A method for enriching intestinal endocrine cells and their subtypes in an adjacent intestinal monolayer system.

Abstract

Herein, we provide a novel strategy, method, and system, described as vasoactive intestinal peptide (VIP)-assisted air-liquid interface (ALI) culture, that significantly increases the number of enteroendocrine (EEC) and enterochromaffin (EC) cells compared to conventional submerged culture, while simultaneously maintaining high monolayer barrier integrity. The novel strategy, method, and system overcomes the limitations of existing EEC enrichment methods by maintaining high cell viability and barrier integrity without the need for complex co-culture or genetic engineering / induction procedures. The resulting EEC-enriched contiguous monolayer platform serves as a robust analytical tool, enabling functional studies of hormone secretion from EEC cells with high signal-to-background ratios and reproducibility.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 004,537, filed on 3 April 2020, which is incorporated herein by reference in its entirety.

[0002] (Government support indicated) This invention was made with government support under DK109559 and DK121580, granted by the National Institutes of Health. The United States Government has certain rights to this invention.

[0003] The subject of this disclosure relates to a method for enriching enteroendocrine cells and their subtypes in an adjacent intestinal monolayer system. [Background technology]

[0004] Enteroendocrine cells (EEC) These constitute less than 1% of the human intestinal epithelium population, yet they produce and secrete more than 20 different hormones that control various important physiological functions (Table 1). 1 . EEC It contains at least 15 different subtypes (e.g., enterochromaffin cells, L cells, K cells, M cells, etc.), with enterochromaffin (EC) cells being the most abundant, and these are found throughout the entire digestive tract. EEC It makes up about 40% of 2,3 EC cells can sense various stimuli from the luminal contents (e.g., nutrients, microbial metabolites, irritants, toxins, infections, and mechanical stimuli) and synthesize and secrete the bioamines serotonin or 5-hydroxytryptamine (5HT). 4,5 Over 90% of the total 5HT in the human body is located in EC cells within the intestinal tract, where 5HT is used to regulate gastrointestinal motility, secretion of digestive enzymes, and other important functions. 5 .

[0005] [Table 1]

[0006] EEC play important roles in human physiology, but their rarity represents a significant barrier to in-depth research on sensory function and hormone secretion. To overcome this challenge, living EEC have been isolated from intestinal specimens, enriched by fluorescence-activated cell sorting (FACS) and maintained in short-term culture for in vitro studies of hormone secretion and signal transduction 6,7 . The main limitation of this approach is the requirement for fresh intestinal specimens for each experiment. Over the last decade, advances in the in vitro culture of intestinal epithelial stem cells (IESCs) have paved the way for the generation of physiologically relevant primary cell-derived in vitro models of the intestine 8~10 . These systems accommodate the expansion of IESCs by culturing them in or on extracellular matrix (ECM) as organoids or monolayers, respectively, in the presence of specific growth factors, thus providing an unlimited supply of cells for in vitro research. Monolayers of differentiated intestinal epithelial cells have been established by seeding IESCs or IESC-containing tissues (such as dissociated organoids) directly onto porous membranes, growing them to confluence, and allowing or inducing differentiation 11~15 . Differentiated monolayers displayed characteristic polarized morphology of the intestinal epithelium with brush border proteins and tight junctions as well as corresponding immunofluorescence markers, and contained multiple intestinal epithelial cell lineages. EEC have been identified in these in vitro systems, including organoids and monolayers, but EEC remain as rare as they are in the native intestine 11,12 .

[0007] In in vitro systems, EEC several strategies have been developed to increase the representation of EC cells. Co-culture of epithelial monolayer systems with neurons and myofibroblasts has been found to promote differentiation into EEC , increasing their proportion from 0.3% to 0.9% 16. In organoid systems, due to forced differentiation induced by a combination of EGFR / Wnt / MAPK inhibitors, EEC is produced at a high rate (about 50%) 17 . However, this method caused high cell death. When this protocol was applied to a monolayer system having 100% surface coverage, after 4 days of treatment with EGFR / Wnt / MAPK inhibitors, less than 10% of the surface containing viable cells remained (unpublished data). Recently, lentiviral transduction has been used to stably engineer IESCs by doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation 3 . The proportion of EECs increased from 0.4% (uninduced) to 40% (induced with doxycycline). While this model is somewhat promising, this method modifies the cell genome, and it is necessary to elucidate the influence of genetic modification on cell behavior. Therefore, this approach is also not ideal.

[0008] The above strategies have several disadvantages such as either low cell viability, low barrier integrity, and / or complicated procedures. What is needed is only a slight modification to currently established monolayer protocols, EEC to establish a simple protocol for enriching. The new protocol should eliminate the limitations of the above strategies by maintaining high cell viability and barrier integrity without requiring complicated procedures of co-culture or genetic engineering / induction. According to the present disclosure, such requirements are satisfied and the obstacles are overcome. Summary of the Invention

[0009] This summary lists several embodiments of the subject matter of the present disclosure, and in many cases lists variations and substitutions of these embodiments. This summary is merely illustrative of a large number of different embodiments. The reference to one or more representative features of a given embodiment is similarly illustrative. Such embodiments can typically exist with or without the referenced features(s), and similarly, these features can be applied to other embodiments of the subject matter of the present disclosure, whether or not they are listed in this summary. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such functions.

[0010] In some embodiments, methods are provided for generating living cell constructs comprising enteroendocrine cells and subtypes of enteroendocrine cells. Such methods, in some embodiments, Enteroendocrine cells (EEC) Stem cells capable of differentiating are cultured on the upper surface of a cell support structure having both an upper and lower surface, until at least a portion of the upper surface of the cell support structure is substantially covered by the stem cells, and by maintaining a thin layer of fluid on the upper surface of the support structure, the stem cells are cultured. EEC This may include differentiating into stem cells, and here, stem cells are EEC and EEC It generates a living cell construct containing a substantially continuous monolayer of cells, including subtypes of the cell type.

[0011] In some embodiments, such methods may further include adding one or more compounds to the growth medium to prevent premature lineage fate determination of stem cells during the growth phase, where optionally, by adding one or more compounds to the growth medium, EEC The formation of can be further enhanced. One or more compounds are selected from Wnt signaling activators and / or Wnt signaling enhancers, where optionally, the signaling activators include CHIR99021, WAY316606, ABC99, IQ1, and / or arylpyrimidines, and the Wnt signaling enhancers include Wnt-3A and / or R-spongin proteins.

[0012] In some embodiments, with respect to the methods, systems, constructs, and devices described herein, the fluid layer includes liquids, slurries, hydrogels, and / or semi-solid materials. The fluid layer on the upper surface can be maintained in a range of approximately 0.001 mm to approximately 10 mm, optionally approximately 0.001 mm to approximately 1 mm, above the luminal side of a cell monolayer. The fluid layer on the upper surface can be maintained by adding hormones and / or compounds to the culture medium composition to induce luminal fluid secretion (optionally, the hormones and / or compounds include chemical substances (e.g., ionomycin), hormones (e.g., vasoactive intestinal peptides, serotonin, gastrin, etc.), cytokines, metabolites, bacteria, and / or bacterial components), and optionally, by further adding hypertonic medium to the luminal side to stimulate fluid secretion toward the luminal side and create a thicker mucus layer. The fluid layer can be maintained by a microfluidic flow mechanism. In some embodiments, high barrier integrity can also be maintained.

[0013] In some embodiments, with respect to the methods, systems, structures, and devices described herein, EEC It can secrete serotonin. EEC It can secrete glucagon-like peptide-1 (GLP-1). EEC It secretes peptide YY (PYY), or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin, and / or secretin. EEC It may also contain L cells, and the density of L cells is approximately 50 cells / mm². 2 Higher, arbitrarily approximately 100 cells / mm 2 Higher.

[0014] In some embodiments, the methods disclosed herein are induced by drugs, metabolites, foods, or compounds. EEC This method can be used to screen for hormone secretion from [the body]. EEC This method can be used to screen for drugs, metabolites, foods, or compounds that block the secretion of hormones from. EECIt can be used to screen for drugs, metabolites, foods, or compounds that enhance the secretion of hormones from the tract. Gastrointestinal epithelial cells may be selected from a group consisting of mammalian cells, avian cells, reptile cells, amphibian cells, and insect cells. The gastrointestinal epithelial cells may be human gastrointestinal epithelial cells.

[0015] In some embodiments, with respect to the methods, systems, constructs, and devices herein, the gastrointestinal epithelial cells may be selected from the group consisting of colon cells, small intestinal cells, gastric cells, esophageal cells, tongue cells, nasopharyngeal cells, oropharyngeal cells, laryngopharyngeal cells, and pancreatic epithelial cells.

[0016] Living cell constructs, EEC and EEC A living cell construct produced by the method of any of the above claims is provided herein, comprising a substantially continuous cell monolayer including a subtype of . EEC It can secrete serotonin. In such structures EEC It can secrete glucagon-like peptide-1 (GLP-1). EEC It may secrete peptide YY (PYY), or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin, and / or secretin. Such constructs may contain L cells, where optionally, the density of L cells is approximately 50 cells / mm². 2 Higher, arbitrarily approximately 100 cells / mm 2 Higher.

[0017] In some embodiments, a living cell culture system comprises a cell support structure having both an upper and a lower surface, with a porous carrier further included on the upper surface, and a culture vessel surrounding the cell support structure and providing a region for containing a culture medium, wherein the culture medium is contained below the lower surface of the cell support structure, and the cell support structure is configured to generate a living cell construct from stem cells seeded on the cell support structure, and the system maintains a thin layer of fluid on the upper surface of the support structure, thereby enabling the growth of stem cells. Enteroendocrine cells (EEC)A living cell culture system is provided herein, configured to induce differentiation, wherein a thin layer of fluid on the upper surface is maintained by hormones and / or compounds in a culture medium contained in a culture vessel that induces luminal fluid secretion. Such a system is, EEC Cells and EEC The system may be configured to produce a substantially continuous cell monolayer containing subtypes. In some embodiments, the fluid layer in such a system may be maintained by microfluidic flow of cell-supporting structures. The culture vessel may include a multiwell plate, culture dish, vial, or tube. The fluid layer on the upper surface may be maintained by hormones and / or compounds in the culture medium that induce luminal fluid secretion (optionally, the hormones and / or compounds include chemical substances (e.g., ionomycin), hormones (e.g., vasoactive intestinal peptides, serotonin, gastrin, etc.), cytokines, metabolites, bacteria, and / or bacterial components), or optionally by further adding hypertonic medium to the luminal side to stimulate fluid secretion toward the luminal side and create a thicker mucous layer.

[0018] In some embodiments, methods are provided herein for screening a test compound or microorganism for toxicological, physiological, or carcinogenic effects, wherein (a) a cell construct according to any one of the above claims is prepared; (b) a test compound or microorganism is brought into contact with the cell construct; and (c) the toxicological, pharmacological, physiological, or carcinogenic effects of the microorganism on cells of the cell construct are optionally detected by comparing the cell construct after contact with a similar cell construct that has not been contacted with the compound or microorganism, and / or by comparing the cell construct after contact with the cell construct before the contact step. The test compound or microorganism may be selected from the group consisting of aromatic organic compounds, aliphatic organic compounds, and mixed aromatic and aliphatic organic compounds. The test compound or microorganism may be selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, yeasts, and molds. Such methods may include screening for pharmacological interventions related to diabetes. Such methods may include screening for pharmacological interventions related to obesity.

[0019] Accordingly, these and other objectives are achieved, in whole or in part, by the subject matter of this disclosure. Furthermore, the aforementioned objectives, other objectives, and advantages of the subject matter of this disclosure will become apparent to those skilled in the art after careful examination of the following detailed description, drawings, and examples.

[0020] The subject matter of this disclosure can be better understood by referring to the following drawings. The components in the drawings are not necessarily to scale, and rather the emphasis is on illustrating (often schematically) the principles of the subject matter of this disclosure. In the drawings, the same reference numbers indicate corresponding parts across different drawings. Further understanding of the subject matter of this disclosure can be obtained by referring to the embodiments shown in the illustrations of the accompanying drawings. The illustrated embodiments are merely illustrative examples of systems for carrying out the subject matter of this disclosure, but both the configuration and operation of the subject matter of this disclosure, along with their further purposes and advantages, can generally be better understood by referring to the drawings and the detailed description below. The drawings are not intended to limit the scope of the subject matter of this disclosure as detailed in the accompanying or subsequently amended claims, but merely to clarify and illustrate the subject matter of this disclosure.

[0021] To better understand the subject matter of this disclosure, the following drawings are described here. [Brief explanation of the drawing]

[0022] [Figure 1A] ~ [Figure 1C] This includes schematic diagrams illustrating the differences between three cell culture mechanisms. Figure 1A shows a liquid culture where the apical surface (or luminal side) is covered with culture medium. Figure 1B shows a gas-liquid interface (ALI) culture where the apical surface is exposed to air and completely dry. Figure 1C shows a VIP-assisted ALI culture where vasoactive intestinal peptide (VIP) stimulates cells to secrete and maintain a thin layer of water on the apical side. This thin layer of liquid hydrates the apical surface. [Figure 2A] ~ [Figure 2C] This study includes experimental results demonstrating that the scarcity of EEC and EC cells in an in vitro monolayer platform reduces the reliability of the 5HT secretion assay. Figure 2A is a fluorescence microscope image of a monolayer produced by conventional liquid culture. The left image is a stitched image showing the entire surface of a 24-well insert (surface area = 33 mm²). The middle and right images are magnified views. Markers are as follows: chromogranin A (ChgA, EEC marker), red; 5HT (EC marker); nuclei stained with Hoechst 33342. Figure 2B shows the quantification of the number of EEC (ChgA+) and EC (5HT+) cells on the monolayer. Figure 2C shows 5-HT secretion from the monolayer after 4 hours of apical exposure to DMSO vehicle and 10 μM forskolin. Three independent experiments were performed. The number of samples was 3 for each condition. Statistical analysis was performed using the t-test. *: p<0.05, NS: not significant. [Figure 3A] ~ [Figure 3B] This shows that the number of EEC and EC cells on a human stem cell-derived intestinal epithelial monolayer depends on the differentiation strategy. Figure 3A is a schematic diagram illustrating the methods or processes for generating a fully differentiated, confluent monolayer derived from primary intestinal epithelial stem cells using four differentiation strategies in liquid (Sub) and gas-liquid interface (ALI) cultures, in or out of VIP. Figure 3B is a representative low-magnification fluorescence microscope image of the monolayer. The inset shows a high-magnification image. Markers are as follows: chromogranin A (ChgA, EEC marker); 5HT (EC marker); nuclei stained with Hoechst 33342. The last strategy, VIP-assisted ALI culture, generated a monolayer containing the most abundant EEC and EC cells. [Figure 4A] ~ [Figure 4F]The results of characterization of EC-enriched monolayers are shown. Figure 4A shows the quantification of the number of EEC(ChgA+) cells on monolayers generated by four differentiation strategies. Figure 4B shows the quantification of the number of EC(5HT+) cells. Figure 4C shows 5HT secretion from monolayers after 4 hours of apical exposure to DMSO vehicle and 10 μM forskolin. Figure 4D shows a box plot of the apparent permeability coefficient (Papp) of the monolayer, which indicates that monolayer integrity is best in samples generated from VIP-assisted ALI cultures. A Papp of 4 × 10⁻⁷ cm / s is a generally accepted threshold for barrier integrity. Figures 4E to 4F show the effect of differentiation period (3 to 7 days) on 5HT secretion (Figure 4E) and barrier integrity (Figure 4F, box plot). In Figures 4D and 4F, the number of samples is 6 for each condition. In the other figures, the number of samples is 3 for each condition. Statistical analysis was performed using t-tests: *p<0.05, **p<0.005. [Figure 5A] ~ [Figure 5C] Figure 5A shows 5HT secretion from monolayers generated from stem cells at various passages. Figure 5B shows a list of stem cell information (age and sex) from five donors. Figure 5C shows 5HT secretion from monolayers generated from transverse colon stem cells from five donors. In the other figures, the number of samples is three for each condition. Statistical analysis was performed using t-tests: *p<0.05, **p<0.005. [Figure 6A] ~ [Figure 6D]The results of a small-scale compound screening for the regulation of 5HT secretion are shown. Figure 6A is a confocal fluorescence microscope image (XY and XZ) showing triangular or pyramidal ECs. EC cells appear yellow due to co-staining with 5HT (green; see arrow) and ChgA (red; see arrow). The nuclei are stained with Hoechst 33342 (blue; see arrow). Figure 6B is a schematic diagram of an EC showing that food compounds can stimulate apical receptors, subsequently releasing 5HT through the basolateral boundary. Figure 6C is a list of 13 food irritants, plant sources, and working concentrations. Figure 6D shows 5HT secretion from the monolayer after 4 hours of apical stimulation with food compounds. Three samples were used for each condition. Statistical analysis against vehicle controls was performed using t-tests: *p<0.05, **p<0.005. [Figure 7] The presence of L cells in the monolayer was confirmed. In the left panel, the monolayer was stained with GLP-1 (green, Santa Cruz, sc-514592, 1:200) and Hoechst 33342 (blue). In the right panel, the monolayer was stained with PYY (red, Abcam, ab22663, 1:200) and Hoechst 33342 (blue). [Figure 8A] ~ [Figure 8B] This shows the optimization of differentiation media and culture format for enriching L cells. Figure 8A is a single-layer fluorescence microscope image. GLP-1: green. Nucleus: blue. Figure 8B is the quantification of GPL-1+ L cells. The sample size is 3 cells. *p<0.05. **p<0.005. # NS. [Figure 9A] ~ [Figure 9C] This shows optimization of differentiation time. Figures 9A and 9B show quantification of L cells using the GLP-1 (Figure 9A) marker and the PYY (Figure 9B) marker. Figure 9C shows monolayer fluorescence microscopy images imaged in the XY and YZ planes. The number of samples is 3. *p<0.05. **p<0.005. # NS. [Figure 10]This study shows the secretion of GLP-1 into the basal compartment following 4-hour apical stimulation with various compounds. Stimulation concentrations: glucose=10 mM, forskolin=10 μM, TGR5=10 μM, IBMX=10 μM, KCl=30 mM, bombesin=10 μM. Three samples were used. *p<0.05. **p<0.005. # NS compared to control. [Figure 11A] ~ [Figure 11B] Figure 11A shows the test Wnt activator (CHIR99021) in EM during the proliferation phase. Figure 11A shows a monolayer fluorescence image showing an increase in the abundance of EC(5HT+) cells when stem cells are cultured in the presence of CHIR and then differentiated in conventional liquid culture. Figure 11B shows the quantification of EC cell density. Three samples were used for each condition. Statistical analysis against vehicle control was performed using a t-test: *p<0.05, **p<0.005. [Modes for carrying out the invention]

[0023] Herein, the subject matter of this disclosure will be described more fully below, but some embodiments will be described, though not all of the subject matter of this disclosure. In fact, the subject matter of this disclosure can be embodied in many different forms, but should not be construed as being limited to the embodiments shown herein, rather these embodiments are provided so as to satisfy the applicable legal requirements of this disclosure.

[0024] In some embodiments, EEC A simple strategy, namely VIP-assisted ALI culture, is provided herein to significantly increase the number of EC cells compared to conventional liquid culture while simultaneously maintaining high monolayer barrier integrity. This novel strategy overcomes the limitations of existing EEC enrichment methods by maintaining high cell viability and barrier integrity without requiring complex co-culture or genetic engineering / induction procedures. The resulting EEC-enriched adjacent monolayer platform exhibits a high signal-background ratio and reproducibility. EEC It functions as a robust analytical tool that enables functional research into hormone secretion.

[0025] General Considerations Cells used to carry out the present invention, such as undifferentiated cells and / or gastrointestinal epithelial cells, may be cells of any species of origin, including, but not limited to, mammals, birds, reptiles, amphibians, and insects. In some embodiments, the cells are mammalian cells, including, but not limited to, gastrointestinal epithelial cells of humans, monkeys, apes, goats, sheep, dogs, cats, horses, cattle, and pigs. In some embodiments, the cells are preferably derived from primary tissue and are not cancer cells or tumor cells. Any type of gastrointestinal epithelial cell may be used, including, but not limited to, colon cells, small intestinal cells, gastric cells, esophageal cells, tongue cells, nasopharyngeal cells, oropharyngeal cells, laryngopharyngeal cells, and pancreatic epithelial cells.

[0026] Using chemical screening of numerous compounds, factors, metabolites, foods, small molecules, and / or drugs, we modulate cellular signaling pathways. EECIn addition to inducing their formation, their secretion can also be blocked or enhanced. Factors, small molecules, and drugs include activators and inhibitors of the Wnt signaling pathway, BMP signaling pathway, GREM1 signaling pathway, GREM2 signaling pathway, and Notch signaling pathway. Non-exclusive examples include CHIR99021 (Wnt activator), IWP (Wnt inhibitor), Y-27632 (Notch inhibitor), Noggin (BMP inhibitor), Jagged 1 (Notch activator), Gremlin (BMP antagonist), cytokines, and food compounds (fiber, butyrate, other fatty acids, metabolites). Other fatty acids include propionates and acetates, which are short-chain fatty acids produced by microbial fermentation of fiber. Additional metabolites include branched-chain fatty acids, bile acids, and microbial secondary bile acids, urea, amines, ammonia, lactates, phenols, indole, sulfur, carbon dioxide, hydrogen, hydrogen sulfide, and methane. Metabolites can also be produced from complex carbohydrates (soluble fiber), beans, and resistant starches, which are produced from the microbiome. Other chemical substances include antidiuretic hormones, laxatives, bacterial endotoxins, hormones (e.g., VIP), and endogenous substances (e.g., bile acids), aldosterone, somatostatin, α2-adrenergic agonists (e.g., clonidine), acetylcholine, nitric oxide, and adenosine triphosphate (ATP).

[0027] Application and Use of This Disclosure proposed EECEnriched surfaces may be planar or intricate, but unlike organoids, which are closed structures, they are characterized by having an open structure. By providing a non-cancerous culture system characterized by an open structure, this disclosure eliminates the limitations of organoid systems, making the culture of EEC-enriched tissues composed of primary cells compatible with conventional tissue culture methods and current robotics used in automated, high-throughput culture and analysis platforms. The open structure and permeable substrate allow for the application of various maturation agents to any of the cell surfaces (basal / apical or lumen), as well as the measurement of secretions from any of the surfaces while culturing cells. EEC Enrichment of the assay enables the secretion of assays with or without high epithelial barrier function and high sensitivity. EEC Interactions with other components of bacteria and microorganisms present on it also become possible here. EEC These ex vivo tissues, including those found in mice, pigs, and humans, can be generated from a variety of species. The ability to create these tissues from healthy and diseased sources, as well as from cells of various genetic backgrounds, is useful for drug screening, disease mechanism research, and EEC This will be important for basic biological research. The addition of various other cell types (e.g., immune cells, fibroblasts, and other cells that have been found to coexist with specific epithelial tissues in vivo) co-cultured on or within biomimetic scaffolds will be important. EEC Interactions and EEC This will be invaluable for understanding the effects of drugs and metabolites on these tissues. These EEC-enriched epithelial tissues are EEC Based on our understanding of physiology, toxicology, and pharmacology, EEC It is superior to the model. Several examples are given below, but this list is not exhaustive. (1) Screening research on drugs, biological agents, toxins, mutagens, food compounds, pathogens, viruses, microbiomes, etc. (2) Disease models using translational animal models or primary cells derived from humans, (3) Pharmacological and pharmacokinetic models for screening, including comprehensive dose-response profiles for drugs, food compounds, etc. (4) EEC -In vitro models for studying nerve interactions, (5) Personalized medicine based on research conducted on specific genetic backgrounds and individual patients, (6) Identification of compounds in screening for therapeutic drugs for metabolic diseases, obesity, and diabetes. (7) EEC Assays of synbiotics, prebiotics, and probiotics for functional effects. (8) EEC The effects of immune cells and their products (antibodies and cytokines) on [the subject].

[0028] Screening method Therefore, as described above, in some embodiments, this disclosure is, EEC by or EEC A method for screening a test compound or microorganism for toxicological, physiological, or pharmacological effects exerted on cells, comprising: (a) preparing a cell construct as described herein; (b) contacting the test compound or microorganism with the construct; and (c) detecting the toxicological, physiological, or carcinogenic effects of the microorganism on cells of the cell construct (for example, by comparing the cell construct after contact with a similar cell construct that has not been contacted with the compound or microorganism, and / or by comparing the construct after the contact step with the cell construct before the contact step).

[0029] In some embodiments, the test compound or microorganism is selected from the group consisting of aromatic organic compounds, aliphatic organic compounds, and mixed aromatic and aliphatic organic compounds. For example, in some embodiments, the compounds for screening are compounds such as natural products, prebiotics, probiotics, foods, food metabolites, carcinogens, drugs, drug metabolites, bacterial metabolites and toxins, irritants, soil compounds, and ingestible toxins.

[0030] In some embodiments, the test compound or microorganism is selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, yeasts, and fungi. For example, in some embodiments, the microorganism is a type of bacteria found in the normal or healthy gut microbiota (or "microbiome") of mammals, particularly the human species. See, for example, U.S. Patent Application Publication No. 2014 / 0093478. In some embodiments, the microorganism is an infectious organism such as Clostridium, cholera, Salmonella, Shigella, nematodes (e.g., tapeworms, pinworms, hookworms), and amoebas (e.g., Giardia). Thus, in some embodiments, the microorganism is an intestinal bacterium or pathogen, including both benign and infectious intestinal bacteria and pathogens.

[0031] Appropriate detection methods, though not limited to them, include ELISA, electrochemistry, immunohistochemistry, PCR for DNA, mRNA expression, RNA sequencing, transepithelial electrical resistance, transport assays (ions, compounds, proteins, etc.), secretion assays, electron microscopy, flow cytometry, mass spectrometry of supernatant or reservoir, and other radiochemical assays, fluorescence-based sensors, and the adhesion of microorganisms to epithelial cells.

[0032] While specific examples of colonic monolayers are shown, it is understood that monolayers from other gastrointestinal epithelial cells, particularly small intestinal cells, intestinal cells, gastric cells, esophageal cells, tongue cells, nasopharyngeal cells, oropharyngeal cells, laryngopharyngeal cells, and pancreatic epithelial cells, can also be formed in a manner similar to that described below, or by variations of such techniques that are obvious to those skilled in the art.

[0033] General Considerations for VIP Support ALI Culture ALI (air-liquid interface) cultures can be prepared by removing the liquid or culture medium from either the apical reservoir, the luminal reservoir, or the luminal reservoir, all of which are used indiscriminately. Unlike conventional liquid cultures, the monolayer apical surface of ALI cultures is exposed to air and completely dry. This situation does not reflect the in vivo intestinal lumen environment because the apical surface does not have a high water content. In fact, the water and electrolyte homeostasis of the colonic mucosa is balanced by the movement of water in and out of the lumen. To improve ALI cultures, this disclosure uses intestinal hormones, vasoactive intestinal peptides (VIPs), to assist in balancing fluid movement across the epithelium, by maintaining a thin layer of fluid (approximately 0.1 mm to 1.0 mm thick, or approximately 0.3 mm to 0.5 mm thick, or optionally approximately 0.4 mm thick) in the apical reservoir to suppress drying. This combination of VIP culture and ALI culture, referred to herein as "VIP-supported ALI culture," provides superior results compared to ALI culture alone or in-liquid culture alone. EEC The number of cells increased significantly. An additional advantage of VIP-assisted ALI culture is improved barrier integrity (see Example 2 for details). Although not bound by any particular theory or mechanism of action, in some embodiments, VIP-assisted ALI culture can maintain a thin layer of fluid at the apical end (approximately 0.1 mm to 1.0 mm thick, or approximately 0.3 mm to 0.5 mm thick, or optionally approximately 0.4 mm thick), thereby increasing oxygen availability and eliminating stress due to drying. This disclosure overcomes the limitations of conventional ALI culture or in-liquid culture.

[0034] Referring to the drawings, Figures 1A to 1C include schematic diagrams illustrating the differences between three cell culture mechanisms. Figure 1A is a schematic cross-section of an in-liquid culture 100 in which the apical surface AS (or luminal side of the culture) of differentiated cells DC is covered with differentiation medium DM. In contrast, Figure 1B is a schematic cross-section of an air-liquid interface (ALI) culture 110 in which the apical surface AS is exposed to air A and completely dried. Finally, Figure 1C is a schematic cross-section of a vasoactive intestinal peptide (VIP)-assisted ALI culture 120 in which the vasoactive intestinal peptide VIP stimulates cells (differentiated cells DC and enterochromaffin cells EC) to secrete and maintain a thin layer of water, i.e., secreted water SW, at the apical surface AS. This liquid, i.e., the thin layer of secreted water SW, hydrates the apical surface AS. In each of Figures 1A to 1C, the culture may be located in a culture well W, or in other suitable culture apparatus, culture vessel, system, or device, including, but not limited to, multiwell plates, agar plates, culture dishes, vials, tubes, etc., as known to those skilled in the art. In each of Figures 1A to 1C, the culture includes a porous membrane PM on which cells (differentiated cells DC and enterochromaffin cells EC) are cultured.

[0035] To explain in more detail, Figure 3A shows a monolayer of intestinal epithelium derived from human stem cells. EEC This indicates that the number of EC cells depends on the differentiation strategy. Figure 3A is a schematic diagram of a method or process for generating fully differentiated confluent monolayers derived from primary intestinal epithelial stem cells (SCs) using four differentiation strategies in liquid (Sub) and gas-liquid interface (ALI) cultures, in or in the presence of VIP. As shown in Figure 3A, stem cells (SCs) were seeded on the extracellular matrix (ECM) on a porous membrane (PM). The cells were grown in EM medium for approximately 4 days. Next, the cells were differentiated for up to 5 days using one of the four differentiation strategies. The results of the four differentiation strategies are shown in Figure 3B. Figure 3B is a representative low-magnification fluorescence microscope image of the monolayer. The inset shows a high-magnification image. The markers are as follows: chromogranin A (ChgA, EEC marker); 5HT (EC marker); nuclei stained with Hoechst 33342. The last strategy, VIP-assisted ALI culture, yielded the most abundant EEC A monolayer containing EC cells was generated.

[0036] A method for enriching intestinal endocrine cells in a single-layer system. In some embodiments, methods for generating a living cell construct comprising a cell monolayer containing enteroendocrine cells and subtypes of enteroendocrine cells are provided herein. Such a method may include differentiating stem cells capable of differentiating into enteroendocrine (EEC) cells by culturing them on the upper surface of a cell support structure having both an upper and lower surface until at least a portion of the upper surface of the cell support structure is substantially covered by the stem cells, and by maintaining a thin layer of fluid on the upper surface, thereby differentiating the stem cells, the stem cells generating a living cell construct comprising a substantially continuous cell monolayer containing enteroendocrine cells and subtypes of enteroendocrine cells. The thin layer of fluid may include a liquid, a slurry, a hydrogel, and / or a semi-solid material. The thin layer of fluid on the upper surface can be maintained in a thickness range of approximately 0.001 mm to 10 mm, optionally 0.001 mm to 1 mm, optionally 0.3 mm to 0.5 mm, or optionally 0.4 mm above the luminal side of the cell monolayer. In some embodiments, the thin layer of fluid on the upper surface is maintained by adding hormones and / or compounds (optionally, hormones and / or compounds include chemical substances (e.g., ionomycin), hormones (e.g., vasoactive intestinal peptides, serotonin, gastrin, etc.), cytokines, metabolites, bacteria and / or bacterial components) to the culture medium composition to induce luminal fluid secretion, and optionally, by further adding hypertonic medium to the luminal side to stimulate fluid secretion toward the luminal side and create a thick mucus layer. Furthermore, in some embodiments, the thin layer of fluid can be maintained by a microfluidic flow mechanism.

[0037] In some embodiments, such methods allow for the maintenance of high barrier integrity during culture. Some EECs (e.g., EC cells) can secrete serotonin. EEC (For example, L cells) can secrete glucagon-like peptide-1 (GLP-1). EECIt may secrete peptide YY (PYY), or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin, and / or secretin.

[0038] In some embodiments, such methods are induced by drugs, metabolites, foods, or compounds. EEC These methods are used to screen for hormone secretion from [the body]. EEC It can also be used to screen for drugs, metabolites, foods, or compounds that block the secretion of hormones from the body. Gastrointestinal epithelial cells may be selected from the group consisting of mammalian cells, avian cells, reptile cells, amphibian cells, and insect cells. In some embodiments, the gastrointestinal epithelial cells are human gastrointestinal epithelial cells. In some embodiments, the gastrointestinal epithelial cells are selected from the group consisting of colon cells, small intestinal cells, gastric cells, esophageal cells, tongue cells, nasopharyngeal cells, oropharyngeal cells, laryngopharyngeal cells, and pancreatic epithelial cells.

[0039] In some embodiments, the above method further comprises adding one or more compounds to the growth medium to prevent premature determination of the lineage fate of stem cells during the proliferation stage, wherein optionally, by adding one or more compounds to the growth medium, EEC The formation of can be further enhanced. One or more compounds may be selected from Wnt signaling activators and / or Wnt signaling enhancers, where optionally, the signaling activators include CHIR99021, WAY316606, ABC99, IQ1, and / or arylpyrimidines, and the Wnt signaling enhancers include Wnt-3A, R-spongin 1, and other Wnt1 to Wnt16 and other R-spongins, such as R-spongin 1 to R-spongin 4 proteins.

[0040] Living cell constructs produced by the methods disclosed herein are also provided. The living cell constructs may include a cell monolayer containing enteroendocrine cells and their subtypes.

[0041] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the subject matter of this disclosure pertains. The terms used herein in describing the subject matter of this disclosure are for the sole purpose of describing specific embodiments and are not intended to limit the subject matter of this disclosure.

[0042] With respect to teachings relating to sentences and / or paragraphs that cite references, all publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety.

[0043] Unless otherwise specified in the context, it is particularly intended that the various features of the subject matter of this disclosure described herein may be used in any combination. Furthermore, in some embodiments of the subject matter of this disclosure, it is also intended that any feature or combination of features shown herein may be excluded or omitted. For example, if a composition is shown herein to include components A, B, and C, it is particularly intended that A, B, or C, or any combination thereof, may be omitted or abandoned individually or in any combination.

[0044] Similar numbers refer to similar elements throughout. In drawings, certain line thicknesses, layers, components, elements, or features may be exaggerated for clarity. When used, dashed lines indicate any feature or operation unless otherwise specified.

[0045] Where used in the detailed description of the subject matter of this disclosure and the appended claims, the singular forms ("a," "an," and "the") are intended to include the plural forms as well, unless otherwise specified in the context.

[0046] Furthermore, as used herein, "and / or" encompasses not only all possible combinations of one or more of the enumerated items in question, but also the absence of any combinations as interpreted by the alternative ("or").

[0047] As used herein, the term “approximately” when referring to measurable values ​​such as quantities or concentrations means that the specified value is included, as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, if X is a measurable value, “approximately X” means that X is included, as well as variations of X of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. The ranges shown herein for measurable values ​​may include any other ranges and / or individual values ​​within them.

[0048] When used herein, phrases such as "between X and Y" and "about X and Y" should be interpreted as including X and Y. When used herein, phrases such as "about X and Y" mean "about X and about Y," and phrases such as "about X to Y" mean "about X to about Y."

[0049] When an element is described as being "on" another element, "attached" to another element, "connected" to another element, "coupled" to another element, or "contacting" another element, that element may be directly on another element, attached to another element, connected to another element, coupled to and / or in contact with another element, or there may be an intervening element. In contrast, when an element is described as being, for example, "directly on" another element, "directly attached" to another element, "directly connected" to another element, "directly coupled" to another element, or "directly contacting" another element, there is no intervening element. It will also be understood by those skilled in the art that any reference to a structure or feature positioned "adjacent" to another feature may have a portion that overlaps with or lies beneath the adjacent feature.

[0050] Spatially relative terms such as "under," "below," "lower," "over," and "upper" may be used in this specification to describe the relationship between a certain element or feature shown in the drawings and one or more other elements or features, for the sake of clarity.

[0051] In this specification, various elements, components, regions, layers, and / or sections may be described using terms such as "first," "second," etc., but it will be understood that these elements, components, regions, layers, and / or sections should not be limited by these terms. Rather, these terms are used solely to distinguish one element, component, region, layer, and / or section from another. Accordingly, a first element, component, region, layer, or section discussed herein may be referred to as a second element, component, region, layer, or section without departing from the teaching of the subject matter of this disclosure. The order of operations (or steps) is not limited to the order shown in the claims or drawings unless otherwise specifically indicated.

[0052] As used herein, the terms “comprise,” “comprises,” and “comprising” identify the presence of a specified feature, integer, process, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof.

[0053] When used herein, the transitional phrase "consisting essentially of" should be interpreted as encompassing materials or processes that do not substantially affect the specified materials or processes enumerated in the claims, as well as any basic and novel properties described in the claims. Therefore, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising" when used in the claims of the present invention.

[0054] As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and similar grammatical variations) describe increases of at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, and 500% or more compared to the control.

[0055] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and similar grammatical variations) describe a reduction of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% compared to a control. In certain embodiments, a reduction may result in no detectable activity or amount at all or essentially none (i.e., a negligible amount, e.g., less than about 10% or even less than 5%).

[0056] In some embodiments, the stem cells useful in this disclosure may include, but are not limited to, epithelial stem cells, intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, respiratory stem cells, gastric stem cells, nasal stem cells, genital duct cells (cervix, vagina, uterus), urethral cells, olfactory cells, oral cells, tongue cells, and / or conjunctival cells. In some embodiments, the stem cells are intestinal epithelial stem cells.

[0057] In some embodiments, a living cell construct comprising a cell monolayer may comprise one or more different cell types (e.g., one, two, three, four, five, or more). As used herein, “cell type” refers to morphologically or phenotypically distinct cell morphologies within a species. In some embodiments, cells placed on a cell-supporting structure may originate from healthy, inflamed, or diseased human or animal tissue. In some embodiments, cells useful for constructing the living cell constructs of the subject matter of this disclosure may originate from human or animal tissue having diseases including, but are not limited to, inflammatory bowel disease, constipation, cystic fibrosis, irritable bowel syndrome, leaky gut syndrome, bacterial overgrowth syndrome, celiac disease, lactose intolerance, excessive gas syndrome, diarrheal diseases, and / or polyps, appendicitis.

[0058] In some embodiments, the cell layer of the subject of this disclosure may be a flat two-dimensional structure, as shown, for example, in Figures 1A to 1C. In some embodiments, the cell monolayer of the subject of this disclosure may be folded into a three-dimensional shape or structure to mimic, for example, the crypt structure or crypt-villous structure of the intestine in vivo.

[0059] In some embodiments, the subject matter of the present disclosure may further include placing an impermeable physical barrier and / or a partially permeable (i.e., semipermeable) physical barrier on the luminal side of or above (and on or above / superimposed on) a cell monolayer containing mucus-producing cells. In some embodiments, water transport can be regulated by controlling the movement of liquid / water or water vapor.

[0060] In some embodiments, the volume of liquid culture medium in the luminal (apical) reservoir (with or without a physical barrier) or on the luminal side of the cell is located above the luminal side of the cell monolayer, in a range of approximately 0.001 mm to approximately 10 mm (for example, approximately 0.001 mm, 0.002 mm, 0.003 mm, 0.004 mm, 0.005 mm, 0.006 mm, 0.007 mm, 0.008 mm, 0.009 mm, 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.2 mm, 0. 3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, or 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm , 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, 4 0.5mm, 4.6mm, 4.7mm, 4.8mm, 4.9mm, 5mm, 5.1mm, 5.2mm, 5.3mm, 5.4mm, 5.5mm, 5.6mm, 5.7mm, 5.8mm, 5.9mm, 6mm, 6.1mm, 6.2mm, 6.3mm, 6.4mm, 6.5mm, 6.6mm, 6.7mm, 6.8mm, 6.9mm, 7mm, 7.1mm, 7.2mm, 7.3mm, 7.4mm, 7.5mm, 7.6mm, 7.7mm, 10mm, or any value or range within these ranges) (for example, approximately 0.005mm to approximately 10mm, 0.01mm to approximately 10mm, The depth may be approximately 0.05mm to 10mm, 0.1mm to 10mm, 0.5mm to 10mm, 1mm to 10mm, 5mm to 10mm, 0.001mm to 1mm, 0.005mm to 1mm, 0.01mm to 1mm, 0.05mm to 1mm, 0.1mm to 1mm, 0.5mm to 1mm, 0.001mm to 0.1mm, 0.005mm to 0.1mm, 0.01mm to 0.1mm, 0.05mm to 0.1mm, 0.1mm to 0.1mm, 0.5mm to 0.1mm, or any value or range within these ranges.

[0061] As used herein, a "partially permeable physical barrier" or "porous carrier" is impermeable to or substantially impermeable to mucin, but water can pass through the barrier. Therefore, in some embodiments, a partially permeable physical barrier may have a molecular weight cutoff (MWCO) of about 100 kDa, i.e., the barrier is impermeable to molecules greater than about 100 kDa. In some embodiments, a partially permeable barrier may have an MWCO of about 100 kDa to about 150 kDa. Mucin has a molecular weight of about 200 kDa to 200 MDa.

[0062] As used herein, the “impermeable physical barrier” is at least substantially, preferably completely impermeable to liquid culture media (e.g., water) and mucin.

[0063] Accordingly, in some embodiments, an impermeable physical barrier or a partially permeable physical barrier (or permeable carrier) can be used to restrain mucin on or near the surface of a mucin-producing cell monolayer. In some embodiments, an impermeable physical barrier and / or a partially permeable physical barrier can be used to prevent or reduce dilution of mucin by the liquid medium as it is produced by the cell monolayer.

[0064] Non-limiting examples of physical barriers include semi-liquid materials (e.g., hydrogels), gas-impermeable membranes, gas-permeable membranes, and hygroscopic materials (honey, glycerin, sugar, nylon, ABS (acrylonitrile / butadiene / styrene), polycarbonate, cellulose, and poly(methyl methacrylate)). In some embodiments, partially permeable (e.g., molecular weight cutoff of about 100 kDa) physical barriers may include, but are not limited to, porous materials including porous membranes, some synthetic polymers, hydrogels (e.g., agarose, gelatin, collagen, Matrigel®, etc.), some oils, and / or meshes (e.g., nylon, photoresist, polydimethylsiloxane, and other synthetic polymers, etc.). In some embodiments, vapor-permeable physical barriers may be used. Non-limiting examples of vapor-permeable membranes useful in the subject matter of this disclosure include polydimethylsiloxane (PDMS) without coatings / fillers, certain synthetic polymers, and / or meshes. Non-limiting examples of impermeable membranes (impermeable to water and mucin) include solid floaters (e.g., wax, plastics, etc.), meshes (e.g., nylon, photoresist, polydimethylsiloxane, and other synthetic polymers), oils (e.g., mineral oil, perfluorocarbon, natural oil, etc.), and / or synthetic polymers.

[0065] As used herein, “cell support structure” can be any structure on which one or more cells and / or tissues can be placed, and can be organic, inorganic, or a composite thereof, including, for example, any porous membrane or mesh membrane.

[0066] In some embodiments, the cell support structure may include an organic polymer such as collagen, typically in combination with other components as discussed below. In some embodiments, the support is porous. As will also be discussed below, the support can be provided with structural support by being placed on or attached to a porous carrier (e.g., a porous membrane, mesh, inorganic grid, hydrogel, or a combination thereof). The support can be any suitable shape or configuration, including flat, tubular, curved, spherical, ellipsoidal, etc., and may include composites (e.g., to mimic macroanatomical structures).

[0067] Accordingly, useful cell support structures in the subject matter of this disclosure include, but are not limited to, membranes, ECM (extracellular matrix), hydrogels, natural or synthetic polymers, and / or two-dimensional or three-dimensional scaffolds, and / or any combination thereof. In some embodiments, for example, the bottom wall of a tubular reservoir may be a cell support structure (e.g., a membrane). In some embodiments, the cell support structure may include microstructures (e.g., features having a size of less than about 1 mm (e.g., a depth of about 100 microns, 200 microns, or 300 microns, a maximum depth of 800 microns or 1000 microns or more, and / or a width of about 10 microns or 50 microns, a maximum width of 100 microns or 200 microns or more), such as microwells, columns, and / or grooves). In some embodiments, the cell support structure may consist of, for example, polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), cellulose acetate, regenerated cellulose, cellulose nitride, nylon, carbon grid, graphene film, glass, bioglass (e.g., 45S5 bioglass), hydroxyapatite, calcium phosphate, silicon, silicon oxide, silicon nitride, titanium oxide, aluminum oxide, gold, nickel, and / or stainless steel, or any combination thereof.

[0068] In some embodiments, non-porous materials useful as cell support structures of the subject matter of this disclosure can be made porous by methods including, but not limited to, sintering, etching, leaching, lithography, and laser microfabrication. For example, porous meshes of silicon and gold can be processed by lithography / etching. In some embodiments, photoreactive polymers such as photoresists, which are processed into films having micropores or nanopores or micromesh or nanomesh by photolithography, can be used as cell support structures. In some embodiments, elastomer films such as polydimethylsiloxane (PDMS) or EcoFlex, which are processed into porous films or micro / nanomesh by soft lithography or molding, can also be used as cell support structures. In some embodiments, the cell support structure may also be a dehydrated or flexible but strong matrix such as a collagen or fibrin film or composite.

[0069] Cells and / or tissues may be placed on cell support structures or scaffolds with or without additional adhesion proteins or extracellular matrix. In some embodiments, the scaffold may include, but is not limited to, extracellular matrix (ECM) materials comprising collagen, gelatin, laminin, elastin, fibronectin, vitronectin, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, gelatinous protein mixtures secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (e.g., Matrigel®, Geltrex®, MaxGel®, etc.), and / or commercially available cell substrates (e.g., CELLstart®, CTS®), and any combination thereof (e.g., collagen / Matrigel® mixture). In some embodiments, two-dimensional or three-dimensional scaffolds can be constructed using hydrogels from natural polymers, synthetic polymers, and hybrid hydrogels. Examples of natural and synthetic polymers include, but are not limited to, chitosan, agarose, alginates (e.g., AlgiMatrix®), fibrin, silk, polyvinyl alcohol, sodium polyacrylate, acrylate polymers, polyethylene glycol (PEG), synthetic peptides, poly-N-isopropylacrylamide, and / or polyacrylamide, and / or any combination thereof. In some embodiments, the surface of the scaffold can be manipulated to promote cell adhesion with ECM molecules, natural or synthetic polymers, or, but not limited to, synthetic peptides including poly-l-lysine, RGD-peptides, and other integrin-recognizing peptide segments. In some embodiments, cell-supporting structures useful in the subject matter of this disclosure can be mixed with cellular materials (immune cells or other cell types, tissues, blood) or non-cellular materials (drugs, polymer beads, magnetic particles, etc.). In some embodiments, the cell-supporting structures may include two-dimensional or three-dimensional micropatterns or microstructures.

[0070] In some embodiments, the cells of a living cell construct may be cultured in a liquid medium containing, for example, additives, compounds, and / or solutions that can contribute to the water balance across the entire cell layer. In some embodiments, additives, compounds, and / or solutions may include, but are not limited to, hormones, chemical additives, food additives, bacterial metabolites, and / or hypertonic salt solutions. In some embodiments, additives, compounds, and / or solutions may be present in / in the luminal reservoir (luminal side of the cell monolayer) and / or the basal reservoir (basal side of the cell monolayer). For example, hormones that stimulate the secretion of water and electrolytes into the intestinal lumen can be added to the basal side (basal reservoir) of the cell monolayer to help balance fluid movement across the cell monolayer. In contrast, food additives and bacterial metabolites can be added to the luminal side (luminal reservoir) of the cell monolayer.

[0071] In some embodiments, the disclosed living cell constructs, and related methods and systems, may include culture wells, or other suitable culture apparatus, culture vessels, systems, or devices known to those skilled in the art, including, but not limited to, multiwell plates, agar plates, culture dishes, vials, tubes, etc., for accommodating cell support structures, porous membranes, etc., and / or for creating luminal reservoirs and / or basal reservoirs described and shown herein (see, for example, Figures 1 and 3).

[0072] In some embodiments, hormones useful in the subject matter of this disclosure include, but are not limited to, vasoactive intestinal peptides (VIPs), 5-hydroxytryptamine (serotonin, 5-HT), substance P, bone morphogenetic protein (BMP), gastrin, cholecystokinin, secretin, ghrelin, motilin, gastric inhibitory polypeptides, leptin, glucagon-like peptides, somatostatin, and / or neurotensin.

[0073] Non-limiting examples of useful chemical additives include, but are not limited to, but but include butyrate, dibenzazepine, gamma-secretase inhibitors (DAPT, LY411575), forskolin, guaifenesin, carbachol, prostaglandins, phorbal ester (phorbal 12-myristate 13-acetate), histamine, and / or N-(1-oxobutyl)-cyclic 3',5'-(hydrogen phosphate)2'-butanoate-adenosine, monosodium salt (i.e., dibutyryl-cAMP, sodium salt) (CAS 16980-89-5).

[0074] Examples of food additives include N-nitrosoanabasin, matairesimol, and / or caffeine.

[0075] In some embodiments, bacterial metabolites may include, but are not limited to, short-chain fatty acids.

[0076] In some embodiments, the salts in the hypertonic salt solution useful in the subject matter of this disclosure include, but are not limited to, sodium, chlorine, potassium, magnesium, phosphates, carbonates, and / or lithium. In some embodiments, the concentration of the salt in the hypertonic solution is approximately 1 mM to approximately 1000 mM (for example, approximately 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM). M, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 26 0mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM, 305mM, 310mM, 315mM, 320mM, 325mM, 330mM, 335mM, 340mM, 345mM, 350mM, 3 55mM, 360mM, 365mM, 370mM, 375mM, 380mM, 385mM, 390mM, 395mM, 400mM, 400mM, 405mM, 410mM, 415mM, 420mM, 425mM, 430mM, 435mM, 440mM , 445mM, 450mM, 455mM, 460mM, 465mM, 470mM, 475mM, 480mM, 485mM, 490mM, 495mM, 500mM, 505mM, 510mM, 515mM, 520mM, 525mM, 530mM, 535 mM, 540mM, 545mM, 550mM, 555mM, 560mM, 565mM, 570mM, 575mM, 580mM, 585mM, 590mM, 595mM, 600mM, 605mM, 610mM, 615mM, 620mM, 625mM, 6 30mM, 635mM, 640mM, 645mM, 650mM, 655mM, 660mM, 665mM, 670mM, 675mM, 680mM, 685mM, 690mM, 695mM, 700mM, 705mM, 710mM, 715mM, 720mM,725mM, 730mM, 735mM, 740mM, 745mM, 750mM, 755mM, 760mM, 765mM, 770mM, 775mM, 780mM, 785mM, 790mM, 795mM , 800mM, 805mM, 810mM, 815mM, 820mM, 825mM, 830mM, 835mM, 840mM, 845mM, 850mM, 855mM, 860mM, 865mM, 870mM It could be 875mM, 880mM, 885mM, 890mM, 895mM, 900mM, 905mM, 910mM, 915mM, 920mM, 925mM, 930mM, 335mM, 940mM, 945mM, 950mM, 955mM, 960mM, 965mM, 970mM, 975mM, 980mM, 985mM, 990mM, 995mM, 1000mM, and any value or range within those. Therefore, in some embodiments, the concentration of the salt in this solution is approximately 5 mM to 50 mM, approximately 5 mM to 100 mM, approximately 10 mM to 100 mM, approximately 10 mM to 250 mM, approximately 10 mM to 500 mM, approximately 10 mM to 1000 mM, approximately 50 mM to 100 mM, approximately 50 mM to 500 mM, approximately 50 mM to 1000 mM, approximately 100 mM to 250 mM, approximately 100 mM to 500 mM, approximately 100 mM to 1000 mM, approximately 200 mM to 50 It can be 0 mM, approximately 200 mM to 1000 mM, approximately 300 mM to 500 mM, approximately 300 mM to 800 mM, approximately 300 mM to 1000 mM, approximately 400 mM to 500 mM, approximately 400 mM to 800 mM, approximately 400 mM to 1000 mM, approximately 500 mM to 750 mM, approximately 500 mM to 1000 mM, approximately 600 mM to 1000 mM, approximately 700 mM to 1000 mM, approximately 800 mM to 1000 mM, and any value or range within these ranges.

[0077] Any substance useful for the growth / maintenance of cells and / or tissues can be introduced into the basal reservoir or luminal reservoir. In some embodiments, the substances may include, but are not limited to, fibronectin, laminin, epidermal growth factor (EGF), R-spongin, noggin, cytokines (e.g., interleukins (e.g., IL-6, IL-17, IL-22), tumor necrosis factor (TNF)), ephrin receptors (e.g., ephrin B, EphB), bone morphogenetic proteins (BMP, BMP-2, BMP-7), Wnt (wingless integration sites) (e.g., Wnt3, Wnt3A, and other Wnt), notch signaling factors (notch receptors), Dll1 / 4, noggin, Grem1, Grem2, acetate, butyrate, propionate, desaminotyrosine, catecholamines (e.g., dopamine, norepinephrine), cytokines, and / or short-chain fatty acids.

[0078] In some embodiments, a method is provided for evaluating the effectiveness of a drug for preventing infection by an organism or reducing the infectivity of an organism, comprising: bringing the luminal side of a living cell construct of the subject matter of this disclosure into contact with an organism; bringing the luminal side of the living cell construct into contact with a drug; and determining whether the organism infects one or more cells in the cell monolayer of the living cell construct, wherein the drug is determined to be effective in preventing infection by the organism or reducing the infectivity of the organism if, compared to a control (i.e., contact with the organism but not with the drug), the organism does not infect one or more cells in the cell monolayer of the living cell construct. In some embodiments, the contact between the luminal side of the living cell construct and the organism occurs before, simultaneously with, or after contact between the luminal side of the living cell construct and the drug. In some embodiments, the drug is present in an amount of approximately 25% to approximately 100% of the organism (for example, approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6%). It may be determined that the killing or injuring occurs at any of the following percentages (3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, and any value or range within these percentages).

[0079] In some embodiments, the subject matter of the present disclosure provides a method for evaluating the immunological response of a cell monolayer to biological invasion, particle contact, and / or chemical / compound contact, the method comprising: contacting the luminal side of a living cell construct of the subject matter of the present disclosure with a biological organism, a particle, and / or chemical / compound; and assaying the cells of the cell monolayer of the living cell construct for the production of markers related to the immune response (e.g., cytokines, chemokines, hormones, neurotransmitters, and / or antimicrobial peptides), thereby evaluating the immunological response of the cell monolayer of the living cell construct to contact with a biological organism, a particle, and / or chemical / compound contact.

[0080] In some embodiments, the chemical substance and / or compound may include, but is not limited to, food metabolites and / or bacterial metabolites such as vitamins or short-chain fatty acids.

[0081] The organisms that can be studied using the methods and living cell constructs of the subject matter of this disclosure may be any organism, such as bacteria, viruses, fungi, protozoa, and / or helminths. Therefore, for example, any bacteria, virus, fungi, protozoa, or helminths can be studied for their ability to infect cells to evaluate the effectiveness of drugs that prevent infection by organisms / reduce the infectivity of organisms, and / or the immunological response of cells in living cell constructs in response to contact by organisms.

[0082] In some embodiments, the organism may be a bacterium. Non-specific examples of bacteria include multiple species of the genera Escherichia, Yersinia, Salmonella, Campylobacter, Clostridium, Helicobacter, Bacteroides, Peptostreptococcus, Vibrio, Shigella, Salmonella, and Listeria. Examples include bacteria from Staphylococcus spp. and multiple species of the genus Staphylococcus. In some embodiments, the bacteria may include, but are not limited to, Acinetobacter baumannii, Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelil, Borrelia recurrentis, Brucella abortus, Brucella canis, and Brucella meritensis. melitensis, Brucella suis, Burkholderia pseudomalayCampylobacter pseudomallei, Chlamydia jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium amycolatum, Corynebacterium diphtheriae, Coxiella burnettii Ehrlichia burnetii, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterotoxigenic Escherichia coli, Enteropathogenic Escherichia coli, Enteroinvasive Escherichia coli, enterohemorrhagic Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori Klebsiella pneumoniae, Legionella pneumophilaPneumophila, several species of the genus Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidus, Parachlamydia, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii, Salmonella bongoli bongori), Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, Vibrio vulnificus Examples include Vibrio vulnificus, Vibrio parahaemolyticus, and / or Yersinia pestis.

[0083] In some embodiments, the organism may be a protozoan. Non-limiting examples of protozoans include those from the phyla Amoebozoa, Excavata, and / or Chromalveolata. In some embodiments, the protozoan may include, but is not limited to, those from the genera Amoeba, Entamoeba, Plasmodium, Giardia, and / or Trypanosoma. In some embodiments, the protozoa may include, but are not limited to, Entamoeba histolytica, Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora cayetanensis, and / or Giardia lamblia.

[0084] In some embodiments, the organism can be a virus. Non-limiting examples of viruses include Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lymphocryptovirus, Rhadinovirus, Adenovirus, Astrovirus, Calicivirus, Mastadenovirus, Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus, Nupapillomavirus, Polyomavirus, Molluscipoxvirus, Orthopoxvirus, Orthopoxvirus, Parapoxvirus, Alphatorquevirus, Betatorquevirus, Gammatorquevirus, Gemycircularviruses, Erythrovirus, Dependovirus, Bocavirus, Coltivirus, Rotavirus, Searnavirus, Hepevirus, Alphacoronavirus, Betacoronavirus, Torovirus, Mamastrovirus, Norovirus, Sapovirus, Flavivirus, Hepacivirus,Pegivirus, Cardiovirus, Cosavirus, Enterovirus, Hepatovirus (e.g., Hepatitis A), Kobuvirus, Parechovirus, Rosavirus, Salivirus, Alphavirus, Rubivirus, Deltavirus, Lyssavirus, Vesiculovirus, Filoviridae, Ebolavirus, Marburgvirus Paramyxoviridae, Henipavirus, Morbilivirus, Respirovirus, Rubulavirus, Metapneumovirus, Pneumovirus, Arenavirus, Peribunyaviridae, Orthobunyavirus, Hantavirus, Nairovirus, Phenuiviridae, Phlebovirus, Influenza A virus A) Examples include influenza B virus, influenza C virus, togotovirus, gammaretrovirus, deltaretrovirus, lentivirus, spumavirus, and / or orthohepadnavirus.

[0085] In some embodiments, the organism may be a helminth, including, but not limited to, enteric flukes, roundworms, pinworms, and / or tapeworms. In some embodiments, the helminth may include, but not limited to, helminths from the genera Ascaris, Ancylostoma, Trichuris, Strongyloides, Necator, Schistosoma, and / or Trichinella. Further non-exclusive examples of helminths include Ascaris lumbricoides (roundworm), Ancylostoma duodenale (hookworm), Necator americanus (hookworm), Strongyloides stercoralis, Trichinella spiralis, and / or Trichuris trichiura (whipworm).

[0086] In some embodiments, the organism may be a fungus. Non-specific examples of fungi include multiple species of the genera Candida, Aspergillus, Mucor, Fusarium, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Lichtheimia, Pneumocystis, and Sporothrix. This includes fungi from the spp. and / or multiple species of the genus Cunninghamella (Cunninghamella spp.). Further non-exclusive examples of fungi include Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Cryptococcus gattii, Pneumocystis jirovecii, and / or Torulopsis glabrata.

[0087] The subject matter of this disclosure is described here with reference to the following embodiments. These embodiments should be understood not as being intended to limit the claims to the subject matter of this disclosure, but rather as examples of certain embodiments. Any variations of the illustrated methods that can be conceived by those skilled in the art are intended to be included within the scope of the subject matter of this disclosure. [Examples]

[0088] The following embodiments are included to further illustrate various embodiments of the subject matter of this disclosure. However, those skilled in the art should understand that many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the subject matter of this disclosure, and similar or equivalent results can still be obtained.

[0089] Example 1 Conventional monolayer culture methods are insufficient for reliable hormone assays. EEC Furthermore, it is not possible to generate EC cells. The culture conditions for the intestinal epithelial monolayer platform have been optimized in previous publications. EEC The optimal culture medium composition for obtaining the maximum expression level of the lineage marker gene ChgA was investigated.11 Nicotinamide and the p38MAP kinase inhibitor (SB202190), two compounds commonly used in intestinal epithelial cell culture, were removed from the medium because they inhibit differentiation into secretory cells. For proper differentiation and cell morphology of the monolayer culture, the inclusion of A8301, a TGF-β receptor signaling inhibitor, is necessary. Here, using the same culture medium composition and conventional liquid culture, in vitro monolayers were generated from human transverse colon-derived stem cells. EEC(ChgA + ) Cells and EC(5HT + Each cell is approximately 22±5mm in size. -2 and 10±3mm -2 It was present at the following density (Figures 2A and 2B). Therefore, the 96-well insert (surface area = 11 mm²) 2 The total number of EC cells in the sample was 110 ± 33. To test whether the number of EC cells was sufficient for a functional assay, the monolayer was stimulated apex with a vehicle (dimethyl sulfoxide [DMSO]) or forskolin (10 μM) for 4 hours, and 5HT released from the basal end was collected and quantified (Figure 2C). Forskolin is a potent stimulant of 5HT release through activation of the cAMP-dependent pathway. 18In the three independent experiments, forskolin-stimulated 5HT secretion was 0.97±0.34 ng / mL, 0.88±0.28 ng / mL, and 0.60±0.23 ng / mL, respectively, corresponding to 3.6-fold, 2.1-fold, and 2.4-fold induction compared to the vehicle control (0.27±0.10 ng / mL, 0.42±0.18 ng / mL, and 0.25±0.07 ng / mL, respectively). Due to the high standard deviation of the 5HT measurements, significant variability was observed between experiments: the results in the first experiment were statistically non-significant (p>0.05, t-test), statistically significant in the second experiment (p<0.05), but again considered statistically non-significant (p>0.05) in the third experiment. The high standard deviation was due to several factors: errors in 5HT measurements by enzyme-linked immunosorbent assay (ELISA), well-to-well variability, 5HT residue remaining in the wells (mainly from fetal bovine serum in the culture medium), and 5HT spontaneously leaching from apoptotic ECs during the assay. To improve the reliability of the 5HT secretion assay and its signal / background ratio, monolayers are used. EEC Furthermore, it is necessary to enrich the number of EC cells.

[0090] Example 2 Novel VIP-supported ALI culture method and system EEC and enrichment of EC cells IESCs and their differentiated cells are generally maintained in liquid culture. Recently, applying gas-liquid interface (ALI) culture to intestinal monolayer culture has improved the differentiation of secretory cell lineages, focusing on goblet cells and mucus formation. 19~21Unlike conventional liquid cultures, the monolayer apical surface of the ALI culture was exposed to air and completely dried. In particular, this situation does not reflect the in vivo intestinal lumen environment because the apical surface does not have a high water content. In fact, the water and electrolyte homeostasis of the colonic mucosa is balanced by the movement of water in and out of the lumen. To improve ALI culture, this disclosure uses intestinal hormones, vasoactive intestinal peptides (VIPs), to assist in the balance of fluid movement across the epithelium, thereby maintaining a thin layer of fluid (approximately 0.1 mm to 1.0 mm, or approximately 0.4 mm thick) in the apical reservoir and suppressing drying. This VIP-assisted ALI culture method significantly increased goblet cell formation and assisted in mucus secretion / accumulation. EEC Since both belong to the secretory cell lineage, these simple methods can be used in an in vitro monolayer system. EEC Furthermore, it is highly likely that this will increase the presence of EC cells.

[0091] To verify whether ALI culture and VIP improve differentiation of the EEC lineage, a monolayer of the colon derived from the human transverse colon was differentiated under the four strategies outlined in Figure 3A, and in the monolayer... EEC The presence of EC cells was revealed by immunofluorescence staining (Figure 3B). In-liquid culture (EEC: 22±5 mm) -2 EC: 10±3mm -2 Compared to conventional in-liquid culture without VIP, ALI alone significantly increased the number of EECs (259%) and ECs (260%). Adding VIP to the liquid culture also increased the number of EECs (177%) and ECs (180%), but to a lower degree compared to ALI. A synergistic effect was observed with the combination of ALI and VIP, and this effect resulted in a significant increase in the number of EECs (377%) and ECs (430%) compared to conventional in-liquid culture without VIP (Figures 4A and 4B). In the monolayer, EECs (83±11mm) -2 ) and EC (43±9mm -2 It had an enriched density. The proportion of EC within the EEC remained in the range of 45% to 51% under these differentiation conditions, which is consistent with the proportion of EC in the body, where EC constitutes over 70% of the EEC population in the proximal colon and decreases to about 40% in the rectum.2 To test whether enriched EC cells increase 5HT secretion, monolayers were stimulated with either a vehicle (DMSO) or forskolin (10 μM). 5HT secretion was found to correlate with the number of EC cells (Figure 4C). Monolayers generated from VIP-assisted ALI cultures boosted 5HT secretion under forskolin stimulation (3.79 ± 0.57 ng / mL), which was 390% higher than monolayers generated from liquid culture (0.97 ± 0.44 ng / mL). The induction was 6.1 times higher than that of the vehicle control (0.62 ± 0.07 ng / mL). Therefore, enriched EC cells improve the 5HT secretion assay by increasing the signal-to-background ratio.

[0092] Example 3 VIP-supported ALI culture resulted in the generation of a monolayer with high barrier integrity. In addition to improved 5HT secretion, an additional benefit of VIP-assisted ALI culture is improved barrier integrity (Figure 4D). Of the four differentiation strategies, only VIP-assisted ALI culture exhibits acceptable barrier integrity and a low permeability coefficient (P). app <4×10 -7 (cm / s, n=6) and high transepithelial electrical resistance (TEER, 842±166Ω·cm²). 2 The study found that n=6 cells had the following characteristics. In liquid culture (in or out of VIP), adjacent monolayers could not be formed, likely due to the oxygen gradient in the growth medium. The combination of cellular respiration and the low diffusion rate of oxygen in the growth medium may lead to reduced oxygen availability to cells. 22 ALI culture can enhance oxygen utilization, but the cells were stressed due to complete drying at the apical surface. As a result, adjacent monolayers could not be formed by ALI culture. In contrast, in VIP-assisted ALI culture, a thin layer of liquid (approximately 0.4 mm thick) was maintained at the apical side, thus enhancing oxygen utilization while eliminating stress due to drying. High monolayer integrity is required to effectively isolate the apical and basal reservoirs and prevent leakage of stimulants and 5HT.

[0093] Since the production and storage of 5HT within EC cell granules depends on cell maturity, the level of 5HT secretion is influenced by the length of monolayer differentiation. To determine the optimal differentiation period, monolayers were subjected to VIP-assisted ALI culture for 3 to 7 days. As expected, forskolin-stimulated 5HT secretion increased with the length of differentiation (due to cell maturation), and unstimulated basal secretion also increased (Figure 4E). Barrier integrity was maintained for all samples, except for barrier integrity at less than 7 days of differentiation, when cells may begin to undergo apoptosis (P app <4×10 -7 (cm / s) (Figure 4F). Since a monolayer with high 5HT induction (5.7 times) and monolayer barrier integrity was generated, 5 days was considered optimal for differentiation. This period coincides with the lifespan of differentiated epithelium in the human colon. Unless otherwise specified, 5 days were used for differentiation in this invention.

[0094] Example 4 Subgeneration and donor changes The passage number of stem cells is an important consideration when designing cell-based assays because stem cells are susceptible to replication senescence when grown in vitro. 23 To investigate whether passage number affects 5HT secretion, stem cells from the human transverse colon were seeded on inserts at various passage numbers (7, 9, 11, 13, and 15). No significant changes in 5HT secretion levels (both vehicle- and forskolin-induced) were observed (Figure 5A). Induction of 5HT secretion (forskolin / DMSO) ranged from 4.1 to 5.7 times. Stem cells from passage 16 onwards were discarded due to the possibility of chromosomal abnormalities.

[0095] Primary stem cells derived from various donors are known to exhibit different behaviors under different culture conditions, depending on the age, sex, and health of the individual from which the tissue originated. 24To study donor variability, we investigated variations in cellular behavior related to 5HT secretion using transverse colon stem cells from five cadaveric donors of different ages and sexes (Figure 5B). Significant variations in 5HT secretion levels (both vehicle- and forskolin-induced) were observed (Figure 5C). Donors 3 and 4 showed low 5HT induction (forskolin / DMSO, <3.6), while donors 1, 2, and 5 showed high 5HT induction (greater than 5.0). Significant donor variability suggests that 5HT secretion behavior is complex and may be related to many factors other than sex and age, such as body mass index, blood glucose levels, dietary habits, and health status. Therefore, a complete investigation of donor variability needs to be established for primary cell-based assays. Cells from donor 2 exhibited most representative behavior and were used in the above study and subsequent screening experiments.

[0096] Example 5 Small-scale screening of irritating food ingredients In vivo, EC cells are characterized by a pyramidal shape with a large basal surface. This pyramidal shape was reproduced in vitro on a monolayer platform (Figure 6A). Similar to other EEC subtypes, EC cells sense luminal contents, particularly food compounds and metabolites, through sensory receptors on their cell surface, and release 5HT into the lamina propria via the basolateral boundary (Figure 6B). 7 The adjacent monolayer enriched with EC allows for independent stimulation and analysis of the EC response at the apical and / or basal ends. This is advantageous over the widely used intestinal organoid system, which has lumens that cannot be reached. To demonstrate the usefulness of the monolayer system, thirteen stimulating plant-based food components (Figure 6C) were tested for their ability to regulate 5HT production in response to lumen stimulation (Figure 6D). Among these compounds, curcumin and forskolin were found to be the most potent in inducing 5HT by 7.3 and 4.9 times, respectively. Cinnamaldehyde and bradykinin slightly increased 5HT secretion by 1.8 and 1.4 times, respectively. Curcumin showed the strongest effect at a median effective dose of 51 μM (E50 This compound stimulated 5HT secretion in a dose-dependent manner. Other compounds did not significantly stimulate 5HT secretion.

[0097] Example 6 Existence of other EEC subtypes Enteroendocrine (EEC) cells in the intestinal epithelium consist of at least 15 different subtypes. Enterochromaffin (EC) cells are the most abundant subtype and are found in the digestive tract. EEC They account for approximately 40% of the total. In addition to EC cells, at least one other important EE subtype is the L cell, which is a major regulator of gut-brain interaction and is important in regulating appetite and glucose homeostasis. 4 L cells co-secrete the peptide hormones GLP-1 and PYY in response to food intake. 5~8 GLP-1 stimulates insulin secretion from pancreatic β-cells. 9,10 It reduces gastric emptying and increases the feeling of fullness. 11 PYY slows down gastrointestinal motility and reduces food intake. 12 Due to their important functions as regulators of postprandial responses to nutrients, L cells are of particular interest in the fields of obesity and diabetes. 13~17 L cells were identified at a density of 12±2 mm² even in a monolayer cultured with VIP-ALI, accounting for approximately 14% of the EE population (Figure 7). However, the density of L cells was still too low to perform a reliable functional assay of GLP-1 / PYY secretion. Therefore, further experiments were conducted to develop an optimal protocol for enriching L cells (see Example 7).

[0098] Example 7 Optimization of enteroendocrine L cell formation in stem cell-derived monolayer systems enabling hormone secretion assays. Optimization of differentiation media and culture methods for enriching L cells Experiments were conducted to systematically optimize four conditions for enriching L cells: (1) presence or absence of ALI, (2) presence or absence of VIP, (3) R-spongin / noggin ratios of 1:4, 1:1, and 4:1 in differentiation medium, and (4) presence or absence of BIMU8 (a potent 5-HT4 agonist). Colon stem cells were seeded on a porous membrane coated with Matrigel and cultured for 3 days in growth factor-enriched medium until the cell monolayer reached confluence. The cells were then differentiated for 4 days and treated with GLP-1. + L cell density was used as the reading (Figure 8A). As expected, based on the data presented herein, ALI culture significantly increased L cell formation compared to liquid culture conditions under all conditions. The addition of VIP and BIMU8 during differentiation also increased L cell formation. See Figure 8B. The R-spongin / norging ratio in the differentiation medium also affected L cell density, with the best results obtained by using a 4 / 1 ratio. Using the conditions disclosed herein (ALI, VIP, R-spongin / norging = 4 / 1, BIMU8), a density of 137 ± 12 cells / mm³ was obtained. 2 GLP-1 + L cells were obtained, with a density of 3±2 cells / mm³ under previously used conditions (in liquid, without VIP, R-spongin / noggin = 1 / 1, without BIMU8). 2 It is approximately 46 times higher than that, and in VIP-ALI culture (with ALI, with VIP, R-spongin / noggin = 1 / 1, without BIMU8) it is 12±2 mm 2 It is 11 times higher than that.

[0099] Based on this investigation, the addition of other compounds, chemicals, hormones, etc. to the culture medium in the disclosed system and method may be used. EEC Furthermore, it can further enhance the growth of EC cells, especially the development of L cells. Further non-limiting examples of compounds and hormones are listed in Table 2 below.

[0100] [Table 2]

[0101] Additional compounds suitable for addition to the culture medium in the disclosed systems and methods for increasing L cell formation include, but are not limited to, short-chain fatty acids. 8、11 oligofructose 10 Secondary bile acids, plant-derived compounds, L-amino acids Phe, Trp, Gln, and Ala, and the dipeptide glycine-sarcosine. 11 These are some examples.

[0102] In some embodiments, the addition of such compounds, hormones, etc. to the disclosed culture method and system is, EEC Furthermore, it can enhance the growth of EC cells, particularly the development of L cells.

[0103] References for the above information:

[0104] Improvement in differentiation time and quantification for both GLP-1 markers and PYY markers. The differentiation period (4-6 days) was further improved to maximize the number of L cells and identify the optimal time for hormone secretion assays (Figures 9A and 9B). At 6 days of differentiation, the most abundant L cells for both GLP-1 and PYY markers were generated (Figure 9C). Not all L cells were co-stained with both GLP-1 and PYY. 86% of GLP-1 cells were co-stained. + Cells coexist with PYY, and 81% of PYY + Cells coexist with GLP-1. The cross-sectional image (Figure 9C, bottom) shows the location of L cells within adjacent monolayers. The reachable apex of the L cells allows stimulation from the apical monolayer side (i.e., replication of physiological stimuli). Next, hormones (GLP-1 and PYY) are secreted from the basal side of the monolayer. Due to the high resistance of the monolayer, it can function as an impermeable barrier, so the secreted hormones remain in the basal reservoir and are not diluted across both the apical and basal reservoirs. This further improves the detection limit. The reachable basal side of the monolayer allows for quantification of secreted hormones along with sample collection from the basal reservoir.

[0105] Hormone GLP-1 secretion assay from L cell platform To validate the platform for hormone secretion assays, we studied hormone (GLP-1) secretion under multiple apical stimuli using an optimized monolayer. The apical surface of differentiated colon monolayers was exposed for 4 hours to seven different stimuli that could stimulate GLP-1 secretion. Culture media were collected at the basal end, and GLP-1 was subjected to enzyme-linked immunosorbent assay (ELISA). Forskolin and bombesin were the most potent in inducing GLP-1 secretion, approximately 6 times higher than the control (DMSO). These data (Figure 10) demonstrate that this system can be used for screening the effects of drugs, bacteria and other microorganisms, foods, microbial and food metabolites, and other chemicals on L-cell hormone secretion.

[0106] Example 8 EEC And increasing EC cells by other approaches: preventing the early determination of the fate of stem cell lineages. Stem cells are highly likely to undergo lineage fate determination during the proliferation stage, towards becoming intestinal cells and goblet cells, which are the major cell types in the intestinal epithelium. By adding signaling molecules such as CHIR99021, it is possible to prevent premature lineage fate determination and force stem cells to remain in their basal state. CHIR99021 activates Wnt signaling by inhibiting GSK3. When stem cells were cultured in EM under 3 μM CHIR99021 and then subjected to conventional liquid culture in DM, the presence of EC cells increased by approximately 200% (Figures 11A and 11B). This result suggests that activation of Wnt signaling prevents premature lineage fate determination. In addition to CHIR99021, other Wnt activators (WAY316606, ABC99, IQ1, arylpyrimidine), as well as recombinant proteins of Wnt-3A and R-spongin (Wnt signaling enhancers), may increase EC cell formation. Although the synergistic effects of Wnt activator (in EM) and VIP support ALI (in DM) were not studied, combining both strategies may lead to... EECFurthermore, it is expected that the formation of EC cells will increase even further.

[0107] Discussion of Examples 1 to 8 This disclosure, including the above examples, is more effective than conventional liquid culture. EEC This document presents a novel strategy and system, described herein as VIP-assisted ALI culture, that significantly increases the number of EC cells while simultaneously maintaining high monolayer barrier integrity. The novel strategy, system, and device overcome the limitations of existing EEC enrichment methods by maintaining high cell viability and barrier integrity without requiring complex co-culture or genetic engineering / induction procedures. The resulting EEC-enriched adjacent monolayer platform exhibits high signal-to-background ratio and reproducibility. EEC It functions as a robust analytical tool that enables functional research into hormone secretion.

[0108] References All references listed herein, including, but not limited to, all patents, patent applications and their publications, scientific journal articles, and database entries (e.g., entries in the GENBANK® database and all annotations available therein), are incorporated herein by reference to the extent that they supplement, explain, provide background to, or teach the methodologies, techniques, and / or compositions used herein.

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[0110] It will be understood that various details of the subject matter of this disclosure may be changed without departing from the scope of the subject matter of this disclosure. Furthermore, the detailed descriptions above are for illustrative purposes only and not intended to limit the scope. [Note 1] A method for generating a living cell construct comprising enteroendocrine cells and subtypes of enteroendocrine cells, The method involves culturing stem cells capable of differentiating into enterocrine (EEC) cells on the upper surface of a cell-supporting structure having both an upper and lower surface, until at least a portion of the upper surface of the cell-supporting structure is substantially covered by the stem cells. By maintaining a thin layer of fluid on the upper surface of the support structure, the stem cells are differentiated into EEC cells, A method comprising, wherein the stem cells generate a living cell construct comprising a substantially continuous cell monolayer comprising EEC cells and subtypes of EEC cells. [Note 2] The thin layer of the fluid comprises a liquid, slurry, hydrogel, and / or semi-solid material, as described in Appendix 1. [Note 3] The method according to Appendix 1 or 2, wherein the thin layer of fluid on the upper surface is maintained in a range of approximately 0.001 mm to approximately 10 mm, and optionally approximately 0.001 mm to approximately 1 mm, above the luminal side of the cell monolayer. [Note 4] The method according to any one of the claims 1 to 3, comprising adding a hormone and / or compound (optionally, the hormone and / or compound includes a chemical substance (e.g., ionomycin), a hormone (e.g., vasoactive intestinal peptide, serotonin, gastrin, etc.), a cytokine, a metabolite, a bacterium and / or bacterial component) to a culture medium composition to induce luminal fluid secretion, and optionally further adding a hypertonic medium to the luminal side to stimulate fluid secretion toward the luminal side, thereby maintaining a thin layer of the fluid on the upper surface by creating a thick mucus layer. [Note 5] Chemical compounds (e.g., diterpenoids (e.g., forskolin), methylxanthines (e.g., IBMX)), prebiotics (e.g., oligofructose and inulin-type fructans), hormones (e.g., serotonin, arginine vasopressin, angiotensin II), receptor agonists (e.g., BIMU8, liraglutide (Saxenda), AM1638 (e.g., FFAR1 agonist), AR231453 (e.g., GPR119 agonist), and GPBAR1 agonist), cytokines (e.g., IL-1β, IL-6, TNF-α), bacteria or food metabolites (e.g., acetates, propionates, and butyrates), bacteria and / or bacterial components (e.g., LPS, Akkermansia muciniphila, multiple species of the genus Bifidobacterium) The method according to any one of the appendices 1 to 4, further comprising adding several species of the genus Lactobacillus (spp.) to the living cell construct to increase the density of the EEC cells. [Note 6] Chemical compounds (e.g., diterpenoids (e.g., forskolin), methylxanthines (e.g., IBMX)), prebiotics (e.g., oligofructose and inulin-type fructans), hormones (e.g., serotonin, arginine vasopressin, angiotensin II), receptor agonists (e.g., BIMU8, liraglutide (Saxenda), AM1638 (e.g., FFAR1 agonist), AR231453 (e.g., GPR119 agonist), and GPBAR1 agonist), cytokines (e.g., IL-1β, IL-6, TNF-α), bacteria or food metabolites (e.g., acetates, propionates, and butyrates), bacteria and / or bacterial components (e.g., LPS, Akkermansia muciniphila, multiple species of the genus Bifidobacterium) The method according to any one of the appendices 1 to 5, further comprising adding several species of the genus Lactobacillus (spp.) to the living cell construct to mimic the differentiation stages of EEC cells and further increase the density of EEC cells. [Note 7] The thin layer of fluid is maintained by a microfluidic flow mechanism, as described in any one of the appendices 1 to 6. [Note 8] A method described in any one of the appendices 1 to 7, which maintains high barrier integrity. [Note 9] The EEC cells described above secrete serotonin, according to any one of the methods described in Appendix 1 to 8. [Note 10] The EEC cells secrete glucagon-like peptide-1 (GLP-1), as described in any one of the appendices 1 to 9. [Note 11] The method according to any one of the appendices 1 to 10, wherein the EEC cells secrete peptide YY (PYY), or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin, and / or secretin. [Note 12] The EEC cells described above include L cells, as described in any one of the methods described in Appendix 1 to 11. [Note 13] The density of the aforementioned L cells is approximately 50 cells / mm². 2 Higher, arbitrarily approximately 100 cells / mm 2 A higher method, as described in Appendix 12. [Note 14] A method according to any one of the appendices 1 to 13, used for screening the secretion of hormones from EEC cells induced by drugs, metabolites, foods, or compounds. [Note 15] The method described in any one of the appendices 1 to 14, for use in screening for drugs, metabolites, foods, or compounds that block hormone secretion from EEC cells. [Note 16] The method described in any one of the appendices 1 to 15, for use in screening drugs, metabolites, foods, or compounds that enhance hormone secretion from EEC cells. [Note 17] The method according to any one of the appendices 1 to 16, wherein the gastrointestinal epithelial cells are selected from the group consisting of mammalian cells, avian cells, reptile cells, amphibian cells, and insect cells. [Note 18] The gastrointestinal epithelial cells are human gastrointestinal epithelial cells, as described in any one of the appendices 1 to 17. [Note 19] The gastrointestinal epithelial cells are selected from the group consisting of colon cells, small intestinal cells, gastric cells, esophageal cells, tongue cells, nasopharyngeal cells, oropharyngeal cells, laryngopharyngeal cells, and pancreatic epithelial cells, according to any one of the claims 1 to 18. [Note 20] The method according to any one of the appendices 1 to 19, further comprising adding one or more compounds to the growth medium to prevent early lineage fate determination of stem cells during the proliferation stage, wherein optionally, the addition of the one or more compounds to the growth medium can further enhance the formation of EEC cells. [Note 21] The method according to Appendix 20, wherein the one or more compounds are selected from Wnt signaling activators and / or Wnt signaling enhancers, wherein optionally, the signaling activator comprises CHIR99021, WAY316606, ABC99, IQ1, and / or arylpyrimidines, and the Wnt signaling enhancer comprises Wnt-3A and / or R-spongin proteins. [Note 22] A living cell construct produced by any one of the methods described in Appendix 1 to 21, comprising a substantially continuous cell monolayer containing EEC cells and subtypes of EEC cells. [Note 23] The aforementioned EEC cells are living cell constructs described in Appendix 22, which secrete serotonin. [Note 24] The aforementioned EEC cells are living cell constructs as described in any one of the appendices 22-23, which secrete glucagon-like peptide-1 (GLP-1). [Note 25] The EEC cells described above are living cell constructs as described in any one of the appendices 22-24, which secrete peptide YY (PYY) or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin, and / or secretin. [Note 26] The aforementioned EEC cells include L cells, where the density of the L cells is approximately 50 cells / mm². 2 Higher, arbitrarily approximately 100 cells / mm 2 A higher-grade living cell construct as described in any one of the appendices 22-25. [Note 27] A cell support structure having both an upper surface and a lower surface, and further comprising a porous carrier on the upper surface, A culture vessel that surrounds the cell support structure and provides a region for containing culture medium, A living cell culture system equipped with, A live cell culture system comprising: a culture medium contained beneath the lower surface of a cell support structure, the cell support structure configured to generate a living cell construct from stem cells seeded on the cell support structure, the system configured to differentiate the stem cells into enteroendocrine (EEC) cells by maintaining a thin layer of fluid on the upper surface of the support structure, the thin layer of fluid on the upper surface being maintained by hormones and / or compounds in the culture medium contained in the culture vessel that induce tubular fluid secretion. [Note 28] The system described in Appendix 27, configured to generate a substantially continuous cell monolayer containing EEC cells and subtypes of EEC cells. [Note 29] The system according to any one of the appendices 27 to 28, wherein the thin layer of fluid is maintained by the microfluidic flow of the cell support structure. [Note 30] The culture vessel is a system as described in any one of the appendices 27 to 29, including a multiwell plate, culture dish, vial, or tube. [Note 31] The system according to any one of the appendices 27 to 30, wherein a thin layer of fluid on the upper surface is maintained by a hormone and / or compound in the culture medium that induces the secretion of luminal fluid (optionally, the hormone and / or compound includes a chemical substance (e.g., ionomycin), a hormone (e.g., vasoactive intestinal peptide, serotonin, gastrin, etc.), a cytokine, a metabolite, a bacterium and / or bacterial component), and optionally, a hypertonic medium is further added to the luminal side to stimulate fluid secretion toward the luminal side, thereby creating a thick mucus layer. [Note 32] A method for screening test compounds or microorganisms for toxicological, physiological, or carcinogenic effects, (a) Prepare a cell construct as described in any one of the appendices 22 to 26, (b) The test compound or microorganism is brought into contact with the cell construct, and then, (c) A method for detecting the toxicological, pharmacological, physiological, or carcinogenic effects of the microorganism on the cells of the cell construct by optionally comparing the cell construct after contact with a similar cell construct that has not been in contact with the compound or microorganism, and / or by comparing the cell construct after contact with the cell construct before the contact step. [Note 33] The method according to Appendix 32, wherein the test compound or microorganism is selected from the group consisting of aromatic organic compounds, aliphatic organic compounds, and mixed organic compounds of aromatic and aliphatic types. [Note 34] The method according to any one of the appendices 32 to 33, wherein the test compound or microorganism is selected from the group consisting of Gram-negative bacteria, Gram-positive bacteria, yeast, and mold. [Note 35] The method described in any one of the appendices 32 to 34, including screening for pharmacological interventions related to diabetes. [Note 36] The method described in any one of the appendices 32 to 35, including screening for pharmacological interventions related to obesity.

Claims

1. A method for producing a living cell construct enriched with enteroendocrine cells (EECs), comprising EECs and EEC subtypes, (a) Culturing stem cells capable of differentiating into EECs on a cell support structure comprising both a first surface configured to contact a liquid or gas-liquid interface and a second surface configured to contact a liquid interface, until at least a portion of the first surface of the cell support structure is substantially covered by the stem cells. (b) Differentiating the stem cells into EECs by exposing them to a vasoactive intestinal peptide (VIP) that induces luminal fluid secretion and maintaining a thin layer of fluid on the first surface of the cell-supporting structure, Includes, A method for generating a living cell construct comprising a substantially continuous EEC-enriched cell monolayer containing EEC and subtypes of EEC, wherein the living cell construct is an EEC-enriched cell monolayer containing EEC and subtypes of EEC.

2. The method according to claim 1, wherein the thin layer of fluid comprises a liquid, a slurry, a hydrogel, and / or a semi-solid material.

3. The method according to claim 1 or 2, wherein the thin layer of fluid on the first surface is maintained in a range of 0.001 mm to 10 mm or 0.001 mm to 1 mm above the cell monolayer.

4. The method according to any one of claims 1 to 3, wherein the VIP is added to the culture medium composition to induce luminal fluid secretion.

5. The method according to any one of claims 1 to 4, further comprising adding 5-hydroxytryptamine (serotonin, 5-HT), substance P, BMP-2, BMP-7, gastrin, cholecystokinin, secretin, ghrelin, motilin, gastric inhibitory polypeptide, leptin, glucagon-like peptide, somatostatin, neurotensin, or a combination thereof, to the first and / or second surface of the living cell construct during differentiation.

6. The method according to any one of claims 1 to 5, further comprising adding IBMX, oligofructose, inulin-type fructan, liraglutide, AM1638, AR231453, IL-1β, IL-6, TNF-α, acetate, propionate, butyrate, LPS, Akkermansia muciniphila, multiple species of the genus Bifidobacterium, multiple species of the genus Lactobacillus, or a combination thereof, to the first surface and / or the second surface of the living cell construct during differentiation.

7. The method according to any one of claims 1 to 6, wherein the thin layer of fluid is maintained by a microfluidic flow mechanism.

8. The method according to any one of claims 1 to 7, wherein high barrier integrity is maintained.

9. The method according to any one of claims 1 to 8, wherein the EEC secretes one or more of the following: serotonin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY), or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin, and / or secretin.

10. The method according to any one of claims 1 to 9, wherein the EEC comprises L cells.

11. The method according to claim 10, wherein the density of the L cells is greater than 50 cells / mm² or greater than 100 cells / mm².

12. (a) Inducing the secretion of hormones from the EEC; (b) Blocking hormone secretion from the EEC; and / or (c) Enhance hormone secretion from the EEC The method according to any one of claims 1 to 11, used for screening drugs, metabolites, foods, or compounds.

13. The method according to any one of claims 1 to 12, wherein the stem cells are gastrointestinal epithelial cells.

14. The method according to claim 13, wherein the gastrointestinal epithelial cells are human gastrointestinal epithelial cells.

15. The method according to claim 13 or 14, wherein the gastrointestinal epithelial cells are selected from the group consisting of colon cells, small intestinal cells, gastric cells, esophageal cells, tongue cells, nasopharyngeal cells, oropharyngeal cells, laryngopharyngeal cells, and pancreatic epithelial cells.

16. The method according to any one of claims 1 to 15, further comprising adding one or more compounds to the growth medium in order to enhance the formation of EEC.

17. The method according to claim 16, wherein the one or more compounds are selected from Wnt signaling activators and / or Wnt signaling enhancers.

18. A living cell construct enriched with enteroendocrine cells (EECs), (a) A first surface configured to be in contact with a liquid or gas-liquid interface, the first surface comprising a porous carrier and stem cells capable of differentiating into EECs disposed on the first surface; (b) A second surface configured to contact the liquid interface; (c) A cell culture medium, (i) The second surface is in fluid contact with the fluid surface, (ii) A cell culture medium that is in fluid contact with the first surface via the porous carrier; The cell culture medium includes a vasoactive intestinal peptide (VIP) that induces luminal fluid secretion, thereby differentiating the stem cells into EECs. The EEC-enriched living cell construct is substantially a continuous EEC-enriched cell monolayer, comprising a cell monolayer containing EEC and subtypes of EEC. EEC-enriched living cell constructs.

19. The EEC-enriched living cell construct according to claim 18, wherein the cell culture medium further comprises vasoactive intestinal peptide (VIP), 5-hydroxytryptamine (serotonin, 5-HT), substance P, BMP-2, BMP-7, cholecystokinin, secretin, ghrelin, motilin, gastric inhibitory polypeptide, leptin, glucagon-like peptide, somatostatin, neurotensin, gastrin, or a combination thereof.

20. The living cell construct enriched with the EEC according to claim 18 or 19, wherein the thin layer of fluid is maintained by microfluidic flow.

21. The EEC-enriched living cell construct according to any one of claims 18 to 20, further comprising a culture vessel that contains the EEC-enriched living cell construct and provides a region for containing a culture medium.

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