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.&#160;This study is funded by the Dutch Heart Foundation, Dekkerbeurs senior clinical scientist award, project no. 2019T065.

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 regulations of the WMA Declaration of Helsinki and institutional guidelines of the Medical Ethical Committee of the VU Medical Center. All the experiments and experimental protocols were performed in accordance with institutional guidelines and approved by the Medical Ethical Committee of the VU Medical Center. For complete information about the control and patient cell lines used, refer to 8.
1. Isolating primary human SMCs from aortic biopsies
NOTE: Perform the following steps under a sterile tissue culture laminar flow hood. Wear gloves and use standard aseptic techniques when handling human blood and human tissue samples. SMCs are cultured in 231 Human Vascular Smooth Muscle Cell Basal Medium supplemented with 100 U/mL penicillin, 100 &#181;g/mL streptomycin, and Smooth Muscle Growth Supplement referred to as SMC medium.
<ol>
	<li>Sterilize two pairs of surgical forceps and a scalpel by immersing them in 70% ethanol and subsequently wiping them dry.</li>
	<li>Pipette 2 mL of SMC medium in a Petri dish in which the tissue dissection will be performed.</li>
	<li>Pipette 2.5 mL of SMC medium into two T25 flasks. Swirl the flasks around so that the small volume of medium covers the whole surface.</li>
	<li>Transport the harvested human aortic wall biopsy from the operating room to the laboratory on ice in a sterile plastic tube with cold, sterile tissue transfer solution (see the Table of Materials) or 0.9% NaCl.</li>
	<li>Open the tube with the tissue inside the tissue culture hood. Take the biopsy out of the tube using the sterilized forceps and place it in the Petri dish (Figure 1A).</li>
	<li>Visually inspect the biopsy to identify the three aortic layers, the tunica intima (inner), media (middle), and adventitia (outer layer). Look for the presence of atherosclerotic plaques on the intima side and slimy connective tissue on the adventitial side (Figure 1B) to distinguish the layers.</li>
	<li>To isolate SMCs from the media, remove the other two layers. Place the tissue with the intima plaque side up first (Figure 1B). Remove the solid plaque by pulling it away from the tissue with forceps while pushing the tissue down with the other pair of forceps. Remove subsequent layers of plaque until the pink, uniform medial layer is visible.</li>
	<li>Flip the tissue (Figure 1C). Repeat the same procedure, as in step 1.7, by pulling off the adventitial layer (Figure 1D). Be sure to remove all visible parts in as many attempts as needed, as this layer will not detach from the media easily. 
	NOTE: It is essential that the intimal and adventitial layers be removed properly to obtain as clean an SMC population as possible.</li>
	<li>Once the medial layer is isolated, cut the tissue into cubes of approximately 1 mm x 1 mm x 1 mm. Press the media down with forceps and cut the tissue in one direction using the scalpel. Do not cut back and forth; make clean, unidirectional cuts to minimize damage. Try to make as many cubes as possible, given the size of the biopsy (Figure 1E).</li>
	<li>Place the tissue pieces in the upper quarter of the T25 flask using the forceps. Place 10-20 cubes per flask if the amount of material allows it (Figure 1F). 
	NOTE: Use smooth forceps to minimize adhesion of the tissue to the ribs of the forceps and easily detach the tissue into the flask.</li>
	<li>Incubate the tissue cubes in the T25 flasks for approximately 10 days at 5% CO2 at 37 &#176;C in an incubator. 
	NOTE: First cell outgrowth is expected around that time. The cells that initially migrate might look smaller than normal SMCs.</li>
	<li>Once cell growth is observed, add 2.5 mL of medium to the flask, making sure not to pipette it onto the tissue pieces to prevent them from detaching.</li>
	<li>After approximately 5 more days, when clusters of cells are observed around the tissue pieces, change the culture medium. If tissue pieces detach, remove them as they will not reattach.</li>
	<li>Once the cells are 80-90% confluent, transfer them to a T75 flask and continue culturing in this format. 
	NOTE: A 1:2 or 1:3 split ratio is recommended. Freeze a backup of the cells at an early passage. The cells generally maintain their properties until passage 10; later passages should not be used for experiments.</li>
</ol>
2. Isolating primary dermal fibroblasts from skin biopsies
NOTE: Perform the following steps under a sterile tissue culture laminar flow hood. Wear gloves and use standard aseptic techniques when handling human blood and human tissue samples. Fibroblasts are cultured in Basal Medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin, referred to as fibroblast medium.
<ol>
	<li>Sterilize two pairs of surgical forceps and a scalpel by dipping them in 70 % ethanol and subsequently wiping them dry.</li>
	<li>Pipette 2 mL of fibroblast medium in a Petri dish in which the tissue dissection will be performed.</li>
	<li>Pipette 2.5 mL of fibroblast medium into two T25 flasks. Swirl the flasks around so that the small volume of medium covers the whole surface.</li>
	<li>Transport the harvested skin biopsy from the operating room to the laboratory on ice in cold, sterile tissue transfer solution (see the Table of Materials) or 0.9% NaCl in a sterile plastic tube.</li>
	<li>Open the tube with the tissue inside the tissue culture hood. Take the biopsy out of the tube using the sterilized forceps and place it in the Petri dish (Figure 2A).</li>
	<li>Visually inspect the biopsy to identify the three skin layers, the epidermis, dermis, and subcutaneous fat. Look for a recognizable skin surface, sometimes with visible hair, to identify the epidermis. On the opposite side, look for the subcutaneous fat, which is often yellow and slimy. Identify the layer between the epidermis and the subcutaneous fat as the dermis-the source of viable fibroblasts (Figure 2B).</li>
	<li>To isolate fibroblasts from the dermis, remove the other two layers and place the tissue on its side so that all three layers are visible. 
	NOTE: Unlike the aortic tissue, skin layers cannot be pulled apart from each other; hence, they must be cut. The tissue is also more rubbery, making it more difficult to cut. Use a sharp scalpel.</li>
	<li>Hold the tissue down with forceps. Cut in parallel to the border between the epidermis and dermis. Cut away the whole epidermis. Try to cut in one clean line and do not move back and forth to avoid tissue damage.</li>
	<li>Flip the tissue. Repeat the same procedure as in step 2.8; this time, cut within the dermis parallel to the border with the subcutaneous fat.</li>
	<li>Once the dermis is isolated, cut the tissue into cubes approximately 1 x 1 x 1 mm3. Press the tissue down with forceps and cut the tissue in one direction using the scalpel. Do not cut back and forth; make clean, unidirectional cuts to minimize damage. Try to make as many cubes as possible, given the size of the biopsy (Figure 2C).</li>
	<li>Place the tissue pieces in the upper quarter of the T25 flask using the forceps. Place 10-20 cubes per flask if the amount of material allows it (Figure 2D). 
	NOTE: Use smooth forceps to minimize the adhesion of the tissue to the ribs of the forceps and easily detach the tissue into the flask.</li>
	<li>Incubate the tissue cubes in the T25 flasks for approximately 10 days at 5% CO2 at 37 &#176;C in an incubator. 
	NOTE: First cell outgrowth is expected around then. The cells that initially migrate might look smaller than normal fibroblasts.</li>
	<li>Once cell growth is observed, add 2.5 mL of medium to the flask, making sure not to pipette it onto the tissue pieces to prevent them from detaching.</li>
	<li>After approximately 5 more days, when clusters of cells can be observed around the tissue pieces, change the culture medium. If tissue pieces detach, remove them as they will not reattach.</li>
	<li>Once the cells are 80-90% confluent, transfer them to a T75 flask and continue culturing in this format. 
	NOTE: A 1:2 or 1:3 split ratio is recommended. Freeze a backup of the cells at an early passage. The cells generally maintain their properties until passage 10; later passages should not be used for experiments.</li>
</ol>
3. Measuring contraction (example SMCs)
<ol>
	<li>Prepare a 96w10e ECIS array (see Figure 3A for a representative image of the array with magnified electrodes and cells seeded on the electrodes). 
	NOTE: Perform the following steps under a sterile tissue culture laminar flow hood.
	<ol>
		<li>Coat a sterile 96w10e array with 200 &#181;L of 10 mM L-cysteine per well for 30 min at room temperature.</li>
		<li>Wash the plate twice with sterile water. Let the plate dry overnight in the tissue culture hood with the lid slightly open. 
		NOTE: Coating the plate with L-cysteine is only necessary when using the plate for the first time.</li>
		<li>The next day, UV-sterilize plate and lid for 30 min. Turn the lid upwards so that the inside is sterilized. Once the plate is sterilized, do not open the plate outside the flow hood.</li>
	</ol>
	</li>
	<li>Seeding cells on the ECIS array
	<ol>
		<li>Prewarm 1% sterile gelatin solution in the water bath for 30 min. 
		NOTE: The solution is stored in the fridge and might solidify, making it difficult to pipette.</li>
		<li>Subsequently, coat the wells with 100 &#181;L of the 1 % gelatin solution per well and incubate the plate at 37 &#176;C for at least 1 h.</li>
		<li>Aspirate the gelatin from the wells.</li>
		<li>Count the cells using an automated cell counter and seed the SMCs in triplicate at a seeding density of 30,000 cells/well in 200 &#181;L of SMC medium.</li>
		<li>Place the plate with the SMCs into the ECIS 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.</li>
		<li>Check if all the electrodes have contact with the holder (green; red if no connection) 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.</li>
		<li>Select the plate type (96-well array) in the same panel by clicking Array type.</li>
		<li>In the right upper panel Data Collection Setup, click on single frequency and change the impedance value to 4000 Hz and the interval to 8 s.</li>
		<li>Click the Start button to start measurements. Wait for a new panel to open, where the ECIS file can be saved.</li>
		<li>Allow the cells to attach and establish a monolayer for 48 h. 
		NOTE: The attachment and spreading of cells on the electrodes generate a baseline resistance value (Figure 3B).</li>
	</ol>
	</li>
	<li>Stimulation of cells to induce contraction
	<ol>
		<li>Induce SMC contraction by stimulating the cells with 10 &#181;g/mL of the calcium ionophore, ionomycin. 
		NOTE: Ionomycin will induce the influx of extracellular Ca2+, activating the contractile cascade; see Figure 4A for representative images of non-stimulated and stimulated contracted cells.</li>
		<li>Dilute 1 mg of ionomycin powder in 100 &#181;L of dimethylsulfoxide to make a stock solution of 10 mg/mL, and store 10 &#181;L aliquots at -20 &#176;C. Before use, dilute the ionomycin solution 1:10 in SMC medium to prepare the working solution to be added to the cells.</li>
		<li>Perform the cell stimulation while the array is still in the array holder inside the cell culture incubator and the electrodes are attached to the system.
		<ol>
			<li>To stimulate the cells, remove the lid and place it on a sterile surface inside the incubator. Prepare two pipettes, set at 2 &#181;L and 150 &#181;L.</li>
			<li>Before starting the stimulation, press Mark in the software and place a comment. 
			NOTE: This will make it easier to find the exact timing of the stimulation when analyzing the data.</li>
			<li>Pipette 2 &#181;L 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. 
			NOTE: Due to the rapid effects, it is not necessary to change tips between different cell lines and conditions. Work quickly while stimulating and mixing because the ionomycin has an acute effect. When stimulating a full plate, stimulate a maximum of 3 columns at once. After every stimulation, wait at least 30 min until the next stimulation to let the cells recover from the temperature and CO2 change caused by the opening of the incubator door.</li>
			<li>Directly after the stimulation is done, press Mark again to add a comment that the stimulation is done.</li>
		</ol>
		</li>
		<li>Finishing the stimulation experiment
		<ol>
			<li>Record the impedance values for approximately 5 min after the ionomycin stimulation. Finish the recording by pressing Finish.</li>
			<li>Reuse the ECIS arrays up to three times: wash the wells twice with sterile water, and incubate the plate at 37 &#176;C for 2-3 h with trypsin or a similar reagent. Repeat steps 3.1.2 and 3.1.3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Exporting the data
	<ol>
		<li>To export the data, click File | Export data | To Excel (Selected). Select a folder to save the file.</li>
		<li>Click Separate when the program asks to combine or separate sheets.</li>
	</ol>
	</li>
	<li>Analyzing the contractile output
	<ol>
		<li>To calculate the contractile response, use the following equation (1), where C indicates contraction (expressed in % of change compared to baseline), prestimulation (PrS) indicates the resistance value (expressed in &#937;) just before ionomycin stimulation and post-stimulation (PoS) indicates the resistance (expressed in &#937;) 2 min after finishing the stimulation. 
		<img alt="Equation 1" src="/files/ftp_upload/63122/63122eq01.jpg" /> (1) 
		NOTE: As shown in equation (1), the impedance value of a well filled with culture medium without any attached cells (value of 290 &#937;) is subtracted from all the results in the final calculation. It is recommended to measure contractile responses in triplicate in each experiment and perform three independent experiments with the same cell line to account for any inter- and intra-experimental variation.</li>
		<li>Once the three independent experiments have been performed, group the data together by calculating the average of the three measurements, including the standard deviation (SD).</li>
	</ol>
	</li>
	<li>Comparing different groups
	<ol>
		<li>To define the range of normal contraction and investigate subsequent specific research questions, use the control group to define &#34;normal contraction&#34; and compare it to the contractile response of patients or treatment groups.</li>
		<li>Calculate the mean and SD of the whole control group, i.e., of the end values for all cell lines, as described in step 3.5.2.</li>
		<li>Use the range of the mean &#177; 2SD to identify cells in the other group that fall outside this range, indicating that they have an altered contractile response. 
		NOTE: The mechanism behind the altered contractile response is subject to specific research questions and is cell- and condition-dependent and will not be discussed in this protocol.</li>
	</ol>
	</li>
</ol>
4. Detecting the presence of SMC specific markers
<ol>
	<li>RNA isolation
	<ol>
		<li>Seed the same patient-specific cell lines used for the contraction measurements in 6-well plates, one well per cell line. Count the cells using an automated cell counter and seed the cells at a seeding density of 200,000 cells/well in SMC medium and allow them to attach overnight.</li>
		<li>Wash the cells once with sterile PBS.</li>
		<li>Lyse the cells and isolate the cells according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>cDNA synthesis
	<ol>
		<li>Perform cDNA synthesis in 20 &#181;L of a reverse transcription reaction mixture. Dilute the concentrations of total isolated RNA according to the instructions provided by the manufacturer.</li>
	</ol>
	</li>
	<li>qPCR
	<ol>
		<li>Measure the gene expression of SMC marker genes, e.g., ACTA2, CNN1, SMTN, and TAGLN to confirm that the isolated cells are indeed SMCs and detect SMC markers. Check for correlation of the results with the contractile output.</li>
		<li>Use at least two housekeeping genes to normalize the data, e.g., YWHAZ and TBP. 
		NOTE: The qPCR run and analysis can be performed using different PCR machines and compatible software, depending on what is available and optimized in the laboratory. See Supplemental Table S1 for primer sequences.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

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 ...

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3.5K Views.  Vrije Universiteit Amsterdam. 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.

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

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 and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

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 and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

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 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.

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)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

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

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

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 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 ...

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 (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

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

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

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 suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

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 processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

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

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

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

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

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