n de Llano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+to+consistently+obtain+endothelial+and+smooth+muscle+cell+cultures+from+umbilical+cord+vessels.">Procedure to consistently obtain endothelial and smooth muscle cell cultures from umbilical cord vessels.</a> Translational Research. 149 (1), 1-9 (2007).</li><li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+endothelial+cells+derived+from+umbilical+veins.+Identification+by+morphologic+and+immunologic+criteria.">Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52 (11), 2745-2756 (1973).</li><li>Cairr&#227;o, E., Santos-Silva, A., Alvarez, E., Correia, I., Verde, I. <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+human+umbilical+artery+smooth+muscle+cells+expressing+functional+calcium+channels.">Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels.</a> In Vitro Cellular &#38; Developmental Biology-Animal. 45 (3), 175-184 (2009).</li></ol>

Die SMC kann als Modell für zukünftige Tests mit constrictor Drogen verwendet werden, zu isolieren und strukturell charakterisieren L-Typ Calcium-Kanälen, um zelluläre und molekulare Aspekte der vaskulären Funktion zu untersuchen und sie in das Tissue Engineering verwenden. SMCs und ECs sind für die Entwicklung von neuen Biomaterialien, die in die Produktion von Halbzeugen synthetische Blutgefäße beim Menschen eingesetzt werden eingesetzt werden können fundamental.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>095K8311</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>Biochrom-AG</td>
<td>0117T</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>3103806</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>067K2302</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 &mu;L/ 1 mL of medium</td>
</tr>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Sterilized</td>
</tr>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diameter of 0.6-1mm</td>
</tr>
<tr>
<td>Syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 - 20 mL</td>
</tr>
<tr>
<td>Physiological Saline Solution (PSS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>20-40 mL per artery</td>
</tr>
<tr>
<td>Collagenase type I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>047K1052</td>
<td>3 mL per artery</td>
</tr>
<tr>
<td>Hank's Buffered Salt Solution(HBSS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diluted collagenase </td>
</tr>
<tr>
<td>Phosphate buffered saline solution (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Warmed to 37&deg;C</td>
</tr>		</tbody>
		</table>
		</div>

isolation of human umbilical arterial smooth muscle cells (huasmc)

Die menschliche Nabelschnur (UC) ist eine biologische Probe, die einfach nur nach der Geburt erreicht werden kann. Diese biologische Probe, die meiste Zeit, verworfen, und ihre Erhebung bedeutet keine zusätzliche Gefahr für das Neugeborene oder die Mutter die Gesundheit. Außerdem keine ethischen Bedenken erhoben werden. Der UC wird von einer Vene und zwei Arterien, von denen beide Endothelzellen (ECs) zusammen<sup&gt; 1</sup&gt; Und glatten Muskelzellen (SMC)<sup&gt; 2</sup&gt; Lassen sich zwei der wichtigsten zellulären Komponenten der Blutgefäße, isoliert werden. In diesem Projekt werden die SMCs wurden nach enzymatischer Behandlung der UC Arterien entsprechend den experimentellen Verfahren zuvor von Jaffe beschrieben, erhalten<em&gt; Et al</em<sup&gt; 3</sup&gt;. Nach Zellisolation waren sie in t-Blitz mit DMEM-F12 mit 5% fötalem Rinderserum und wurden für mehrere Passagen kultiviert gehalten. Zellen behielten ihre morphologischen und andere phänotypische Merkmale in den verschiedenen Generationen. Das Ziel dieser Studie war es, Zellen der glatten Muskulatur zu isolieren, um sie als Modelle für zukünftige Tests mit constrictor Drogen, zu isolieren und strukturell charakterisieren L-Typ Calcium-Kanälen, um zelluläre und molekulare Aspekte der vaskulären Funktion zu untersuchen<sup&gt; 4</sup&gt; Und sie in das Tissue Engineering verwenden.

Watch this Scientific Journal Video about Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC at JoVE.com

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

Die Nabelschnur werden verwendet, um Zellen der glatten Muskulatur durch verschiedene Arten zu isolieren. In dieser Arbeit verwendet die enzymatische Behandlung zu isolierten glatten Muskelzellen.

Isolierung humaner Nabelschnur arteriellen glatten Muskelzellen (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

isolation-of-human-umbilical-arterial-smooth-muscle-cells-huasmc

Research

JoVE Journal

Biology

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

13.9K Aufrufe.  Universidade da Beira Interior. Die Nabelschnur werden verwendet, um Zellen der glatten Muskulatur durch verschiedene Arten zu isolieren. In dieser Arbeit verwendet die enzymatische Behandlung zu isolierten glatten Muskelzellen.

Serie: Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolierung humaner Nabelschnur-Endothelzellen (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Forschung

Biologie

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca 2 + Transient-Erkennung in der glatten Muskulatur

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Exploring arteriellen glatten Muskelzellen KV7 Kalium Kanal Funktion mit Patch-Clamp-Elektrophysiologie und Druck Myographie

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

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;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

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

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Isolation der Lungenarterie glatten Muskelzellen aus neugeborenen Mäusen

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Zwei-Photonen-Imaging von intrazellulären Ca<sup&gt; 2+</sup&gt; Transport- und Stickoxid-Produktion in Endothelzellen und glatte Muskelzellen eines isolierten Rattenaorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Die Messung von Calcium Schwankungen innerhalb des Retikulum von kultivierten glatten Muskelzellen Mit FRET-basierten konfokale Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

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.

Die Isolierung von murinen Coronary glatten Gefäßmuskelzellen

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

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 &#176;C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

Vorbereitung und Nutzung von frisch isolierten menschlichen Detrusor glatte Muskelzellen für die Charakterisierung von 9-Phenanthrol-Sensitive KationStröme

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...