Isolation and primary culture of human abdominal aorta smooth muscle cells from brain-dead donors: an experimental model for vascular diseases.

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.

Role of Preservation Solution in Human Aneurysmatic Aorta Harvest and Transport: A Comparative Analysis of Different Solutions for Tissue Injury Protection.

INTRODUCTION: Human aortic tissues in vitro are tools to clarify the pathophysiological mechanisms of the cardiovascular system, cell culture, and transplants. Therefore, this study aims to analyze and compare the preservation of human aneurysmatic aortic tissues in three different solutions. METHODS: Six human abdominal aortic aneurysms were obtained from patients after surgical ablation. The aorta samples were incubated in different solutions - 0.9% normal physiological saline solution, Ringer's lactate solution, and histidine-tryptophan-ketoglutarate solution (Custodiol®). Segments were collected at 0, 6, 24, and 48 hours. Creatine kinase and nitrate/nitrite were quantified for each incubation time. The tissue's alpha-smooth muscle actin was analyzed by immunofluorescence. RESULTS: There was a significant increase in creatine kinase formation in the normal saline group at 0 and 48 hours and in the Ringer's lactate group at 0 and 48 hours (P=0.018 and P=0.028). The lower levels of creatine kinase and nitrate/nitrite and the aortic tissues' morphological integrity show that histidine-tryptophan-ketoglutarate has better tissue protection. These data suggest that histidine-tryptophan-ketoglutarate induces a protective effect on smooth muscle cells, with less tissue depletion in the aortic aneurysm. CONCLUSION: This study compared three preservation solutions with the potential for human abdominal aortic aneurysm tissue preservation. The histidine-tryptophan-ketoglutarate solution reduced tissue injury and improved tissue preservation in human abdominal aortic aneurysm tissue samples.