0172 Porcine intestinal explants as ex vivo/in vitro model to study gastrointestinal disease

Abstract

Intestinal diseases play an important role in livestock animals especially in pigs. To gain more knowledge about pathological processes during intestinal disease usually animal experiments are needed. However, alternatives to animal testing are highly recommended. Ex vivo cultivation of explants might provide an alternative tool to investigate intestinal diseases in pigs. We therefore evaluated the cultivation of porcine intestinal explants. Intestinal tissue from pigs was obtained from a local abattoir. About 10 cm of the jejunum were transported in pre-warmed PBS to the lab. Intestinal tissue was flushed with PBS and was cut open longitudinally. Thereafter, tissue was washed again with PBS and cut into small pieces. Explants were placed into 12-well plates (mucosa facing upward) prefilled with 1.5 mL cultivation medium (D-MEM containing antibiotics and 10% FBS) and were incubated for up to 72 h at 39°C and 5% CO2. Viability was measured with the water soluble tetrazolium (WST) −1 assay after 2, 4, 24, and 72 h (n = 6). In addition, explants were frozen in liquid nitrogen after incubation for 0, 2, and 4 h and stored at −80°C (n = 9). Gene expression of three different pro-inflammatory cytokines (TNF-α, IL-6, and IL-8) was measured via RT-qPCR. Statistical evaluation was performed with IBM SPSS Statistics software. If data were normally distributed ANOVA was performed. If data were not normally distributed, the Kruskal Wallis Test was used as non-parametric test. Viability was already significantly decreased after 4 h of incubation compared with fresh explants. Furthermore, there was a significant increase of TNF-α expression after 4 h (25-fold) and IL-6 expression after 2 and 4 h (50 and 320-fold) compared with fresh explants. No effect of incubation time was seen for IL-8 expression. Our study highlights the importance of measuring viability when cultivating intestinal explants. In addition, dissection of the tissue and isolation of explants seem to stimulate expression of certain pro-inflammatory cytokines. We can therefore conclude that ex vivo cultivation of intestinal explants might be an alternative screening tool. However, optimization of dissection and culture conditions is needed to prolong possible incubation time.