Abstract
Primary cell cultures are essential tools for elucidating the physiopathological mechanisms of the cardiovascular system. Therefore, a primary culture growth protocol of cardiovascular smooth muscle cells (VSMCs) obtained from human abdominal aortas was standardized. Ten abdominal aorta samples were obtained from patients diagnosed with brain death who were organ and tissue donors with family consent. After surgical ablation to capture the aorta, the aortic tissue was removed, immersed in a Custodiol® solution, and kept between 2 and 8 °C. In the laboratory, in a sterile environment, the tissue was fragmented and incubated in culture plates containing an enriched culture medium (DMEM/G/10% fetal bovine serum, L-glutamine, antibiotics and antifungals) and kept in an oven at 37 °C and 5% CO2. The aorta was removed after 24 h of incubation, and the culture medium was changed every six days for twenty days. Cell growth was confirmed through morphological analysis using an inverted optical microscope (Nikon®) and immunofluorescence for smooth muscle alpha-actin and nuclei. The development of the VSMCs was observed, and from the twelfth day, differentiation, long cytoplasmic projections, and adjacent cell connections occurred. On the twentieth day, the morphology of the VSMCs was confirmed by actin fiber immunofluorescence, which is a typical characteristic of VSMCs. The standardization allowed VSMC growth and the replicability of the in vitro test, providing a protocol that mimics natural physiological environments for a better understanding of the cardiovascular system. Its use is intended for investigation, tissue bioengineering, and pharmacological treatments.
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The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.
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Acknowledgements
Sources of Funding: The authors of this article are grateful to the São Paulo Research Foundation (FAPESP), FAEPA, and CAPES for funding this research.
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Christiane Becari (2017/21539-7; 2018/23718-8), Mauricio S. Ribeiro (2019/11485-1), and Jessyca M. Barbosa (2019/21721-4) were supported by the São Paulo Research Foundation (FAPESP).
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The Research Ethics Committee of the Hospital das Clínicas, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), defends the project under CAAE No. 82879518.6.0000.5440.
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Supplementary file 1: Videomicrograph of the morphological characterization and development of subconfluent VSMCs, showing proliferative and multinucleated myoblasts, showing typical spindle-shaped morphology with long projections connecting adjacent cells, and showing mitosis and phagocytosis. Cells were visualized and filmed under an inverted microscope (Axio Observer, LSM 780 MP). Image superimposition (merge) was performed using the ImageJ Fiji program. Bar = 50 µm. (n = 1). Author name and videographer: Carlos Alexandre Curylofo Corsi; Length (min): 01:11 and size (MB): 29.6 (AVI 30404 KB)
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Corsi, C.A.C., Sares, C.T.G., Mestriner, F. et al. Isolation and primary culture of human abdominal aorta smooth muscle cells from brain-dead donors: an experimental model for vascular diseases. Cell Tissue Bank 25, 187–194 (2024). https://doi.org/10.1007/s10561-023-10091-3
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DOI: https://doi.org/10.1007/s10561-023-10091-3