Development and evaluation of a porcine in vitro colon organ culture technique

Abstract

The intestinal mucosa comprises a complex assemblage of specialized tissues that interact in numerous ways. In vitro cell culture models are generally focused on recreating a specific characteristic of this organ and do not account for the many interactions between the different tissues. In vitro organ culture (IVOC) methods offer a way to overcome these limitations, but prolonging cell viability is essential. This study aimed to determine the feasibility and optimal conditions for in vitro culture of swine colonic mucosa for use as an enteric pathogen infection model. Explants (n = 168) from commercial pigs (n = 12), aged 5 to 10 wk, were used to assess the impact of various culture protocols on explant viability. Explants were cultured for up to 5 d and formalin fixed at 24-h intervals. Following establishment of the culture protocol, explants (n = 208) from 13 pigs were evaluated at Day 0 and 5 of culture. Assessment of viability was based on histological changes (tissue architecture evaluated by H&E, immunostaining of cell proliferation marker Ki-67) and expression of genes encoding IL-1α, IL-8, TNF-α, IFN-γ, and e-cadherin. After 5 d in culture, 20% of explants displayed over 80% of epithelial coverage, whereas 31% of explants had more than 50% of their surface covered by columnar epithelium, and 81% had crypts but with a decreased number of Ki-67-positive cells when compared to Day 0. Notably, large variability in explant quality was observed between donor pigs. Best possible explants were obtained from the distal colon of pigs, processed immediately after euthanasia, cultured at the liquid-tissue-gas interface in media supplemented with a mixture of antibiotics and antifungals and an oxygen-rich gas mix.