Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy.
Intestinal epithelial cells (IECs) not only have a critical function in the absorption of nutrients, but also act as a physical barrier between our body and the outside world. Damage and death of the epithelial cells lead to the breakdown of this barrier function and inflammation due to access of the immune system to compounds of the intestinal flora. Intestinal epithelial damage is frequently associated with various inflammatory disorders, chemo- and radiotherapy as well as drug-mediated toxicity. Until recently, intestinal epithelial-damaging activities of drugs and treatments could be tested only in vivo in animal models because of the poor survival rate of primary IECs ex vivo. The three-dimensional culture and outgrowth of intestinal crypt stem cells into organoids have offered new possibilities to culture and study IECs ex vivo. Here we demonstrate that intestinal organoids are a useful and physiologically relevant model system to study cell death and survival in IECs. We further describe a number of microscopy-based as well as colorimetric methods to monitor and score survival and death of intestinal organoids. Finally, the comparison of organoids isolated from gene-deficient mice and wild-type mice allows investigating the role of specific genes in the regulation of IEC death. Owing to their comparable structure and behavior, intestinal organoids may serve as an interesting and physiologically relevant surrogate system for large- and mid-scale in vitro testing of intestinal epithelium-damaging drugs and toxins, and for the investigation of cell death pathways.
Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Konstanz, Germany.
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Grabinger
Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Konstanz, Germany.
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Luks
Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Konstanz, Germany.
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Kostadinova
Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
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Zimberlin
Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
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Medema
Doerenkamp-Zbinden Chair of In-Vitro Toxicology and Biomedicine, Department of Biology, University of Konstanz, Konstanz, Germany.
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Leist
Chair of Biochemical Pharmacology, Department of Biology, University of Konstanz, Konstanz, Germany.
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Brunner
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000172:deficiency / Q000235:genetics; D000072224:Bcl-2-Like Protein 11; D001681:Biological Assay; D018938:Caco-2 Cells; D002166:Camptothecin / Q000031:analogs & derivatives / Q000633:toxicity; D002945:Cisplatin / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007410:Intestinal Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D007421:Intestine, Small / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D000077146:Irinotecan; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009940:Organoids; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D014409:Tumor Necrosis Factor-alpha; D000077146:Irinotecan; D002945:Cisplatin; D002166:Camptothecin