Enteroids: Promising in Vitro Models for Studies of Intestinal

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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

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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

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chemicals have been screened in human enteroids, demonstrating that mycophenolic

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acid (MPA) is able to potently inhibit rotavirus infection (8). Interestingly, in human

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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

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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

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enteroid growth (51), and the same group demonstrated that a synthetic Toll-like

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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

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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

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(1). At this point in time, explorative studies in enteroids to investigate the effect of

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chemicals on animal disease and production performance in most other farm animals

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(e.g. cattle, sheep, goat, horse) are still in a design stage and have not been reported

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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

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husbandry.

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CONCLUSIONS AND PROSPECTS

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To date, enteroids of almost all of livestock species including pigs, chicken,

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bovine, sheep and horse have been successfully cultured. Similar to enteroids cultured

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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

18


Page 19 of 40

382


(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).

19



Page 20 of 40

404

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

20


Page 21 of 40


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

21



Page 22 of 40

424

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

22


Page 23 of 40


440

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33



Page 34 of 40

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

34


Page 35 of 40


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

35



Zhan et al Palmarini et al

2017 1999

Page 36 of 40

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

36


Page 37 of 40


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

37



Page 38 of 40

Table of Contents Graphic (TOC)

38


Page 39 of 40


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)



Schematic of the morphology of farm animal enteroids and their applications. 250x125mm (96 x 96 DPI)


Page 40 of 40

Enteroids: Promising in Vitro Models for Studies of Intestinal

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