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.
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.
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 TGFβ 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.
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 µg/mL streptomycin, and Smooth Muscle Growth Supplement referred to as SMC medium.
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 µg/mL streptomycin, referred to as fibroblast medium.
3. Measuring contraction (example SMCs)
4. Detecting the presence of SMC specific markers
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 experiment and stimulated simultaneously show practically the same contractile response curve (Figure 4C). To validate whether the shown response is contraction and not osmotic stress because of ionomycin stimulation, the medium was replaced after stimulation, and the behavior of the cells was recorded. This shows that the stimulated cells started spreading over the electrode again (Figure 4D).
As the first application of this novel method, the contractile response in SMCs derived from healthy, non-dilated aortas (n=6) and abdominal aortic aneurysm (AAA) patients (n=21; Figure 5A) was measured, as shown previously8. The median response of the control group was 61% (46-77%), compared to the median response, 52% (15-75%), of the patient group. The values were not significantly different, and a higher variability was noticed in the patient group. Due to the novelty of the method, the control group was used to determine "normal" contraction. Figure 5B demonstrates the mean and ± 2SD range of the control group and how this range is used to identify four patients who have lower contraction values than the "normal" values. Determining the cause of impaired contraction is the objective of a separate mechanistic study. A western bot analysis was performed to determine if the SMC used for the experiment express SMC contractile markers. The western blot analysis (Figure 6A) and subsequent quantification (Figure 6B) confirm that the cells express SMC markers. The marker expression throughout the groups was variable and did not correlate with the contractile output. To determine the proliferative capacity of the SMC, qPCR for Ki67 was performed (Figure 6C). Ki67 expression was detectable in all cells but did not correlate with the contractile output.
Figure 1: SMC isolation from aortic AAA tissue. (A) Materials needed for SMC isolation from aortic tissue: aortic tissue in a Petri dish, SMC medium, two T25 flasks filled with SMC medium, scalpel, and two pairs of surgical forceps. (B) Intimal side of the aortic wall showing atherosclerotic plaques. (C) Adventitial layer of the aortic wall. (D) Separation of the tunica media and tunica adventitia by pulling with the forceps. (E) After the tunica media is isolated, tissue is cut into small pieces using forceps and the scalpel. (F) Tissue pieces placed in the T25 flask. Abbreviations: AAA = abdominal aortic aneurysm ; SMC = smooth muscle cell. Please click here to view a larger version of this figure.
Figure 2: Fibroblast isolation from dermal tissue. (A) Materials needed for fibroblast isolation from dermal tissue: dermal tissue in a Petri dish, fibroblast medium, two T25 flasks filled with fibroblast medium, scalpel, and two pairs of surgical forceps. (B) Skin biopsy showing subcutaneous fat on the upper side. (C) After the dermis is isolated, tissue is cut into small pieces using forceps and the scalpel. (D) Tissue pieces placed in the T25 flask. Please click here to view a larger version of this figure.
Figure 3: ECIS plate setup and graphic representation of aortic SMC contraction. (A) Left; a 96-well ECIS plate. Middle; magnification of one well of the ECIS plate, showing the ten electrodes at the bottom of the well. Right; light microscope image of the SMCs seeded on the ECIS plate; magnification 10x. (B) Resistance curves measured by ECIS after seeding of control SMCs in triplicate. Thick blue line represents the mean of the triplicate, and dotted lines represent the SD of the triplicates. In the first 4-5 h after seeding, SMCs spread out over the entire surface, and high resistance (~1000 Ω) is measured. Once a stable SMC monolayer is formed, resistance reduces over time to around 500 Ω. This figure is modified from 8. Abbreviations: ECIS = electric cell-substrate impedance sensing; SMC = smooth muscle cell; SD = standard deviation. Please click here to view a larger version of this figure.
Figure 4: Measuring SMC contraction using ECIS. (A) Left; SMC monolayer of control SMCs prior to stimulation for contraction. Right; control SMCs after stimulation for contraction. Magnification 10x. (B) Difference plot demonstrating interexperimental measurement reproducibility between two separate contraction measurements. Dotted lines represent the 95% confidence interval, and the thick medial line represents the mean of two interexperimental measurements. Each data point represents the deviation from the mean of two independent measurements of the whole dataset of controls and patients (Purple ●; n = 27). (C) Intraexperimental reproducibility of ECIS contraction measurements. Resistance curves generated by control SMC seeded in two wells. Vertical dotted line marks the stimulation. (D) Cell recovery post stimulation of contraction. Thick blue line represents the resistance value of an unstimulated control SMC. Dotted blue line represents the resistance value of the same control SMC line, after stimulation marked by the vertical dotted black line. Approximately 1 h post stimulation (thick vertical dotted black line), the medium in both conditions was refreshed to remove the stimulus, and the recovery of the cells was tracked for another 3.5 h. Resistance values were normalized to the values prestimulation to monitor the behavior of cells post stimulation. This figure is modified from 8. Abbreviations: ECIS = electric cell-substrate impedance sensing; SMC = smooth muscle cell. Please click here to view a larger version of this figure.
Figure 5: SMC contraction in control and AAA patients' SMCs upon ionomycin stimulation. (A) Mean contractile response of control (Blue ●; n = 6) and AAA patient (Red ●; n = 21) SMCs upon ionomycin stimulation derived from multiple experiments. (B) Mean contractile response of Control (Blue ●; n = 6), Normal contracting (Red ●; n = 15), and Low contracting (Red ●; n = 6) AAA patients' SMCs derived from multiple experiments. Contraction is expressed in percentages of decrease compared to baseline value prior to stimulation. Horizontal black line represents the mean contractile response of control SMCs. Dotted horizontal lines mark two SDs above and below the mean of the control group. The low contracting group is defined as contraction lower than two SDs below the mean of the control group. Boxplot is shown as median with range. This figure is modified from 8. Abbreviations: AAA = abdominal aortic aneurysm; ECIS = electric cell-substrate impedance sensing; SMC = smooth muscle cell; SD = standard deviation. Please click here to view a larger version of this figure.
Figure 6: SMC marker expression. SMC contractile and proliferative markers were analyzed in SMC of control (Δ; n = 6), normal contracting, (●; n = 15), and low contracting (○; n = 6). (A) Western blot analysis of SMC used for the contraction experiments. aSMA, calponin, and SM22 were quantified, and Tubulin was used as loading control. (B) Quantification of western blot depicted in (A). (C) qPCR analysis of Ki67 proliferation marker. This figure is modified from 8. Abbreviations: SMC = smooth muscle cell; aSMA = alpha Smooth Muscle Actin; qPCR = quantitative PCR. Please click here to view a larger version of this figure.
Supplemental Table S1: Primer sequences. Forward and reverse qPCR primer sequences of analyzed housekeeping and SMC marker genes. This table is modified from 8. Abbreviations: SMC = smooth muscle cell; qPCR = quantitative PCR. Please click here to download this Table.
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 tissue in vivo. Additionally, the explant protocols presented here are not innovative but are included to present a complete workflow from, in this case, patient-specific aortic aneurysm material to contraction measurements. Culturing the cells in vitro might also affect some of their properties due to the lack of systemic factors and different substrate stiffness. This might account for the variability in SMC marker expression. Due to the absence of reliable fibroblast markers, fibroblasts are usually distinguished based on the absence of markers compared to other cells. We have thus not characterized the fibroblasts isolated with this protocol. Another limitation is that measuring contraction as a factor of the surface remains somewhat indirect. The possible consequences are that the output comes from averaging the response of the whole well containing thousands of cells. Thus, it cannot be determined whether all cells contract less or whether there are cells that lower the average.
SMC contractility was previously studied using different techniques. Compared to traction force microscopy4, ECIS has multiple advantages, primarily, higher throughput and time efficiency. As stated in the limitations, the tradeoff is that the contraction measurements are obtained indirectly, making ECIS more suitable for screening larger numbers of cell lines and groups and measuring traction force for more in-depth single-cell mechanistic studies. Calcium gradients6 were previously used as indicators of contraction; ECIS provides a more reliable contraction measurement as it measures the locomotion of the cell. Quantification of images of collagen wrinkles caused by the contraction of the cells seeded inside the gel5 provides an average of the whole population of seeded cells, similar to ECIS. However, the measurements obtained through ECIS are far more precise and provide real-time monitoring during the whole course of observation, making them significantly more informative.
In conclusion, this paper describes a novel application for the ECIS system, which can be used to measure the contraction of adherent cells, such as SMCs and fibroblasts. This method can be used to study multiple cell lines in a timely and efficient fashion, making it suitable for patient or drug screening. In line with research on aortic aneurysms, this method could also be used to screen SMCs of patients with other cardiovascular disorders, such as dissections and atherosclerosis.
The authors have nothing to disclose.
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.
96-well Array | Applied Biophysics | 96W10idf PET | Array used to measure contraction in the ECIS setup |
Custodiol | Dr. Franz Höhler Chemie GmbH | RVG 12801 | Solution used to transfer tissue in from surgery room to laboratorium |
Dimethyl sulfoxide | Sigma-Aldrich | 472301 | Solution used to dilute ionomycin |
Fetal Bovine Serum | Gibco | 26140079 | Addition to cell culture medium |
Ham's F-10 Nutrient Mix | Gibco | 11550043 | Medium used to culture skin fibroblasts |
Human Vascular Smooth Muscle Cell Basal Medium (formerly ''Medium 231'') | Gibco | M231500 | Medium used to culture smooth muscle cells |
Invitrogen countess II | Thermo Fisher Scientific | AMQAX1000 | Automated cell counter |
Ionomycin calcium salt from Streptomyces conglobatus | Sigma-Aldrich | I0634-1MG | Compound used for contraction stimulation |
NaCl 0.9% | Fresenius Kabi | B230561 | Solution used to transfer tissue in from surgery room to laboratorium |
Penicillin-Streptomycin | Gibco | 15140122 | Antibiotics used for cell culture medium |
Phospathe buffered saline | Gibco | 10010023 | Used to wash cells |
Quick-RNA Miniprep Kit | Zymo Research | R1055 | Kit used for RNA isolation |
Smooth Muscle Growth Supplement (SMGS) | Gibco | S00725 | Supplement which is added to smooth muscle cell culture medium |
SuperScript VILO cDNA Synthesis Kit | Thermo Fisher Scientific | 11754250 | Kit used for cDNA synthesis |
SYBR Green PCR Master Mix | Thermo Fisher Scientific | 4309155 | Reagent for qPCR |
Trypsin-EDTA | Gibco | 15400-054 | Used to trypsinize cells |
ZTheta | Applied Biophysics | ZTheta | ECIS instrument used for contraction measurements |