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.
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References
Abbott A (2003) Cell culture: biology’s new dimension. Nature 424:870–872
Abud HE, Watson N, Heath JK (2005) Growth of intestinal epithelium in organ culture is dependent on EGF signalling. Exp Cell Res 303:252–262
Appleton GV, Wheeler EE, Challacombe DN, Williamson RC (1991) Validation of organ culture in colonic adaptation to surgical manipulation. Gut 32:1027–1030
Arena ET, Campbell-Valois FX, Tinevez JY, Nigro G, Sachse M, Moya-Nilges M, Nothelfer K, Marteyn B, Shorte SL, Sansonetti PJ (2015) Bioimage analysis of Shigella infection reveals targeting of colonic crypts. Proc Natl Acad Sci U S A 112:E3282–E3290
Autrup H, Barrett LA, Jackson FE, Jesudason ML, Stoner G, Phelps P, Trump BF, Harris CC (1978a) Explant culture of human colon. Gastroenterology 74:1248–1257
Autrup H, Stoner GD, Jackson F, Harris CC, Shamsuddin AK, Barrett LA, Trump BF (1978b) Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens. In Vitro 14:868–877
Best A, La Ragione RM, Clifford D, Cooley WA, Sayers AR, Woodward MJ (2006) A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection. Vet Microbiol 113:63–72
Browning TH, Trier JS (1969) Organ culture of mucosal biopsies of human small intestine. J Clin Invest 48:1423–1432
Bullwinkel J, Baron-Luhr B, Ludemann A, Wohlenberg C, Gerdes J, Scholzen T (2006) Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells. J Cell Physiol 206:624–635
Collins JW, Coldham NG, Salguero FJ, Cooley WA, Newell WR, Rastall RA, Gibson GR, Woodward MJ, La Ragione RM (2010) Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella enterica serovar Typhimurium SL1344. Appl Environ Microbiol 76:6645–6657
Costa MO, Hill JE, Fernando C, Lemieux HD, Detmer SE, Rubin JE, Harding JC (2014) Confirmation that “Brachyspira hampsonii” clade I (Canadian strain 30599) causes mucohemorrhagic diarrhea and colitis in experimentally infected pigs. BMC Vet Res 10:129
Dame MK, Veerapaneni I, Bhagavathula N, Naik M, Varani J (2011) Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation. In Vitro Cell Dev Biol Anim 47:32–38
Danielsen EM, Sjostrom H, Noren O, Bro B, Dabelsteen E (1982) Biosynthesis of intestinal microvillar proteins. Characterization of intestinal explants in organ culture and evidence for the existence of pro-forms of the microvillar enzymes. Biochem J 202:647–654
Deschner E, Lewis CM, Lipkin M (1963) In Vitro study of human rectal epithelial cells. I. Atypical zone of H3 thymidine incorporation in mucosa of multiple polyposis. J Clin Invest 42:1922–1928
Dobbins WO, Herrero BA, Mansbach CM (1968) Morphologic alterations associated with neomycin induced malabsorption. Am J Med Sci 255:63–77
Drake RL, Vogl W, Mitchell AWM, Gray H, Gray H (2010) Gray’s anatomy for students. Churchill Livingstone/Elsevier, Philadelphia
Duvigneau JC, Hartl RT, Groiss S, Gemeiner M (2005) Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. J Immunol Methods 306:16–27
Eckmann L, Jung HC, Schurer-Maly C, Panja A, Morzycka-Wroblewska E, Kagnoff MF (1993) Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin 8. Gastroenterology 105:1689–1697
Fell HB (1963) Studies of development in organ culture: summary, correlation, and speculation. Natl Cancer Inst Monogr 11:73–80
Fletcher PS, Elliott J, Grivel JC, Margolis L, Anton P, McGowan I, Shattock RJ (2006) Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides. AIDS 20:1237–1245
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710–1715
Girish V, Vijayalakshmi A (2004) Affordable image analysis using NIH Image/ImageJ. Indian J Cancer 41:47
Jacobson M, Fellstrom C, Lindberg R, Wallgren P, Jensen-Waern M (2004) Experimental swine dysentery: comparison between infection models. J Med Microbiol 53:273–280
Kesisoglou F, Schmiedlin-Ren P, Fleisher D, Roessler B, Zimmermann EM (2006) Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors. J Gene Med 8:1379–1392
Kik MJ, Koninkx JF, van den Muysenberg A, Hendriksen F (1991) Pathological effects of Phaseolus vulgaris isolectins on pig jejunal mucosa in organ culture. Gut 32:886–892
Konstantinov SR, Favier CF, Zhu WY, Williams BA, Kluss J, Souffrant WB, de Vos WM, Akkermans AD, Smidt H (2004) Microbial diversity studies of the porcine gastrointestinal ecosystem during weaning transition. Anim Res 53:317–324
Levy J (2000) The effects of antibiotic use on gastrointestinal function. Am J Gastroenterol 95:S8–S10
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408
Mitchell JD, Mitchell J, Peters TJ (1974) Enzyme changes in human small bowel mucosa during culture in vitro. Gut 15:805–811
Onuma H, Mastui C, Morohashi M (2001) Quantitative analysis of the proliferation of epidermal cells using a human skin organ culture system and the effect of DbcAMP using markers of proliferation (BrdU, Ki-67, PCNA). Arch Dermatol Res 293:133–138
Paszti-Gere E, Csibrik-Nemeth E, Szeker K, Csizinszky R, Jakab C, Galfi P (2012) Acute oxidative stress affects IL-8 and TNF-alpha expression in IPEC-J2 porcine epithelial cells. Inflammation 35:994–1004
Pernodet N, Maaloum M, Tinland B (1997) Pore size of agarose gels by atomic force microscopy. Electrophoresis 18:55–58
Pie S, Lalles JP, Blazy F, Laffitte J, Seve B, Oswald IP (2004) Weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets. J Nutr 134:641–647
Quinlan JM, Yu WY, Hornsey MA, Tosh D, Slack JM (2006) In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes. BMC Dev Biol 6:24
Reiss B, Williams GM (1979) Conditions affecting prolonged maintenance of mouse and rat colon in organ culture. In Vitro 15:877–890
Resau JH, Sakamoto K, Cottrell JR, Hudson EA, Meltzer SJ (1991) Explant organ culture: a review. Cytotechnology 7:137–149
Rubin JE, Costa MO, Hill JE, Kittrell HE, Fernando C, Huang Y, O'Connor B, Harding JCS (2013) Reproduction of mucohaemorrhagic diarrhea and colitis indistinguishable from swine dysentery following experimental inoculation with “Brachyspira hampsonii” strain 30446. PLoS ONE 8:e57146
Schiff LJ (1975) Organ cultures of rat and hamster colon. In Vitro 11:46–49
Shamsuddin AK, Barrett LA, Autrup H, Harris CC, Trump BF (1978) Long-term organ culture of adult rat colon. Pathol Res Pract 163:362–372
Thomson D (1914) Controlled growth en masse (somatic growth) of embryonic chick tissue in vitro. Proc R Soc Med 7:71–75
White RL (2001) What in vitro models of infection can and cannot do. Pharmacotherapy 21:292S–301S
Wlodarska M, Willing B, Keeney KM, Menendez A, Bergstrom KS, Gill N, Russell SL, Vallance BA, Finlay BB (2011) Antibiotic treatment alters the colonic mucus layer and predisposes the host to exacerbated Citrobacter rodentium-induced colitis. Infect Immun 79:1536–1545
Yu QC, Mergner WJ, Vigorito RD, Resau JH (1990) Postmortem viability and early changes in organ culture of human and rabbit aortic endothelial cells. Pathobiology 58:138–145
Zachrisson K, Neopikhanov V, Wretlind B, Uribe A (2001) Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa. Cytokine 15:148–155
Acknowledgments
This work was funded by the Alberta Livestock and Meat Agency. MOC was supported by a University of Saskatchewan-devolved scholarship. The authors would like to thank Dr. Michael Dame (University of Michigan Medical School) for sharing his valuable knowledge on explant culture, Roman Nosach and Courtney Ek for animal care assistance, and Scott dos Santos for his assistance with qPCR. Primers for porcine IL-8, TNF-α, and IFN-γ qPCR were designed by Cole Enns and generously provided by Dr. Matthew Loewen (Department of Veterinary Biomedical Sciences, University of Saskatchewan). Experiments were designed and conducted in accordance with the Canadian Council for Animal Care and approved by the University of Saskatchewan Animal Research Ethics Board (Protocol 20130034).
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Costa, M.O., Harding, J.C.S. & Hill, J.E. Development and evaluation of a porcine in vitro colon organ culture technique. In Vitro Cell.Dev.Biol.-Animal 52, 942–952 (2016). https://doi.org/10.1007/s11626-016-0060-y
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DOI: https://doi.org/10.1007/s11626-016-0060-y