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

Summary

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.

Abstract

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.

Introduction

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 aneurysm....

Protocol

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 experime....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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