Development and evaluation of a porcine in vitro colon organ culture technique
- 23 June 2016
- journal article
- research article
- Published by Springer Science and Business Media LLC in In Vitro Cellular & Developmental Biology – Animal
- Vol. 52 (9), 942-952
- https://doi.org/10.1007/s11626-016-0060-y
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.Keywords
This publication has 44 references indexed in Scilit:
- Reproduction of Mucohaemorrhagic Diarrhea and Colitis Indistinguishable from Swine Dysentery following Experimental Inoculation with “Brachyspira hampsonii” Strain 30446PLOS ONE, 2013
- Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium -Induced ColitisInfection and Immunity, 2011
- Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiationIn Vitro Cellular & Developmental Biology – Animal, 2010
- Response of Porcine Intestinal In Vitro Organ Culture Tissues following Exposure to Lactobacillus plantarum JC1 and Salmonella enterica Serovar Typhimurium SL1344Applied and Environmental Microbiology, 2010
- Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectorsThe Journal of Gene Medicine, 2006
- Ki‐67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cellsJournal of Cellular Physiology, 2005
- Microbial diversity studies of the porcine gastrointestinal ecosystem during weaning transitionAnimal Research, 2004
- Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT MethodMethods, 2001
- Validation of organ culture in colonic adaptation to surgical manipulation.Gut, 1991
- Postmortem Viability and Early Changes in Organ Culture of Human and Rabbit Aortic Endothelial CellsPathobiology, 1990