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...

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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 ...

This method allows researchers to study the cell contraction of multiple patient cell line simultaneously. It allows us to study diseases quicker and more efficiently. This technique allows us to quantify cell contraction in real time.Within an hour, we can obtain the contraction values of dozens of cells. We demonstrated for the first time that muscle cell contraction is decreased in a group of patients with aortic aneurysms. This can be a new biomarker or target for medical therapy.Begin by sterilizing two pairs of surgical forceps and a scalpel by immersing them in 70%ethanol and subsequently wiping them dry. Then pipette two milliliters of SMC medium in a Petri dish in which the tissue dissection will be performed. Next pipette 2.5 milliliters of SMC medium into two T-25 flasks.Swirl the flasks around so that the small volume of medium covers the whole surface. Visually inspect the biopsy, identifying the three aortic layers by the presence of atherosclerotic plaques on the intima side and slimy connective tissue on the a adventitial side to distinguish the layers. To isolate SMCs from the media, remove the other two layers by placing the tissue with intima plaque side up first, then remove the solid plaque by pulling it away from the tissue with forceps while pushing the tissue down with the second pair of forceps until the pink uniform medial layer is visible.Now flip the tissue and repeat the same procedure by pulling off the adventitial layer, being sure to remove all visible parts in as many attempts as needed as this layer will not detach from the media easily. Once the media layer is isolated, cut the tissue into cubes by pressing the media down with forceps and cutting the tissue in one direction using the scalpel making clean unidirectional cuts to minimize damage. Place 10 to 20 of these tissue pieces in the upper quarter of the T-25 flak using forceps.After sterilizing two pairs of surgical forceps and a scalpel as described previously, pipette two milliliters of fibroblast medium in a Petri dish in which the tissue dissection will be performed. Then pipette 2.5 milliliters of fibroblast medium into two T-25 flasks and swirl the flasks around so that the small volume of medium covers the whole surface. Visually inspect the biopsy for identifying the epidermis with a recognizable skin surface, sometimes with visible hair, then look for yellow and slimy subcutaneous fat on the opposite side.The layer between the epidermis and the subcutaneous fat is the dermis, the source of viable fibroblasts. To isolate fibroblasts from the dermis, remove the other two layers by placing the tissue on the dermis side to make all three layers visible. Then hold the tissue down with forceps and cut away the whole epidermis in one clean line without damaging the tissue.Now flip the tissue and repeat the same procedure by cutting within the dermis parallel to the border with the subcutaneous fat. Once the dermis is isolated, cut the tissue into cubes pressing the tissue down with forceps and cutting the tissue in one direction using the scalpel. Then place 10 to 20 of these tissue pieces in the upper quarter of the T-25 flask using the forceps.Count the cells using an automated cell counter and seed the SMCs in triplicate at a seeding density of 30, 000 cells per well in 200 microliters of SMC medium in the gelatin coated ECIS plate. Place the plate with the SMCs into the ECIS's 96-well holder in the cell culture incubator. Double-click on the ECIS Applied BioPhysics software to open the program and press the Setup button.Check if all the electrodes have contact with the holder in the left lower panel labeled Well Configuration. If the electrodes are not properly connected, adjust the plate in the holder before starting the measurement. Now select the plate type in the same panel by clicking Array type.Next, prepare two pipettes set at two microliter and 150 microliter volume. Before starting the stimulation, press Mark in the software and place a comment. To stimulate the cells, remove the lid and place it on a sterile surface inside the incubator.Then induce SMC contraction by pipetting two microliters of the ionomycin working solution into each well as quickly as possible. Once all the cells have been stimulated, mix the medium in the wells using the second pipette. To determine interexperimental measurement reproducibility, two independent measurements of all included control and patient cell lines were plotted as a Bland-Altman plot, demonstrating that this method did not show variability outside the confidence interval, except for one outlier cell line.Two wells seeded with the same experiment and stimulated simultaneously showed practically the same contractual response curve. For validating whether the shown response was a contraction and not osmotic stress because of ionomycin stimulation, the medium was replaced after stimulation and the behavior of the cells was recorded, which showed that the stimulated cells started spreading over the electrode again. The contractile response in SMCs derived from healthy, non-dilated aortas showed a median response of 61%compared to the median response of 52%from abdominal aortic aneurysm patients.The mean contractile response of the control group was used to identify four patients who had lower contraction values than the normal values. The Western blot analysis and subsequent quantification confirmed that the cells express SMC markers. For determining the proliferative capacity of the SMC, qPCR was performed for Ki67.Ki67 expression was detectable in all cells, but did not correlate with the contractile output. When stimulating the cells for contraction, remember not to pipet too many wells at the same time and tried to pipet as fast as possible. This procedure may need some practice.Using this method, we have divided the patients based on their contraction and to lower normal contracting which allowed us to investigate these patient and the links to their clinical characteristics such as smoking.

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