Viability of coronary arteries cultured in an ex-vivo vascular bioreactor
Authors: Razzi F , Van Der Giessen A , Duncker DJ , Stijnen M , Van Steijn V , Van Beusekom HMM 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ZonMW

Introduction
Cardiovascular diseases (CVD) are the leading cause of death globally. The CVD that causes most of the deaths annually is atherosclerosis [1]. The treatment of choice for acute symptoms like myocardial infarction is percutaneous coronary intervention with the implantation of a stent. Despite successful clinical results, there are still complications [2]. Current pre-clinical research to assess safety and efficacy of stents is primarily based on healthy animal models [3]. However, these are limited to the number of simultaneous experiments and ethical concerns. Several in-vitro and ex-vivo models are available to evaluate and assess medical devices and treatments. In particular, ex-vivo systems represent an important class, because the presence of native tissue guarantees biological response as a result of cell-cell interaction within a physiological extracellular matrix.

Purpose
The main goal of this research is to culture coronary arteries long-term in order to maintain them viable for assessment of safety and efficacy of stents, by mimicking physiological conditions.

Methods
The ex-vivo vascular bioreactor was used and data were acquired as described before [4]. Culture experiments were performed on porcine right coronary arteries (RCA) (n=9). Duration of culture was set to 2 (n=3), 4 (n=3), and 9 days (n=3). Hearts harvested from a local slaughterhouse were kept on Krebs-buffer (4°C) until dissection within 2 hours. The bioreactor containing the dissected RCA was placed in a 38°C incubator with 100% humidity and 5% CO2. The RCA was cultured in a blood-mimicking culture medium [4]. To assess cultured blood vessel morphology, diameter and structure during the culture period, ultrasound imaging was performed. Pressure (60-100 mmHg), flow rate (± 60 ml/min), and diameter measurements were monitored every day. The flow rate was adjusted to ensure a physiological peak endothelial shear stress of around 0.8 – 1.8 Pa. After 2, 4 or 9 days of culture, the RCA was fixed in buffered formaldehyde for 24 hours, embedded in paraffin and sectioned. Histology was performed using hematoxylin and eosin (H&E) and resorcin fuchsin (RF).

Results
Ultrasound imaging showed that the morphology and structure of cultured arteries was maintained during the cultures (average diameter 3.5±1.1 mm). Extracellular matrix was preserved as shown by histology (RF stain, Figure 1). Endothelial cells (ECs) coverage was incomplete at 2, but after 4 days it was completely restored. After 9 days of culture, a minimal layer of newly formed neointima was visible (Figure 1).

Conclusions
The cultured RCAs maintained their physiological morphology up to 9 days of culture. The endothelium was completely restored after 4 days, making the system suitable for ex-vivo interventions like stenting. The investigated model represents a useful tool for testing vascular therapies and devices, supporting the translational phase between in-vitro and ex-vivo studies.