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Enteroids: promising in vitro models for studies of intestinal physiology and nutrition in farm animals Yuebang Yin, Songge Guo, Dan Wan, Xin Wu, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06908 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019
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Journal of Agricultural and Food Chemistry
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Enteroids: promising in vitro models for studies of intestinal
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physiology and nutrition in farm animals
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Yue-Bang Yin†,‡, Song-Ge Guo†,ǂ, Dan Wan†, Xin Wu†*, Yu-Long Yin†
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† Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of
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Subtropical Agriculture, Chinese Academy of Sciences; Hunan Provincial
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Engineering Research Center for Healthy Livestock and Poultry Production;
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Scientific Observing and Experimental Station of Animal Nutrition and Feed
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Science in South-Central, Ministry of Agriculture; Hunan Provincial Key
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Laboratory of Animal Nutritional Physiology and Metabolic Process, Changsha,
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Hunan, 410125, China.
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‡ Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands ǂ College of bioscience and biotechnology, Hunan Agricultural University, Changsha, Hunan, 410128, China
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Correspondence to:
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*
[email protected] (X.W.); Institute of Subtropical Agriculture, Chinese Academy of
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Sciences, Changsha, 410125, China.
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Word count: Abstract, 151 words; Text, 4276 words.
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Running title: Enteroids for studies of farm animals
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Keywords: Enteroid, Intestinal studies, Livestock, Veterinary sciences, Animal
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nutrition, Farm animals
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Abbreviations:
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GI: Gastrointestinal; 2D: 2 dimensional; FABPs: Fatty acid-binding proteins; IP:
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Intestinal peptidase; LGR5+: Leucine-rich repeat-containing G-protein coupled
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receptor 5 positive; ISCs: Intestinal stem cells; EGF: Epidermal growth factor;
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HuNoVs: Human noroviruses; ISEMF: Intestinal subepithelial myofibroblasts; GSK3i:
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Glycogen synthase kinase 3 inhibitor; LPS: Lipopolysaccharides; TLR4: Toll-like
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receptor 4; TLRs: Toll-like receptors; MAP: Mitogen-activated protein.
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ABSTRACT
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The lack of sophisticated in vitro models limits our current understanding of
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gastrointestinal functions in farm animals. Conventional 2D cell lines or primary cells
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fail to recapitulate the physiology of in vivo intestinal epithelium. In contrast stem
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cell-derived, non-transformed 3D enteroids partially recreate the villus-crypt anatomy
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of the native intestine and comprise most if not all intestinal cell types including
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enterocytes, enteroendocrine cells, goblet cells, Paneth cells and stem cells. This
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review summarizes the techniques used for generating and culturing enteroids of
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various farm animal species, focuses on important factors influencing the longevity of
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enteroids, and provides an overview of their current applications in modeling
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veterinary pathogens and in developing chemicals and bioactives for treating animal
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disease and improving production performance. It also mentions current limitations of
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enteroid models and potential solutions, and highlights the opportunities for using
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these enteroids as a platform in studies regarding veterinary sciences and animal
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nutrition.
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INTRODUCTION
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The gastrointestinal (GI) tract is the predominant site for exposure of the body to
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a variety of food, nutrients, drugs and metabolites, toxins and bacteria (1), and thus GI
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homeostasis is crucial for maintaining health of animals. Within the GI tract, the
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intestinal epithelium contains a variety of cell types including enterocytes,
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enteroendocrine cells, enterochromaffin cells, Paneth cells, goblet cells, and intestinal
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stem cells (2, 3). This complex structure allows the intestinal epithelium to carry out
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multiple functions, including nutrient and electrolyte absorption, metabolism,
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electrolyte and hormone secretion, pathogen-host interaction, and innate immunity.
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To study such functions in vitro, many different 2-dimensional (2D) cultures of
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intestinal tumor-based cell lines or immortalized epithelial cells have been developed
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(4). However several shortcomings of the tumor-based cell lines or immortalized
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epithelial cells have limited their application: for instance, they only contain a single
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cell type, lack the complexity and physiology of native epithelium, and often
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accumulate mutations (5). Freshly isolated primary cells and explanted intestinal
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tissues are thought to mimic in vivo physiology more accurately, but they cannot be
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maintained in culture for a long term, which limits their application in developmental,
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physiological and regenerative studies (1). Hence, there is an urgent need for the
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development of more suitable in vitro models.
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In 2009, Dr. Toshiro Sato and Dr. Hans Clevers firstly reported the culturing of
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an in vitro 3D-intestinal model from single intestinal stem cells, which was named
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intestinal organoid, enteroid, or mini-gut (6). The enteroids in part recapitulate the
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intestinal villus-crypt anatomy, and harbour most if not all of the various intestinal
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cell types, a major advantage in comparison with single cell type-based cell lines (6).
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Following a fast growing number of studies use enteroids as in vitro models in various
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fields, including virology (7-9), bacteriology (10), oncology (11), and pharmacology
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(12). Importantly, because enteroids derived from a single animal can be expanded for
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multiple passages and at an almost unlimited scale, the introduction of this new
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intestinal model has strongly reduced the number of experimental animals sacrificed
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for intestinal studies, with positive ethical implications (1).
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A proper health and homeostasis of the GI tract is of prime importance for the
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technical performance of farm animals. However, until shortly a lack of reliable in
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vitro experimental models hampered the understanding of molecular mechanisms
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involved in nutrition, physiology, and pathogen-host interactions of farm animals.
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Two-D cultures of cell lines have been used frequently in studies of pig intestine, such
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as the porcine intestinal epithelial cell lines SD-PJEC (13) and IPEC-J2 (14).
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However, to the best of our knowledge, very few studies using intestinal epithelial
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cell lines of poultry, cattle, and other farm animals have been reported. Intestinal
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primary cells are frequently exploited as a model in livestock studies, but the culturing
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process is often complicated, and the failure to achieve long-term passaging is a major
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hurdle (15). Therefore culturing enteroids from farm animals, though technically in its
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infancy, may offer an attractive alternative, considering the mature state of the
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technique pioneered for human and mouse enteroids. Indeed, a rapidly expanding list
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of studies have recognized the importance of enteroids in livestock studies, and
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enteroids of pigs (16-20), chicken (3, 19, 21), cows (4, 16, 19), sheep (19), and horses
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(19) have been successfully cultured. In this review we will discuss the current in
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vitro intestinal epithelial models used in livestock studies, and highlight the
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importance and potential applications of enteroids in the livestock field.
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CURRENT IN VITRO INTESTINAL EPITHELIAL MODELS
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DEVELOPED FOR LIVESTOCK STUDIES
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The IPEC-J2 cell line consists of non-transformed intestinal epithelial cells,
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cultured originally from the jejunal epithelium of a newborn piglet. The model has
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been broadly used in studies of animal nutrition and in veterinary applications. The
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cell line was developed by Dr Helen Berschneider at the North Carolina State
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University in 1989 (22). As a nontumorigenic cell line, IPEC-J2 is better able to
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mimic in vivo physiology than many other tumor-based cell lines (14). In livestock
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studies, IPEC-J2 has been used in a variety of studies including the effects of nutrients
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on intestinal function (23-25), bioactives (26, 27), probiotics (28), nutrient-pathogen
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interaction (29), barrier and transport (30, 31), cellular signaling (32), toxicity and
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viability (33, 34), and veterinary medicine (35), etc (Table 2). However, IPEC-J2 has
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turned out to lack brush border enzyme activity and the expression of genes
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controlling major gel-forming mucin (i.e. Muc2) (14). A subclone of the IPEC-J2
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cells, named SD-PJEC was developed by Sun and colleagues (13), and has been used
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to investigate infections of swine influenza virus (13, 36). Recently, Kaiser et al
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established a primary intestinal epithelial cell model based on the primary crypt cells
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of specific-pathogen-free layer-type chickens, which showed the characteristics of in
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vivo epithelial cells, and was thought to be a suitable model for probing pathogen-host
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interactions (37). However, this model fails to regenerate (37), thus limits its
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application in biological studies. Primary bovine intestinal epithelial cells were
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successfully cultured from different intestinal sections of Chinese Holstein cows,
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which recapitulated prime features of in vivo bovine intestine including the expression
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of fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP), while the
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cells showed signs of senescence after 5 passages (15). Ovine intestinal carcinoma
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(ST6) cell lines were used to model infection of jaagsiekte sheep retrovirus that cause
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pulmonary adenomatosis in sheep (38). Human colorectal cancer lines including
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Caco-2 and HT29 cells are also used as a model in many livestock studies (39).
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However these human cell models are hampered by the same limitations as exist for
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animal cell lines. In conclusion, considering the inherent shortcomings of 2D cell
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lines and a lack of reliable in vitro models for many livestock species, more suitable
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in vitro models for GI tract studies of farm animals are desperately needed.
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BRIEF INTRODUCTION OF ENTEROIDS
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The long term culture of primary intestinal epithelial cells as enteroids have
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become feasible only following the discovery of Leucine-rich repeat-containing
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G-protein coupled receptor 5 positive (LGR5+) as a true marker of adult intestinal
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stem cells (ISCs) residing at the bottom of intestinal crypts, and the identification of a
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broad collection of growth factors needed for stem cell proliferation and preventing
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anoikis (40). These growth factors allow ISCs to grow and expand permanently in the
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absence of a tumorogenic mutation (40). Enteroids were successfully cultured first
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from mouse (6) and human intestine (41). The key signaling pathways involved in
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enteroid growth are the Wnt/β-catenin, EGF/Ras/Raf/MEK/ERK, Notch, and bone
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morphogenetic protein (BMP) pathway (42). Wnt signaling plays a key role in
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maintaining stemness and self-renewal ability of ISCs, which is through to engage the
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cognate Frizzled receptors serving to stabilize β-catenin and thus to activate
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downstream target genes supporting stemness (42). Epidermal growth factor (EGF)
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binds to its receptor EGFR, to activate Ras/Raf/MEK/ERK axis to regulate mitosis of
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ISCs (42). EGF is also capable of activating the PI3K/Akt and ErbB pathway
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involved in regulating proliferation (1). The Notch pathway is crucial for supporting
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the multipotency and proliferation of stem cells and maintaining the secretory
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differentiation (43). BMP is mainly active in the villus domain of intestinal epithelium,
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and exerts the promotion of spatially arranged differentiation of ISCs, and inhibitory
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effects on the β-catenin activity, thus playing an important role as a regulator of the
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number of ISCs in crypts (43). Suppressing the BMP pathway is therefore beneficial
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for the stemness of ISCs. Based on the machinery of regulating ISCs, supplements of
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key factors are required for enteroid growth, including Wnt3a, EGF, R-spondin1
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(agonist of WNT signaling), and Noggin (inhibitor of BMP protein). Other elements
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involved in the stemness machinery are also of benefit for enteroid growth including
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B27 and N2 supplement, CHIR99021 (agonist of WNT signaling), N-acetylcysteine,
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nicotinamide (inhibitor of sirtuins), A83-01 (TGF-β receptor 1 inhibitor), and
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Y-27632 (Rock inhibitor) (1, 6, 41).
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Enteroids have several clear advantages in comparison with cell lines (Table 1).
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They are cultured from intestinal stem cells, generate villus-crypt structures, and
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spontaneously differentiate into multiple cell lineages, including enterocytes,
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enteroendocrine cells, Paneth cells, goblet cells and tuft cells, thus they exquisitely
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resemble their organ of origin in architecture and function (6, 42). In contrast cell
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lines are often tumor-based, immortalized and contain a single cell type, thus failing
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to resemble the complicated structure and mutual interactions of distinct cell types in
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the native epithelium. Enteroids can be passaged for multiple generations without
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genomic mutation (44), while cell lines do more readily acquire genomic mutations
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after repeated passaging (45). Thus the enteroid model appears to circumvent the
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inherent drawbacks of 2D cells. In humans, several studies have demonstrated that
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results obtained using enteroid models more closely reflect in vivo results compared
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with outcomes obtained in intestinal cell lines. For example, Yin and colleagues
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demonstrated that a broadly used antiviral agent, interferon ɑ (IFNɑ), was less
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effective against rotavirus infection in human enteroids than in Caco2 cells, which
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was consistent with observations in the clinic (7). Another example is that human
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noroviruses (HuNoVs) are confirmed to have tropism for tuft cells (CD300lf receptor)
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in mice (46), whilst HuNoVs are demonstrated to have tropism for enterocytes in
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human enteroids (47). Most recently, de Winter-de Groot et al isolated enteroids from
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34 infants diagnosed with cystic fibrosis (CF), and used confocal microscopical
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measurements of forskolin-induced swelling (FIS) to assess the residual activity of
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mutated CFTR chloride channels in the CF enteroids. They found that enteroid based
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FIS showed a strong correlation with clinical outcome parameters and biomarkers of
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CF at the individual level, including sweat chloride concentration (SCC) and intestinal
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current measurement (ICM), thereby confirming the ability of enteroids to mimic in
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vivo physiology (48). In summary these studies clearly indicate that enteroids function
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as a more advanced in vitro model in comparison with cell line models.
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ENTEROIDS CULTURED FROM FARM ANIMALS
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Enteric disease and homeostasis play important roles in the technical
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performance of farm animals. Since 2D cell line models and primary cell isolation
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approaches are not available for many of the species, the enteroid technique offers an
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attractive alternative for recreating intestinal epithelium in vitro. Currently enteroids
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have been successfully cultured from pig, chicken, bovine, horse, and sheep intestine
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(Table 3). These enteroids may provide a promising new platform in animal science
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and veterinary studies.
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Porcine enteroids. The successful isolation of porcine enteroids was reported first by
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Gonzalez et al, who applied a minor modification of the culture protocol for mouse
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and human enteroids in which they used purified recombinant Noggin, R-Spondin1,
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and Wnt3a to replace conditioned media of these factors (17). Porcine enteroids
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exhibited clear budding (indicating crypt-like structure) and contained distinct
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intestinal cell types including enteroendocrine cells, absorptive enterocytes, goblet
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cells, and stem/progenitor cells (17). They were able to polarize and to form
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well-defined tight junctions (17). Porcine enteroids were also amenable for
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transduction by lentiviruses (18), offering a robust tool for in vitro studies. Enteroids
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cultured with this technique could be passaged at least 8 times for a total of 4.5
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months (17). In two other studies, porcine enteroids were cultured with supplements
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of purified EGF, Noggin, and R-spondin1 (ENR medium), together with Wnt3a- or
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intestinal subepithelial myofibroblasts (ISEMF)-conditioned medium, and in the
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presence of glycogen synthase kinase 3 inhibitor (GSK3i) (18, 49). These enteroids
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could be passaged up to 10 times, but after this stage growth retardation occurred and
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further passaging was not feasible (18). However, porcine enteroids could be
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successfully passaged up to 49 times by supplementation of Wnt3a, R-Spondin1 and
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Noggin conditioned media (19). Van der Hee et al used conditioned media of
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recombinant Noggin (15% v/v), R-spondin1 (15% v/v), and Wnt3A (30% v/v) to
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culture porcine enteroids, which could be maintained for at least several months (11).
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A recent study indicated that porcine enteroids could be kept viable for up to 13
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passages (3 months) using a commercial culture medium named IntestiCult (Stem
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Cell Technologies) (16), however the composition of this medium is not specified in
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detail. Together, it is plausible that Wnt3a, R-Spondin1 and Noggin conditioned
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medium perform better for growth of porcine enteroids than purified recombinant
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surrogates.
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One practical limitation of the use of 3D enteroids is the orientation of their
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apical border facing the organoid lumen, implying that food components,
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microorganisms, chemicals, drugs, bioactive and toxic compounds added to the
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medium are poorly exposed to the luminal side of the organoids (44).This is of
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relevance because apical and basolateral membranes of intestinal epithelial cells have
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different expression level for numerous receptors such as Toll-like receptors (TLRs)
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and cytokine receptors (20). Though microinjection is able to deliver substances into
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the lumen of a single enteroid (50), the heterogeneity in enteroid size, difficulties of
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synchronous exposure and variability in injected volumes compromise the validation
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of this method (20). To circumvent these limitations, van der Hee et al developed a
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novel approach to culture enteroids two-dimensionally, in which 3D enteroids were
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enzymatically-dissociated into single-cell suspensions, and were then transferred on
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Matrigel pre-coated culture plates or Transwell filters (20). Two-D enteroids are
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capable of forming tight junctions with high transepithelial electrical resistance in
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three days, thus representing a robust platform for exploring intestinal permeability
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and intestinal functions including drug and nutrient absorption and hormone secretion
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(20). These 2D enteroids are also suitable for studying innate immunity, infection and
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disease-associated polymorphisms (20). Thus, 2D porcine enteroids may compensate
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for certain limitations of 3D enteroids.
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Chicken enteroids. Chicken enteroids were cultured successfully for the first time by
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Pierzchalska et al, in which they used a mixture of purified R-spondin1 and Noggin,
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and other enteroid growth factors (51). These chicken enteroids could be cultured and
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passaged for more than 5 weeks (51). Chicken enteroids were shown to express
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markers of various intestinal cell types including enteroendocrine cells, absorptive
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enterocytes, goblet cells, and stem/progenitor cells (51). Li et al also used purified
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R-spondin1 and Noggin to culture chicken enteroids, and they mentioned that these
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enteroids could grow for at least one week, while no further tracking was reported
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(50). Other studies reported that Wnt3a, R-Spondin1 and Noggin conditioned media
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allowed chicken enteroids to grow up to 35 passages for 125 days (19). Interestingly,
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chicken embryo enteroids consist of both epithelial cells and myofibroblasts, which is
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different from mouse, human, porcine and other mammalian enteroids (2).
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Collectively, as concluded previously for porcine enteroids, R-spondin1 and Noggin
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condition media might be superior in comparison with purified growth factors for the
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long-term culture of chicken enteroids. Chicken enteroids might provide an important
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platform for studying infection, immunity, nutrition, and nutrient transport in chicken
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Enteroids from cattle and other farm animals. Three-D bovine enteroids were
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cultured with supplementation of Wnt3a, R-Spondin1 and Noggin conditioned media,
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and could be passaged up to 45 times for 165 days (19). Bovine enteroids could also
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be cultured with commercial IntestiCult Organoid Growth Medium (STEMCELL
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Technologies, UK), but these enteroids could only be maintained in culture for 11
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days, and then failed to expand after passaging (19). Again, the exact composition of
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the commercial media is not publicly available but it was reported that supplementing
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additional inhibitors including Rho-associated kinase inhibitor (Y27632), p38
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mitogen-activated protein (MAP) kinase inhibitor (SB202190) and TGF-βR inhibitor
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(LY2157299) allowed bovine enteroids cultured in IntestiCult Organoid Growth
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Medium to be passaged up to 5-8 times for 2 months without morphological changes
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(19). Another study showed that a mixture of commercial IntestiCult Medium and
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Wnt3a conditioned media supplemented with purified recombinant R-spondin1,
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Noggin and other inhibitors allowed bovine enteroids to be cultivated for over 12
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passages (16). Thus, it seems that Wnt3a, Noggin and R-spondin1 condition media
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and inhibitors controlling stemness-related pathways might be useful for increasing
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culture longevity of bovine enteroids.
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Sheep and horse enteroids were also successfully cultured with supplementation
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of Wnt3a, R-Spondin1 and Noggin conditioned media, which could be passaged 66
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times for 239 days, and 44 times for 168 days, respectively (19). Recently, Stewart
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and colleagues published a protocol for the cultures of horse enteroids, which were
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confirmed to contain four distinct cell types including ISCs, enteroendocrine cells,
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goblet cells, and Paneth cells (52). It is expected that these enteroid models will turn
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out to be highly useful in providing new information about intestinal characteristics
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and function in these two species.
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A combined protocol for the culture of farm animal enteroids. Based on
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information from the available recent studies, a culture protocol for farm animal
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enteroids could be composed, which largely resembles the protocol for culturing
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mouse and human enteroids (6, 7, 19-21, 52). The intestine samples are longitudinally
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opened, and the muscle layers are peeled away with forceps in case of adult animals;
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it is not necessary to separate epithelial layer and muscular layer for young animals.
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Then, the villi are removed by gently scraping mucosal surface using a glass slide,
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followed by cutting tissue into small pieces (5 × 5 mm). The tissue pieces are then
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washed with cold PBS until the supernatant looks clear (the remaining villi on the
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tissue can be visualized clearly by eyes), followed by incubation with 2.5 - 10 mM
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(depending on species) ethylenediaminetetraacetic acid (EDTA) and 1 mM
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dithiothreitol (DTT) in PBS for half an hour (small intestine) or one hour (large
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intestine) at 4°C on a 50 rpm rocker. The supernatant, containing villi and debris, is
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discarded, and 15 mL cold PBS is added, followed by repeated vortexing (3x 10
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seconds). Crypts are collected by filtering the supernatant through a 100 μm cell
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strainer. This procedure is performed thrice, followed by spinning down at 1200 rpm
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for 2 min at 4°C. Then the crypt pellet is resuspended in Dulbecco's Modified Eagle
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Medium: Nutrient Mixture F-12 (DMEM/F12) containing 25 µg/mL gentamicin and
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100 U/mL penicillin/streptomycin depleted of growth factors (Noggin, R-spondin1,
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Wnt3A and extra inhibitors), followed by spinning down at 600 rpm for 2 min at 4°C.
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Then the crypt pellet is resuspended in DMEM/F12 containing 25 µg/mL gentamicin
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and 100 U/mL penicillin/streptomycin without growth factors, and the number of
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crypts is counted under a light microscope. Crypts (200-500) are resuspended in
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growth factor reduced Matrigel and settled in the wells of a 24-well plate. Following
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Matrigel solidification, 500 μL culture medium (DMEM/F12 containing gentamicin,
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P/S, growth factors including Noggin, R-spondin1, Wnt3A, EGF, and extra inhibitors
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including Y27632, SB202190 and LY2157299) is added, followed by placing the
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24-well plate in cell culture incubator. The development of the enteroid structure is
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illustrated in Figure 1. During the first day in culture, crypts will quickly seal at their
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upper opening and grow into a spherical shape (day 1), then the spheroids undergo
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continuous budding events (day 3-4) to finally become a mature enteroid containing a
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multiplicity of intestinal cell types (day 5).
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CURRENT APPLICATIONS OF FARM ANIMAL ENTEROIDS
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Enteroids of farm animals model pathogen infections. A lack of suitable in vitro
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models hampers our understanding of the pathogenesis of veterinary infections and
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the development of effective treatments. Toxoplasma gondii or Salmonella
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typhimurium were confirmed to be able to infect porcine enteroids (16). It is also
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found that bovine enteroids were sensitive for infections of Toxoplasma gondii and
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Salmonella typhimurium (16). Although the application of enteroids to model
331
pathogen infections in other farm animals has not been reported yet, this model should
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open up new avenues for studying veterinary pathogens in distinct species.
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Enteroids of farm animals model the effects of chemicals on animal disease and
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production performance. As an exquisite in vitro model, enteroids are expected to
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be suitable for developing chemicals with benefit for treating disease or improving
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body function. Human enteroids have already been used to screen a G-protein coupled 16
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receptor (GPCR)-modulating compound library, showing that β2-adrenergic receptor
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agonists are capable to induce cystic fibrosis transmembrane conductance regulator
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(CFTR) function in the GI tract of CF patients (53). A set of immunosuppressive
341
chemicals have been screened in human enteroids, demonstrating that mycophenolic
342
acid (MPA) is able to potently inhibit rotavirus infection (8). Interestingly, in human
343
enteroids a plant extract of tannins, isolated from Syzygium guineense, was found to
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exert an anti-cancer effect depending on the Wnt signaling pathway (54). Enteroids
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cultured from farm animals have been used for investigating the effect of chemicals
346
on animal production performance, although the available data are still sparse.
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Pierzchalska et al found that prostaglandin E2 was able to boost chicken embryo
348
enteroid growth (51), and the same group demonstrated that a synthetic Toll-like
349
receptor (TLR) 2 agonist promoted the growth of chicken embryo enteroids (3).
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Melatonin, an important hormone generated by the pineal gland, was shown to
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increase amino acid transport using chicken enteroids (55). In porcine enteroids, it
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was found that lipopolysaccharides were able to internalize TLR4 to the early
353
endosome (56). Interestingly, since pig and human intestine have many genetic,
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anatomical and physiological properties in common, the porcine enteroid model is
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also thought to be a proper platform for developing medicines treating human diseases
356
(1). At this point in time, explorative studies in enteroids to investigate the effect of
357
chemicals on animal disease and production performance in most other farm animals
358
(e.g. cattle, sheep, goat, horse) are still in a design stage and have not been reported
359
yet. However we anticipate that enteroids will turn out to become excellent in vitro
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models to explore the utilization of both synthetic and natural chemicals in animal
361
husbandry.
362 363
CONCLUSIONS AND PROSPECTS
364
To date, enteroids of almost all of livestock species including pigs, chicken,
365
bovine, sheep and horse have been successfully cultured. Similar to enteroids cultured
366
from humans and mice, farm animal enteroids recapitulate the physiology of in vivo
367
intestinal epithelium composed of villus and crypt domains containing a large variety
368
of intestinal cell types (Figure 2). It seems that Wnt3a, Noggin and R-spondin1
369
condition medium are superior to purified recombinant factors for long-term culture
370
of enteroids, and inhibitors controlling stemness related pathways are necessary for
371
increasing their longevity. Farm animal enteroids are expected to become a suitable
372
model for investigating immunity, inflammation, infections, pathogen-host interaction,
373
and intestinal barrier functions (Figure 2). Of note, Wu et al recently used human
374
ileal enteroids to confirm that human milk oligosaccharides (HMOs) were capable of
375
increasing enteroid crypt budding (57). Moreover mouse enteroids have been used for
376
evaluating nutrient sensing, transport, and incretin secretion (58). Thus, this novel
377
model raises expectations as a powerful platform for studying animal nutrition and
378
food technology. So far however, applications in this direction are rare, thus more
379
efforts should be invested into this field of research. Enteroids also provide the option
380
to study nutrition at an individualized level, since they are capable of retaining
381
individual variations in genetics (polymorphisms) and maintain an epigenetic memory
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Journal of Agricultural and Food Chemistry
(59).
383
Although enteroids offer a great potential in biological studies, there are also
384
several shortcomings that may limit their application: (1) The cost of culturing
385
enteroids is still considerable, in particular due to the Matrigel component (2).
386
Moreover, Matrigel is undefined, and the amount of growth factors and other
387
important components in Matrigel may differ considerably between batches (2). To
388
circumvent these problems, a hanging drop culture was developed for enteroid culture,
389
in which only 5% Matrigel and smaller amount of culture media were needed as
390
compared with the approach of immersing enteroids in Matrigel (2). In addition, a
391
hanging drop culture avoids the time cost of Matrigel solidification and speeds up the
392
culture procedure (2). In the future, more creative approaches should be developed to
393
reduce the culture cost and simplify culture precedure. (2) A standardized operation
394
procedure (SOP) for farm animal enteroid culture is lacking, which may result in
395
variations in quality and properties of enteroids cultured by different groups (1), thus,
396
more efforts should be invested to generate a standardized protocol. (3) Enteroids do
397
not contain the complex immune and neural systems that exist in vivo, which reduces
398
their ability to faithfully mimic bodily physiology. However the co-culturing of
399
enteroids with immune or neural cells may in part circumvent this pitfall (60). (4)
400
Spherical morphology and their embedding in Martrigel may prevent lumen
401
penetration of chemicals or bioactives and washout; however intraluminal
402
micro-injection of the 3D organoids or converting them into a 2D monolayer culture
403
may overcome most of these hurdles (18, 20).
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Collectively, although enteroid models still face several unsolved technical
405
difficulties and limitations, in our opinion they should receive more attention as useful
406
new exploratory models in intestinal studies of farm animals, and may potentially lead
407
to important new insights in the research area for livestock studies.
408 409
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411
Figure legends
412
Figure 1: A schematic diagram illustrating morphology changes of enteroids as a
413
function of time. Each crypt quickly seals the upper opening, followed by growing
414
into a spherical shape (day 1), then the spheroid structure undergoes continuous
415
budding events (day 3-4) to finally become a mature enteroid containing various
416
intestinal cell types (day 5), followed by accumulation of a large body of dead cells in
417
the lumen (day 6). The picture was taken by the first author to illustrate the real
418
morphology of porcine enteroids.
419
Figure 2: Schematic of the morphology of farm animal enteroids and their
420
applications.
421 422
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ACKNOWLEDGEMENT
425
Funding Resources
426
This work was supported by National Key Research and Development Program of
427
China (2016YFD0500504), the earmarked fund for China Agriculture Research
428
System (CARS-35), Science and Technology Service Network Initiative program of
429
Chinese Academy of Sciences, Agricultural innovation project of Hunan Province
430
(2018ZD12), and CAS President’s International Fellowship for Visiting Scientists
431
(2019VBA0015).
432
Conflict of Interest
433
The authors declare no competing financial interest.
434
Author contributes
435
YB. Y., X. W., and YL. Y. designed and structured the article, S. G. and YB. Y. were
436
involved in searching literature, S. G. and D. W. were involved in discussion, and X.
437
W. checked and revised manuscript, YB. Y. wrote the manuscript.
438
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O.; Su, C. W.; Smillie, C.; Shekhar, K.; Chen, Z.; Wu, C.; Ordovas-Montanes, J.;
661
Alvarez, D.; Herbst, R. H.; Zhang, M.; Tirosh, I.; Dionne, D.; Nguyen, L. T.;
662
Xifaras, M. E.; Shalek, A. K.; von Andrian, U. H.; Graham, D. B.;
663
Rozenblatt-Rosen, O.; Shi, H. N.; Kuchroo, V.; Yilmaz, O. H.; Regev, A.; Xavier,
664
R. J., T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and
665
Differentiation. Cell 2018, 175, 1307-1320 e22.
666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697
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698 699 700 701 702 703 704 705
Table 1. Comparison between enteroids and intestinal cell lines. Enteroids
Intestinal cell lines
Origin
Adult stem cell
Cell type
Multiple
Tumor-based, immortalized cells or non-transformed tumorigenic cells Single
Morphology
3D, and containing villus 2D or mono-layer without and crypt domains villus and crypt domain No Yes
Mutation 706 707
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Table 2. Cell models for intestinal studies of livestock animals (selected). Author
Year
Type of model
Species
Application/finding
Jeong Gu et al
2019
IPEC-J2 cells
Pig
Hydrolyzed Fumonisin B1 has less ability in induction of inflammatory responses compared with Fumonisin B1.
Lee et al
2019
IPEC-J2 cells
Pig
N-acetylcysteine could govern intestinal inflammation, permeability, and wound healing to improve livestock intestinal health.
An et al
2018
IPEC-J2 cells
Pig
Investigated the effects of the pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase alpha 1 (PDHA1) pathway on amino acid consumption.
Omonijo et al
2018
IPEC-J2 cells
Pig
Thymol is benefit for improving barrier function and inflammatory responses during lipopolysaccharide (LPS)-induced inflammation
Liu et al
2017
IPEC-J2 cells
Pig
Detected effect of Lactobacillus reuteri I5007 on intestinal endogenous host defense peptides expression.
Liao et al
2017
IPEC-J2 cells
Pig
Studied the effect of deoxynivalenol on apoptosis, barrier function, and expression levels of genes involved in nutrient transport, mitochondrial biogenesis and function.
Pan et al
2017
IPEC-J2 cells
Pig
Studied the effects of soybean agglutinin (SBA) on cell proliferation and cell cycle progression.
Paszti-Gere et al
2015
IPEC-J2 cells
Pig
Investigated the effect of oxidative stress on barrier integrity and localization of transmembrane serine proteinase 2.
Springler et al
2016
IPEC-J2 cells
Pig
Investigated the effect of fusarium-derived metabolites on the intestinal barrier integrity.
Sun et al
2012
SD-PJEC
Pig
Studied features of a porcine intestinal epithelial cell line for influenza virus production.
Tang et al
2018
IPEC-J2 cells
Pig
Epidermal growth factor protect IPEC-J2 Cells from lipopolysaccharides-induced apoptosis via alleviating oxidative stress.
Thomas et al
2018
SD-PJEC
Pig
Compared porcine airway and intestinal epithelial cell lines for the susceptibility and expression of pattern recognition receptors upon influenza virus infection.
Jung et al
2018
IPEC-J2 cells
Pig
Investigated porcine deltacoronavirus (PDCoV) infection.
Yang et al
2018
IPEC-J2 cells
Pig
Investigated the anti-inflammatory activity of low-molecular-weight chitosan oligosaccharide.
Zhai et al
2018
IPEC-J2 cells
Pig
Cecropin A regulated the expression level of tight junction-related protein and enhanced the intestinal barrier function by
Kaiser et al
2017
Primary intestinal
Chicken
inhibiting the MEK/ERK pathway. Successfully established an in vitro primary intestinal epithelial cell of chicken.
epithelial cell
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Zhan et al Palmarini et al
2017 1999
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Primary intestinal
Holstein
Developed a method of establishment of primary bovine intestinal epithelial cells.
epithelial cells
cows
Intestinal
Ovine
Studied infection of jaagsiekte sheep retrovirus
Human
Investigated the mechanisms of the transport of antihypertensive tripeptides LKP and IQW derived from egg white.
carcinoma cells Xu et al
2017
Caco-2 and HT29
Table 3. Enteroids of different livestock species and origins of growth factors. Author
Year
Species
Enteroid type
Intestinal
Passages/time
Noggin
R-spondin1
Wnt3a type
13/3 months
IntestiCult
IntestiCult
IntestiCult
segment Derricott et al
2018
Pig
3D
Proximal jejunum
Stewart et al
2018
Pig
3D
Jejunum
Not mentioned
Purified
Purified
Purified
Van der Hee et al
2018
Pig
3D and 2D
Ileum
Several months
Conditioned
Conditioned
Conditioned
Powell and Behnke
2017
Pig
3D
Terminal ileum
49 passages
Conditioned
Conditioned
Conditioned
Khalil et al
2016
Pig
3D
Mid-jejunum
10 passages
Purified
Purified
Conditioned
Gonzalez et al
2013
pig
3D
The duodenum,
8/4.5 months
Purified
Purified
Purified
jejunum, ileum, proximal
and
distal colon Li et al
2018
Chicken
3D
Whole jejunum
One week
Purified
Purified
Not mentioned
Powell and Behnke
2017
Chicken
3D
Terminal ileum
35/125 days
Conditioned
Conditioned
Conditioned
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Pierzchalska et al
2012
Chicken
3D
Small intestines
5 weeks
Purified
Purified
Not mentioned
Derricott et al
2018
Bovine
3D
Proximal
12 passages
IntestiCult
IntestiCult
Conditioned
jejunum Hamilton et al
2018
Bovine
3D
Ileum
11 days
IntestiCult
IntestiCult
IntestiCult
Powell and Behnke
2017
Bovine
3D
Terminal ileum
45/165 days
Conditioned
Conditioned
Conditioned
Powell and Behnke
2017
Sheep
3D
Terminal ileum
66/239 days
Conditioned
Conditioned
Conditioned
Stewart et al
2018
Horse
3D
Jejunum
Not mentioned
Purified
Purified
Purified
Powell and Behnke
2017
Horse
3D
Terminal ileum
44/168 days
Conditioned
Conditioned
Conditioned
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Table of Contents Graphic (TOC)
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A schematic diagram illustrating morphology changes of enteroids as a function of time. Each crypt quickly seals the upper opening, followed by growing into a spherical shape (day 1), then the spheroid structure undergoes continuous budding events (day 3-4) to finally become a mature enteroid containing various intestinal cell types (day 5), followed by accumulation of a large body of dead cells in the lumen (day 6). The picture was taken by the first author to illustrate the real morphology of porcine enteroids. 229x155mm (96 x 96 DPI)
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Schematic of the morphology of farm animal enteroids and their applications. 250x125mm (96 x 96 DPI)
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