Biology
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Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro
Chapters
Summary February 15th, 2022
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.
Transcript
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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