Name: isolation of primary patient-specific aortic smooth muscle cells and semiquantitative real-time contraction measurements in vitro
Uploaded: 2022-02-15T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro at JoVE.com

We would like to gratefully acknowledge Tara van Merrienboer, Albert van Wijk, Jolanda van der Velden, Jan D. Blankensteijn, Lan Tran, Peter L. Hordijk, the PAREL-AAA team, and all vascular surgeons of the Amsterdam UMC, Zaans Medisch Centrum, and Dijklander hospital for providing materials and support for this study.

NOTE: Aortic biopsies were obtained during open aneurysm repair in Amsterdam University Medical Centers, VU University Medical center, Amsterdam, Zaans Medisch Centrum, Zaandam and Dijklander hospital, Hoorn, the Netherlands. Control aortic tissue was obtained from the piece of the aorta attached to the renal artery harvested for kidney transplants. Only patients above the age of 18 were included, and all patients gave their informed consent to participate in the study. All material was collected in accordance with the r...

<ol>
	<li>Milewicz, D. 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=Genetic+basis+of+thoracic+aortic+aneurysms+and+dissections:+focus+on+smooth+muscle+cell+contractile+dysfunction.">Genetic basis of thoracic aortic aneurysms and dissections: focus on smooth muscle cell contractile dysfunction.</a> Annual Review of Genomics and Human Genetics. 9, 283-302 (2008).</li><li>Milewicz, D. 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=Altered+smooth+muscle+cell+force+generation+as+a+driver+of+thoracic+aortic+aneurysms+and+dissections.">Altered smooth muscle cell force generation as a driver of thoracic aortic aneurysms and dissections.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (1), 26-34 (2017).</li><li>Groeneveld, M. 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=Betaglycan+(TGFBR3)+up-regulation+correlates+with+increased+TGF-&#946;+signaling+in+Marfan+patient+fibroblasts+in+vitro.">Betaglycan (TGFBR3) up-regulation correlates with increased TGF-&#946; signaling in Marfan patient fibroblasts in vitro.</a> Cardiovascular Pathology. 32, 44-49 (2018).</li><li>Chen, J., Li, H., SundarRaj, N., Wang, J. H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-smooth+muscle+actin+expression+enhances+cell+traction+force.">Alpha-smooth muscle actin expression enhances cell traction force.</a> Cell Motility and the Cytoskeleton. 64 (4), 248-257 (2007).</li><li>Peyton, S. R., Putnam, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+governs+smooth+muscle+cell+motility+in+a+biphasic+fashion.">Extracellular matrix rigidity governs smooth muscle cell motility in a biphasic fashion.</a> Journal of Cellular Physiology. 204 (1), 198-209 (2005).</li><li>Williams, D. A., Fogarty, K. E., Tsien, R. Y., Fay, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+gradients+in+single+smooth+muscle+cells+revealed+by+the+digital+imaging+microscope+using+Fura-2.">Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2.</a> Nature. 318 (6046), 558-561 (1985).</li><li>Wu, 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=NLRP3+(nucleotide+oligomerization+domain-like+receptor+family,+pyrin+domain+containing+3)-caspase-1+inflammasome+degrades+contractile+proteins:+implications+for+aortic+biomechanical+dysfunction+and+aneurysm+and+dissection+formation.">NLRP3 (nucleotide oligomerization domain-like receptor family, pyrin domain containing 3)-caspase-1 inflammasome degrades contractile proteins: implications for aortic biomechanical dysfunction and aneurysm and dissection formation.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (4), 694-706 (2017).</li><li>Bogunovic, N., 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=Impaired+smooth+muscle+cell+contractility+as+a+novel+concept+of+abdominal+aortic+aneurysm+pathophysiology.">Impaired smooth muscle cell contractility as a novel concept of abdominal aortic aneurysm pathophysiology.</a> Scientific Reports. 9 (1), 1-14 (2019).</li><li>Hurst, V., Goldberg, P. L., Minnear, F. L., Heimark, R. L., Vincent, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearrangement+of+adherens+junctions+by+transforming+growth+factor-&#946;1:+role+of+contraction.">Rearrangement of adherens junctions by transforming growth factor-&#946;1: role of contraction.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 276 (4), 582-595 (1999).</li><li>Hu, N., 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=Comparison+between+ECIS+and+LAPS+for+establishing+a+cardiomyocyte-based+biosensor.">Comparison between ECIS and LAPS for establishing a cardiomyocyte-based biosensor.</a> Sensors and Actuators B: Chemical. 185, 238-244 (2013).</li><li>Peters, M. F., Lamore, S. D., Guo, L., Scott, C. W., Kolaja, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell-derived+cardiomyocytes+in+cellular+impedance+assays:+bringing+cardiotoxicity+screening+to+the+front+line.">Human stem cell-derived cardiomyocytes in cellular impedance assays: bringing cardiotoxicity screening to the front line.</a> Cardiovascular Toxicology. 15 (2), 127-139 (2015).</li><li>Zhang, S., Yang, Y., Kone, B. C., Allen, J. C., Kahn, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-stimulated+cyclic+guanosine+monophosphate+inhibits+vascular+smooth+muscle+cell+migration+by+inhibiting+Ca/calmodulin-dependent+protein+kinase+II.">Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/calmodulin-dependent protein kinase II.</a> Circulation. 107 (11), 1539-1544 (2003).</li><li>Halterman, J. A., Kwon, H. M., Zargham, R., Bortz, P. D. S., Wamhoff, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+factor+of+activated+T+cells+5+regulates+vascular+smooth+muscle+cell+phenotypic+modulation.">Nuclear factor of activated T cells 5 regulates vascular smooth muscle cell phenotypic modulation.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 31 (10), 2287-2296 (2011).</li><li>Bass, H. M., Beard, R. S., Cha, B. J., Yuan, S. Y., Nelson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombomodulin+induces+a+quiescent+phenotype+and+inhibits+migration+in+vascular+smooth+muscle+cells+in+vitro.">Thrombomodulin induces a quiescent phenotype and inhibits migration in vascular smooth muscle cells in vitro.</a> Annals of Vascular Surgery. 30, 149-156 (2016).</li><li>Burger, J., 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=Molecular+phenotyping+and+functional+assessment+of+smooth+muscle+like-cells+with+pathogenic+variants+in+aneurysm+genes+ACTA2,+MYH11,+SMAD3+and+FBN1.">Molecular phenotyping and functional assessment of smooth muscle like-cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.</a> Human Molecular Genetics. , (2021).</li><li>Yeung, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transdifferentiation+of+human+dermal+fibroblasts+to+smooth+muscle-like+cells+to+study+the+effect+of+MYH11+and+ACTA2+mutations+in+aortic+aneurysms.">Transdifferentiation of human dermal fibroblasts to smooth muscle-like cells to study the effect of MYH11 and ACTA2 mutations in aortic aneurysms.</a> Human Mutation. 38 (4), 439-450 (2017).</li></ol>

This paper presents a method to measure SMC contraction in vitro, based on the changes in impedance and surface occupation. First, the isolation, culturing, and expansion of patient-specific primary human SMCs and skin fibroblasts is described, followed by how to use them for contraction measurements. 
A limitation of the study is related to obtaining the cells through an explant protocol. The cells that proliferate from the biopsy might have different properties than the original tis...

Smooth muscle cells (SMCs) are the predominant cell type in the aortic medial layer, the thickest layer of the aorta. Within the wall, they are radially oriented and are involved in, among other functions, vasoconstriction and vasodilation1. The SMC contractile machinery is involved in the transmission of force in the aorta through the functional link with the extracellular matrix2. Mutations in genes encoding for the proteins of the SMC contractile apparatus, such as smooth muscle myosin heavy chain (MYH11) and smooth muscle actin (ACTA2), have been related to cases of familial thoracic aortic aneurysms, underscoring the relevance of SMC contraction in maintaining the structural and functional integrity of the aorta1,2. Furthermore, mutations in the TGF&#946; signaling pathway are also associated with aortic aneurysms, and their effects in aortic aneurysm pathophysiology can also be studied in skin fibroblasts3.
High-throughput measurement of SMC contraction in vitro is challenging. As SMC contractility cannot be measured in vivo in humans, in vitro assays on human cells present a feasible alternative. Moreover, abdominal aortic aneurysm (AAA) development in animal models is either chemically induced by, for example, elastase perfusion, or caused by a specific mutation. Therefore, animal data are not comparable to AAA development in humans, which mostly has a multifactorial cause, such as smoking, age, and/or atherosclerosis. In vitro SMC contractility has so far been mainly measured by traction force microscopy4,5, quantification of Fura-2 fluorescence intracellular calcium fluxes6, and collagen wrinkling assays7. While traction force microscopy provides invaluable numeric insight into the forces generated by a single cell, it is not suitable for high-throughput screening due to the complex mathematical data processing and the analysis of one cell at a time, meaning that it is very time-consuming to measure a representative number of cells per donor. Fura-2 dye and collagen wrinkling assays allow the superficial determination of contraction and do not give a precise numerical output, making them less suitable for discriminating patient-specific differences. Impaired SMC contraction in cells derived from the aorta of abdominal aortic aneurysm patients was demonstrated for the first time by optimizing a novel method for measuring SMC contraction in vitro8. This was done by repurposing the electric cell-substrate impedance sensing (ECIS) method. ECIS is a real-time, medium-throughput assay for the quantification of adherent cell behavior and contraction9,10,11 such as SMC growth and behavior in wound-healing and migration assays12,13,14. The exact method is described in the protocol section. In this optimized way, the ECIS can also be used to study fibroblast contraction due to their similar size and morphology.
The aim of this paper is to provide a stepwise description of the method for measuring SMC contraction in vitro using ECIS8 and comparing the contraction between control and patient SMCs. First, the isolation and culturing of primary SMCs from control and patient aortic biopsies is explained, which can be used for contraction measurement. Second, contraction measurements and analysis, alongside the verification of SMC marker expression, are described. Furthermore, this paper describes the method for the isolation of patient-specific dermal fibroblasts whose contraction can be measured using the same methodology. These cells can be used for patient-specific studies focused on aortic aneurysm or other cardiovascular pathologies15 or prognostic studies using a transdifferentiation protocol that allows contraction measurement prior to aneurysm surgery16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well Array</td>
			<td>Applied Biophysics</td>
			<td>96W10idf PET</td>
			<td>Array used to measure contraction in the ECIS setup</td>
		</tr>
		<tr>
			<td>Custodiol</td>
			<td>Dr. Franz H&#246;hler Chemie GmbH</td>
			<td>RVG 12801</td>
			<td>Solution used to transfer tissue in from surgery room to laboratorium</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td>Solution used to dilute ionomycin</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>Addition to cell culture medium</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-10 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11550043</td>
			<td>Medium used to culture skin fibroblasts</td>
		</tr>
		<tr>
			<td>Human Vascular Smooth Muscle Cell Basal Medium (formerly &#39;&#39;Medium 231&#39;&#39;)</td>
			<td>Gibco</td>
			<td>M231500</td>
			<td>Medium used to culture smooth muscle cells</td>
		</tr>
		<tr>
			<td>Invitrogen countess II</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Ionomycin calcium salt from Streptomyces conglobatus</td>
			<td>Sigma-Aldrich</td>
			<td>I0634-1MG</td>
			<td>Compound used for contraction stimulation</td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Fresenius Kabi</td>
			<td>B230561</td>
			<td>Solution used to transfer tissue in from surgery room to laboratorium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Antibiotics used for cell culture medium</td>
		</tr>
		<tr>
			<td>Phospathe buffered saline</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Used to wash cells</td>
		</tr>
		<tr>
			<td>Quick-RNA Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>R1055</td>
			<td>Kit used for RNA isolation</td>
		</tr>
		<tr>
			<td>Smooth Muscle Growth Supplement (SMGS)</td>
			<td>Gibco</td>
			<td>S00725</td>
			<td>Supplement which is added to smooth muscle cell culture medium</td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>11754250</td>
			<td>Kit used for cDNA synthesis</td>
		</tr>
		<tr>
			<td>SYBR Green PCR Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4309155</td>
			<td>Reagent for qPCR</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Used to trypsinize cells</td>
		</tr>
		<tr>
			<td>ZTheta</td>
			<td>Applied Biophysics</td>
			<td>ZTheta</td>
			<td>ECIS instrument used for contraction measurements</td>
		</tr>
	</tbody>
</table>

isolation of primary patient-specific aortic smooth muscle cells and semiquantitative real-time contraction measurements in vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

To test the reproducibility of this method, the method was first validated using control SMCs only. To determine interexperimental measurement reproducibility, two independent measurements of all included control and patient cell lines were plotted as a Bland-Altman plot (Figure 3B). The plot demonstrated that this method does not show variability outside the confidence interval, except for one outlier cell line. Furthermore, these results demonstrated that two wells seeded within the same e...

Watch this Scientific Journal Video about Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro at JoVE.com

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

Research

JoVE Journal

Biology

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

Isolation of Human Umbilical Arterial Smooth Muscle Cells 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 and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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 ...

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 ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...