Invistro cell culture mucus system

Abstract

This invention provides an in vitro cell culture comprising a cell monolayer containing mucus-producing cells and a mucus layer, as well as a method for preparing and using the same. [Solution] The method involves culturing mucus-producing cells on a cell support structure under conditions necessary to establish a mucus layer on the luminal side of a cell monolayer, thereby producing a living cell construct containing a cell monolayer with mucus-producing cells and a mucus layer. The mucus layer may be substantially impermeable to minute objects and may have a thickness of approximately 1 micron to 1 cm.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 768,259, filed November 16, 2018, the disclosure thereof incorporated in its entirety by reference.

[0002] Appeal for government support This invention was made with government support under grant number DK109559, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0003] This disclosure relates to an in vitro cell culture comprising a cell monolayer containing mucin-producing cells and a mucinous layer, and a method for preparing and using the same. [Background technology]

[0004] The inner surfaces of the small and large intestines are protected by a thick blanket of mucus. The small intestine produces a single layer of mucus 50 to 450 μm thick, while the mucus coating of the large intestine consists of two structurally distinct layers (Non-Patent Literature 1). 1 The outer mucosal layer, 400 to 800 μm thick, is loosely packed and provides a comfortable environment for bacteria to live in, while the inner mucosal layer, 100 to 300 μm thick, is impermeable to bacteria and remains firmly attached to the epithelium (Non-Patent Documents 2 and 3). 2,3 In both organs, mucus is continuously produced and secreted by goblet cells lining the intestinal epithelium. It is estimated that the mucus layer above the cells is replaced on an order of several hours (Non-Patent Literature 4). 4 Mucin 2 (Muc2) is the most abundant gel-forming component of intestinal mucus (Non-Patent Literature 5). 5 The mucous layer is essential for maintaining homeostasis of the colonic mucosa in vivo. Its main functions are to act as a barrier protecting the underlying epithelium from pathogen invasion and to keep the colonic lining lubricated and moist due to the high concentration of water (approximately 97%) present in the mucus (Non-Patent Documents 6 and 7).6,7 The mucus layer also acts as a barrier to prevent the diffusion of molecules and their metabolites derived from food or bacteria, and traps molecules such as secretory IgA (sIgA) and antibacterial peptides secreted by the intestine (Non-Patent Document 8). 8 .

[0005] In vitro intestinal epithelial models provide valuable tools for studying the highly complex intestinal epithelial system in a controlled manner. Adenocarcinoma cell lines, including Caco-2 and HT-29, are widely used as in vitro models of the intestinal epithelium, but they do not generate a high-density mucus layer (Non-Patent Document 9). 9 . HT29-MTX, a stable and homogeneous subpopulation of HT-20 derived after treatment with methotrexate, is often used as a mucus-secreting cell model (Non-Patent Document 10). 10 . However, these tumor cells do not possess normal signaling pathways, physiological mucus secretion, or appropriate responses to external stimuli. In recent years, breakthroughs in intestinal stem cell biology have made it possible to construct in vitro models based on primary intestinal epithelial stem cells (Non-Patent Document 11). 11 . The organoid culture model has realized, for the first time, the in vitro expansion culture and lineage manipulation of adult intestinal epithelial stem cells. The created "mini-guts" organoids possess the diversity of cell lineages found in the in vivo epithelium, including goblet cells that secrete mucus (Non-Patent Documents 11 to 14). 11-14 . The cells are polarized such that their apical surfaces face the lumen that is closed, and their basal sides are attached to Matrigel (registered trademark) or other ECM components (Non-Patent Document 15). 15 . However, mucus is secreted and accumulated into the lumen of the organoid surrounded by a layer of cells and a high-density hydrogel such as Matrigel (registered trademark) or collagen. This mucus secretion is not easily quantified or manipulated, and mucus also does not act as a barrier to external stimuli.

[0006] In attempts to expose the luminal surface, a monolayer model has been established by culturing intestinal epithelial stem cells (e.g., from isolated crypts or dissociated organoid fragments) on a porous membrane coated with either a thick or thin layer of extracellular matrix (Non-patent documents 16-23). 16-23 The cells proliferated in the presence of growth factors (Wnt-3A, R-spondin, and noggin) and differentiated in the absence of growth factors to form continuous monolayers possessing physiological transepithelial electrical resistance (TEER). Due to their open luminal surfaces, monolayers possess inherent advantages compared to organoids. This allows for easy access to the apical epithelium for assaying the impact, absorption, or metabolism of food components, microorganisms, bioactive metabolites, drugs, and toxic compounds. The monolayer model is related to IgA transcytosis (Non-Patent Literature 16). 16 , co-culturing with bacteria (Non-patent documents 17 and 18) 17,18 , Iron transport (Non-patent document 19) 19 , hormone secretion (Non-patent document 19) 19 , co-culture with macrophages (Non-patent document 20) 20 Co-culture with myofibroblasts and intestinal neurons (Non-patent Literature 21) 21 , and cytokine secretion (Non-patent documents 20 and 21) 20,21 It is used to study this. In all of the above-reported monolayer models of primary cells, the cells were in an immersion culture system, i.e., aqueous medium was placed in the base and lumen reservoirs. Trace amounts of mucus were identified by staining with Muc2 antibody (Non-Patent Literature 18). 18 The thickness of the mucus is measured by layering fluorescent microbeads onto the cells and measuring the distance between the cells and beads that are too large to permeate the mucus (Non-Patent Literature 16). 16 However, the mucus layer is not continuous (Non-Patent Document 18). 18 The layer was not thick enough to separate the microbeads from the epithelium (Non-Patent Literature 16). 16Therefore, the beads appear to be in contact with the apical epithelial surface within the resolution of the microscopic image. However, Muc2 was detected in the supernatant above the cells, suggesting that some amount of Muc2 is synthesized and secreted, but not in sufficient quantity or density to reproduce the mucus layer (Non-Patent Literature 24). 24 .

[0007] Therefore, a dense mucus layer with a controllable thickness that is substantially impermeable to microbeads or bacteria has not yet been achieved in any in vitro intestinal epithelial model constructed from primary intestinal epithelial cells. Such an in vitro model is necessary to reflect in vivo conditions. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Allen A. and Carroll NJH, "Adherent and Soluble Mucus in the Stomach and Duodenum," Digestive Diseases and Sciences, 1985; Vol. 30: p. 55S. [Non-Patent Document 2] Pelaseyed T, Bergstrom JH, Gustafsson JK, Ermund A, Birchenough GMH, Schutte A, van der Post S, Svensson F, Rodriguez-Pineiro AM, Nystrom EEL, Wising C, Johansson MEV, Hansson GC. "The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system". Immunological reviews, 2014; Vol. 260: p. 8-20 [Non-Patent Document 3] Murgia X, Loretz B, Hartwig O, Hittinger M, Lehr CM. "The role of mucus on drug transport and its potential to affect therapeutic outcomes". Advanced Drug Delivery Reviews, 2018; Vol. 124: p. 82-97 [Non-Patent Document 4] Lehr CM, Poelma FGJ, Junginger HE, Tukker JJ, "An estimate of turnover time of intestinal mucus gel layer in the rat in situ loop", International Journal of Pharmaceutics, 1991; Vol. 70: p. 235-240

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0009] This summary lists several embodiments of the subject matter of the present disclosure, and in many cases lists variations and permutations of these embodiments. This summary merely illustrates a number and variety of embodiments. References to one or more representative features of a given embodiment are likewise illustrative. Typically, such embodiments may exist with or without the referred features(single or multiple); similarly, such features may apply 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 features.

[0010] In some embodiments, a method is provided herein for producing a living cell construct comprising a cell monolayer containing mucinous cells and a mucus layer, comprising culturing stem cells (e.g., intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, and the like) capable of differentiating into mucinous cells 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 covered by stem cells; and further culturing the stem cells to produce a cell monolayer containing mucinous cells (e.g., goblet cells GC) and other cell types (OC, including enterocytes, enteroendocrine cells, Paneth cells, stem cells, etc.), wherein the cell monolayer has a basal side and a luminal (apical) side, and the mucinous cells of the cell monolayer establish a mucus layer on the luminal side of the cell monolayer, thereby producing a living cell construct comprising a cell monolayer containing mucinous cells and a mucus layer. The mucus layer may be substantially or completely impermeable to microscopic objects. In some embodiments, the thickness of the mucus layer may range from about 1 micron to about 1 cm. In some embodiments, the thickness of the mucus layer may range from about 30 microns to about 1 cm. The GC to OC ratio may range from about 0.1% to about 99.9% in some embodiments.

[0011] In some embodiments, a basal reservoir is located below the basal side of a cell monolayer containing mucinous cells, and a luminal reservoir is located above the luminal side of the cell monolayer containing mucinous cells, with the basal and luminal reservoirs each containing a liquid culture medium. The method further includes: removing the liquid culture medium from the luminal reservoir to create a gas-liquid interface on the luminal side of the cell monolayer containing mucinous cells, and / or adjusting the volume of the liquid culture medium in the luminal reservoir to a depth of approximately 0.001 mm to approximately 10 mm, optionally, approximately 0.001 mm to approximately 1 mm, above the luminal side of the cell monolayer (the mucinous layer is located between (or develops) the liquid culture medium and the cell monolayer). In some embodiments, the method may further include placing an impermeable physical barrier and / or a partially permeable physical barrier on or above the luminal side of the cell monolayer containing mucinous cells. In some embodiments, an impermeable physical barrier and / or a partially permeable physical barrier is in direct contact with the luminal side of the cell monolayer containing mucus-producing cells and / or the mucus layer produced by the mucus-producing cells of the cell monolayer. The liquid medium may be between the impermeable physical barrier and / or the partially permeable physical barrier and the luminal side of the cell monolayer containing mucus-producing cells and / or the mucus layer, and the depth of the liquid medium is approximately 0.001 mm to approximately 10 mm, and optionally in the range of approximately 0.001 mm to approximately 1 mm. In some embodiments, the liquid medium contains hormones, chemical additives, food additives, bacterial metabolites, and / or hypertonic salt solutions (hormones, chemical additives, food additives, bacterial metabolites, and / or hypertonic salt solutions). In some embodiments, the stem cells are epithelial stem cells, intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, respiratory stem cells, gastric stem cells, nasal cavity stem cells, reproductive organ 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. The mucus layer may be substantially impermeable to beads or microorganisms in the size range of about 1 to about 100 microns.

[0012] In some embodiments, a force is applied parallel to the surface of a cell monolayer. In some embodiments, the force includes the application of surface tension forces or mechanical forces. In some embodiments, the mechanical force is a stirrer, a semi-solid material moving parallel to the cell surface, and / or the circulation of slurry above the cell surface.

[0013] In some embodiments, a living cell construct is provided herein that includes a cell monolayer and a mucus layer, which include mucus-producing cells produced by a method disclosed herein. In some embodiments, the mucus layer includes a basal side and a luminal side, with the basal side being adjacent to and below the mucus-producing cells.

[0014] In some embodiments, a living cell construct comprising a cell monolayer containing mucus-producing cells and a mucus layer is provided herein, wherein the mucus layer is substantially impermeable to micro-objectives. The mucus layer may have a thickness ranging from about 1 micron to about 1 cm. The mucus layer may be impermeable to micro-objectives in the size range of about 1 micron to about 100 microns. In some embodiments, the mucus layer may include a basal side and a luminal side, with the basal side being adjacent to and below the mucus-producing cells.

[0015] In some embodiments, methods for determining the ability of an organism, drug, or particle to traverse (permeate) the mucous layer of a cell monolayer are provided herein, the methods comprising bringing the luminal side of the mucous layer of a living cell construct disclosed herein into contact with the organism, drug, or particle, measuring the distance the organism, drug, or particle moves into the mucous layer, and thereby determining the ability of the organism, drug, or particle to traverse (permeate) the mucous layer of a cell monolayer of a living cell construct.

[0016] In some embodiments, methods for studying and evaluating the ability of an organism to infect a cell monolayer including a mucus layer are provided herein, comprising bringing the luminal side of the mucus layer of a living cell construct disclosed herein into contact with the organism, and determining whether the organism traverses the mucus layer and comes into contact with the cell monolayer of the living cell construct. When it is determined that the organism traverses the mucus layer and comes into contact with the cell monolayer, the organism is determined to have the ability to infect a cell monolayer including a mucus layer.

[0017] In some embodiments, methods for evaluating the effectiveness of a drug for preventing or reducing infection by an organism are provided herein, comprising: bringing the luminal side of the mucus layer of a living cell construct disclosed herein into contact with an organism; bringing the luminal side of the mucus layer of the living cell construct into contact with a drug; and determining whether the organism permeates the mucus layer of the cell monolayer and / or infects one or more cells of the cell monolayer of the living cell construct. The drug is determined to be effective in preventing or reducing infection if, compared to a control (i.e., contact with the organism but not with the drug), the organism does not permeate the mucus layer and / or infect one or more cells of the cell monolayer of the living cell construct. If the organism permeates the mucus layer and / or infects one or more cells of the cell monolayer of the living cell construct, the drug is determined to be ineffective. Contacting an organism with the luminal side of the mucus layer of a living cell construct may precede, coincide with, or follow contacting a drug with the luminal side of the mucus layer of a living cell construct.

[0018] In some embodiments, methods are provided herein for evaluating the immunological response of cells including a mucous layer to biological invasion, particle contact, and / or chemical / compound contact, comprising: contacting the luminal side of the mucous layer of a living cell construct disclosed herein with a biological organism, particle, and / or chemical / compound; and assaying the cells of the cellular 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 cellular monolayer of the living cell construct to contact with a biological organism, particle, and / or chemical / compound. The biological organism may be bacteria, viruses, fungi, protozoa, and / or helminths.

[0019] In some embodiments, methods for evaluating mucin dysregulation in an in vitro cell system are disclosed herein, comprising studying the mucinous layer of a living cell construct disclosed herein, wherein the cultured stem cells are from subjects having a disease associated with mucin dysregulation, and / or the cultured stem cells are from a healthy subject and have been gene-edited to represent stem cells from a disease associated with mucin dysregulation; the study comprises evaluating the mucinous layer of the living cell construct for thickness, composition, viscosity, permeability by microorganisms, ability to infect microorganisms, and / or responsiveness to drugs; thereby evaluating mucin dysregulation in an in vitro cell system. In some embodiments, diseases associated with mucin dysregulation include 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.

[0020] The aforementioned and other subjects and aspects of this disclosure are described in detail in the specification presented below.

[0021] Embodiments of the subject matter of this disclosure, achieved in whole or in part by the subject matter of this disclosure, are claimed above in this specification. Other embodiments will become apparent as the description progresses, when combined with the accompanying examples best described below in this specification.

[0022] 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 a constant scale, and instead the emphasis is on illustrating (often schematically) the principles of the subject matter of this disclosure. Similar reference figures in the drawings specify parts that consistently correspond from different viewpoints. Further understanding of the subject matter of this disclosure can be obtained by referring to the embodiments presented in the illustrations in the accompanying drawings. The illustrated embodiments merely illustrate systems for carrying out the subject matter of this disclosure, but both the method and configuration of the subject matter of this disclosure, along with their further objectives and advantages, can generally be better understood by referring to the drawings and the following description. The drawings are intended to clarify and illustrate the subject matter of this disclosure, not to limit the scope of the subject matter of this disclosure as specified and presented in the attached or subsequently amended claims.

[0023] For a more complete understanding of the subject matter of this disclosure, the following drawings are provided for reference. [Brief explanation of the drawing]

[0024] [Figure 1]Figures 1A to 1E provide schematic diagrams of systems and methods according to this disclosure for producing an in vitro mucus layer. Figure 1A provides a gas-liquid interface (ALI) culture. The apical side is exposed to air, and the liquid or culture medium is removed, allowing the mucus to accumulate in a compacted form. Figure 1B illustrates a modified ALI culture. The volume of liquid at the apical side is controlled, allowing the mucus to accumulate in a compacted form and with increased hydration. Figure 1C illustrates an example of using a scaffold as an alternative to a porous membrane. Figure 1D provides an example of using a partially permeable physical barrier (separator) to contain the accumulated mucus. Figure 1E illustrates an example of using an impermeable physical barrier (blocker) to control mucus accumulation. [Figure 2] Figures 2A to 2E provide a schematic example (ALI culture) of creating a compact mucinous layer by removing fluid from the apical epithelial side, and the data obtained. Figure 2A is a schematic diagram showing the immersion and ALI culture formats, including stem cells (SC), mucin-producing goblet cells (GC), other non-mucin-producing cells (OC), and mucin (or diluted mucin) (M). Figure 2B shows immunofluorescence staining of a paraffin-embedded monolayer section (Muc2 and nuclei are labeled). Arrows separate goblet cells. A: apical side; B: basal side. The lower panels (i, ii, and iii) show higher magnification areas marked by corresponding dotted lines in the two upper panels. Figure 2C shows apical surface topography of a human colon monolayer examined by SEM. Upper panel: immersion culture. Lower panel: ALI culture. In Figures 2D and 2E, 1 μm red fluorescent beads (Figure 2D) or GFP-expressing E. coli (Figure 2E) were overlaid on the mucus layer for visualization using confocal microscopy. The nuclei of the intestinal cells were stained with Hoechst 33342. The dotted lines indicate the boundary between the mucus and the microbeads or E. coli. [Figure 3]Figures 3A to 3G show that the hydrated mucus layer separates bacteria or microbeads from the vasoactive intestinal peptide (VIP) incubated epithelium. Figure 3A is a schematic diagram of the culture format. VIP facilitates the movement of water into the lumen or luminal LS, which hydrates the mucus layer. Figure 3B is a graphical representation of concentration-dependent water secretion by VIP after 24 hours of incubation. Figure 3C is an image of the hydrated mucus layer lifted from the epithelium with forceps. Figure 3D includes representative lateral confocal micrographs showing tissue with mucus accumulation separating bacteria at days 0, 2, 4, and 6, respectively. GFP-expressing E. coli and nuclei are labeled. Figure 3E is a plot of mucus thickness against the duration of ALI. Figure 3F is a representative lateral confocal micrograph showing that the mucus layer separated 1 μm red fluorescent beads from the epithelium. Figure 3G shows an image of the apical surface topography of epithelium examined by SEM. The mucous layer was partially removed to reveal the epithelium (dotted line). The upper right panel shows bacteria (rod-shaped structures) above the mucous layer and absent from the epithelial surface. [Figure 4] Figures 4A to 4F show the effects of C. difficile A toxin on human colon epithelium in the absence or presence of a VIP-enhanced mucus layer. Figure 4A is a schematic example showing the cell culture system. Figures 4B and 4C show epithelial permeability (Figure 4B) and IL-8 secretion (Figure 4C) after 4 hours of exposure to A toxin. Figure 4D shows confocal microscopy images, showing the F-actin architecture of the apical membrane (upper panel) and tight junctions stained with ZO-1 (lower panel). Figures 4E and 4F show epithelial permeability (Figure 4E) and IL-8 secretion (Figure 4F) after 8 hours of exposure to A toxin. Unpaired t-test: *P<0.05; **P<0.005; #Not statistically significant. N=3 samples per condition. Scale bar=20μm. [Figure 5]Figures 5A to 5E show cytokine production after 24-hour co-culture of GFP-expressing Escherichia coli (GFP-EC), epithelium, and PBMCs in the absence or presence of a VIP-enhanced mucus layer. Figure 5A is a schematic example showing the co-culture setup. The graphical illustrations in Figures 5B to 5E show basal cytokine production over 24 hours: (Figure 5B) IL-8, (Figure 5C) TNF-α, (Figure 5D) IL-6, and (Figure 5E) IL-1β. Unpaired t-test: **P<0.005; #Not statistically significant. N=3 samples per condition. [Modes for carrying out the invention]

[0025] Herein, the subject matter of this disclosure is described in more detail hereafter, and therein only some embodiments, not all, of the subject matter of this disclosure. In fact, the subject matter of this disclosure can be implemented in many different forms and should not be construed as being limited to the embodiments presented herein; rather, these embodiments are provided so as to satisfy the applicable legal requirements of this disclosure.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by practitioners of the art to which the subject matter of this disclosure pertains. The terminology used herein to describe the subject matter of this disclosure is for the purpose of describing specific embodiments only and is not intended to be limiting to the subject matter of this disclosure.

[0027] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety with respect to the teachings that correspond to the sentences and / or paragraphs in which the references are submitted.

[0028] Unless the context indicates otherwise, it is particularly intended that the various features of the subject matter of this disclosure described herein may be used in any combination. Furthermore, it is also intended that in some embodiments of the subject matter of this disclosure, any feature or combination of features presented herein may be excluded or omitted. For example, if the specification claims that a composition includes components A, B, and C, it is particularly intended that A, B, or C, or any combination thereof, may be excluded individually or in any combination, without claim.

[0029] Similar numbers consistently refer to similar elements. In diagrams, the thickness of certain lines, layers, components, elements, or features may be exaggerated for clarity. Where used, dashed lines illustrate any feature or function unless otherwise specified.

[0030] The singular forms "a," "an," and "the" used in the subject matter of this disclosure and the attached claims are intended to include the plural form unless the context clearly indicates otherwise.

[0031] Furthermore, as used herein, "and / or" refers to and encompasses any of the related listed items, all possible combinations of one or more of them, and, when interpreted selectively ("or"), the absence of any combination.

[0032] As used herein when referring to measurable values ​​such as quantities or concentrations, the term "about" means to include a specified value and variations of that specified value within ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. For example, "about X" means to include X and variations of X within ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% where X is a measurable value. The ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​within them.

[0033] In this specification, phrases such as "between X and Y" and "about X and Y" should be interpreted as encompassing X and Y. In this specification, 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."

[0034] When an element is said to be "on top of," "attached to," "connected to," "linked to," or "in contact with" another element, it will be understood that it may be directly on top of, attached to, connected to, linked to, and / or in contact with the other element, or that there may be an intervening element. In contrast, when an element is said to be, for example, "directly on top of," "directly attached to," "directly connected to," "directly linked to," or "in direct contact with" another element, there is no intervening element. It will also be understood by those skilled in the art that a structure or feature positioned "adjacent to" another feature may have a portion that overlaps with or is underneath the adjacent feature.

[0035] Spatially relative terms, such as "below," "below," "on the lower side," "above," "on the upper side," and similar terms, may be used herein for the ease with which they can be used to describe the relationship of one element or feature to another element(s) or feature(s)(s)(s)(s)(s), as illustrated in the figures.

[0036] The terms "first," "second," etc., may be used herein to describe various elements, components, regions, layers, and / or sections, 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. Therefore, 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 teachings of the subject matter of this disclosure. The sequence of operations (or steps) is not limited to the order in which they are presented in the claims or figures unless otherwise indicated.

[0037] As used herein, the terms “comprise,” “comprises,” and “comprising” specify the existence of the claimed feature, complete, step, action, element, and / or component, but do not preclude the existence or addition of one or more other features, complete, step, action, element, component, and / or groups thereof.

[0038] As used herein, the transitional phrase "essentially consisting of" should be interpreted as encompassing the specified materials or steps described in the claim and things that do not substantially affect the fundamental and novel nature (singular or plural) of the claimed invention. Therefore, the term "essentially consisting of" is not intended to be interpreted as equivalent to "including" when used in the claims of the present invention.

[0039] As used herein, the terms “increase,” “increase,” “increased,” “strengthen,” “strengthened,” “strengthen,” and “strengthen” (and their grammatical variations) refer to an increase of at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to the control.

[0040] As used herein, the terms “reduce,” “reduce,” “reduce,” “reduce,” “decrease,” and “decrease” (and their grammatical variations) refer to a reduction of, for example, 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, the reduction may be without or essentially without any detectable activity or quantity (i.e., it may result in an insignificant amount, e.g., about 10% or even less than 5%).

[0041] As used herein, the term "micro-object" refers to an object having dimensions of approximately 0.1 to 100 μm, which may be used to determine the permeability of a mucus layer. Examples of micro-objects include, but are not limited to, microorganisms (e.g., bacteria) and fine particles (e.g., beads).

[0042] This disclosure describes the discovery of a method for creating a living cell construct comprising a cell monolayer containing mucus-producing cells and a mucus layer, which outlines, for example, the in vivo mucus layer present in the intestinal tract and / or reproductive organs of human and animal subjects. A thick in vivo-like mucus barrier may be important for the successful reproduction of the gut environment for the microbiome and can also serve as an in vitro tool for studying mucosal drug and particle delivery. Although mucus production has been previously observed in in vitro cell systems, these systems have not been able to produce mucus in sufficient quantity or density to reproduce a mucus layer that mimics the mucus layer found in vivo. Without being limited to any particular theory, it is assumed herein that the mucus of these previous in vitro cell systems may be rapidly diluted after being secreted from goblet cells in immersion culture systems and therefore lack the ability to accumulate and form a dense layer (substantially impermeable to microorganisms and particles). As described herein, the subject matter of this disclosure overcomes these limitations.

[0043] In some embodiments, and as shown in Figures 1A to 1E, the Disclosure provides a living cell construct in a reservoir R comprising a cell layer containing a combination or mixture of myxoprofen goblet cells (GC) and other non-mucus-producing cells (OC), such as enterocytes, enteroendocrine cells, Paneth cells, stem cells, etc. (the cells have a luminal LS or apical and basal BS relative to the cell, and a porous matrix (PM) / scaffold / base below the cell); and a method for facilitating mucus accumulation above the apical / luminal LS of the cell to form a high-density in vivo-like mucus layer. The GC to OC ratio may range from about 0.1% to about 99.9% in some embodiments. Therefore, the cell monolayer of the living cell construct disclosed herein may, in some embodiments, comprise a mixture of myxoprofen and non-mucus-producing cells.

[0044] To create a cell layer containing mucus-secreting cells, epithelial stem cells are cultured on the surface of a porous membrane. To support the cells, the membrane can be coated with a thin (e.g., about 1 to 1,000 nanometers) extracellular matrix (ECM) or other chemicals, or with a thick (e.g., about 1 to about 100,000 micrometers) hydrogel scaffold material (ECM or synthetic hydrogel). The cells are grown in the culture medium until a portion of the surface is covered by the cells. The stem cells (SCs) may be allowed to differentiate spontaneously as they begin to cover the surface and / or consume nutrients in the adjacent culture medium. Alternatively, cells may be switched to different media that promote the formation of mucin-producing cells (e.g., goblet cells (GC)) and other cell types (OC), or chemical additives to induce differentiation may be incorporated into the media (e.g., butyrate, bone morphogenetic protein (BMP), gamma-secretase inhibitors (e.g., DAPT, LY411575, dibenzazepine), etc.).

[0045] Then, a mucus layer can be established on the apical cell side / luminal cell side LS using different methods, as illustrated in Figures 2A to 2E. For example, as shown in Figure 2A, a gas-liquid interface (ALI) culture can be prepared, in which liquid or culture medium is removed from the apical reservoir or the luminal side LS or luminal reservoir. ALI conditions allow for mucus accumulation as a dense, compacted layer or compacted mucus layer CML along the apical surface of the luminal side LS (opposite to the basal side BS) above the mucus-producing goblet cells GC and / or OC (which form the epithelium). In some embodiments, ALI conditions can allow the accumulation of a high-density or concentrated mucus layer (CML) by (a) minimizing dilution of secreted mucus, (b) increasing forces on the cell surface due to surface tension or other effects (which promote mucus production and / or secretion), and / or (c) increasing local culture medium osmorality above the cells. The movement of water through mucus-producing goblet cells (GC) due to evaporation on their surfaces can also stimulate mucus production. See Example 1.

[0046] As another example, a modified ALI culture can be prepared as shown in Figure 1B. This approach provides control over the height of the liquid medium above the apical / luminal LS of the mucus-producing cell monolayer so that the mucus accumulated above the cell monolayer is dense, hydrated, and in vivo-like, i.e., a hydrated mucus layer. A liquid or medium depth ranging from approximately 0.001 mm to approximately 10 mm can be provided and maintained above the apical or luminal cell LS so that the mucus accumulating over time is more hydrated relative to that of ALI. The volume of liquid / medium above the luminal LS of the reservoir R can be controlled to produce mucus with a wide range of mucus densities and compactness, depending on experimental needs.

[0047] In some embodiments, one or more materials or substances may be added to the apical or luminal surface of cells to help maintain an aqueous film above the apical cell surface, and may also be used to help program or maintain a desired fluid height over time. For example, semi-liquid masses (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)) may be placed on the apical surface. Chemical reagents, hormones, food metabolites, bacterial products, and other compounds may also be added to the culture system to help program a desired fluid height and consequently the thickness and density of the mucus. See Example 2, in which a hormone is added to the basal medium to promote luminal water secretion.

[0048] Turning to Figure 1C, in addition to the use of a porous membrane PM on which a cell monolayer, such as differentiated epithelial cells, can be cultured, in some embodiments such cells can be cultured or supported on a porous or non-porous scaffold. Examples of scaffolds supporting a cell monolayer include, but are not limited to, hydrogels (natural and synthetic), porous materials, non-porous materials, plastics, ceramics, etc. Other examples include inorganic materials or composites of organic and inorganic materials. Examples of inorganic materials suitable for supports include, but are not limited to, glass, hydroxyapatite, bioglass such as 45S5 bioglass, calcium phosphate, silicon, silicon oxide, titanium oxide, gold, aluminum oxide, etc. Where these materials are not inherently porous, they can be made porous by various methods including, but not limited to, sintering, etching, leaching, lithography, etc. For example, porous meshes of silicon and gold can be fabricated by lithography / etching. Such scaffolds can be supported on or placed adjacent to a porous membrane PM.

[0049] Figure 1D illustrates the use of a separator (a semipermeable / partially permeable physical barrier) positioned above the cell monolayer on the luminal side LS within the reservoir R to prevent mucin dilution and aid in the formation of a high-density layer of mucus. The separator may be impermeable to mucin but permeable to, for example, water. Examples of these separators include, but are not limited to, porous membranes, hydrogels (e.g., agarose, gelatin, collagen, Matrigel®), porous materials, semi-liquid masses, oils (e.g., mineral oil, perfluorocarbons), solid floaters (e.g., wax, plastics), and meshes (e.g., nylon, photoresist, polydimethylsiloxane, and other synthetic polymers). The separator may act as a diffusion barrier to aqueous media (one or more) or mucus components.

[0050] Alternatively, in some embodiments, an impermeable physical barrier (blocker) may also be used, as shown in Figure 1E, to facilitate the accumulation of a high-density mucus layer. The impermeable physical barrier may be positioned above (upper) the mucus-producing cell monolayer in the reservoir R, i.e., on the luminal side LS. The impermeable physical barrier or blocker may be impermeable to mucin and water so as to contain the mucus accumulation. Examples of blockers include, but are not limited to, hydrogels (e.g., agarose, gelatin, collagen, Matrigel®), porous materials, oils (e.g., mineral oil, perfluorocarbons), solid floaters (e.g., wax, plastics), and meshes (e.g., nylon, photoresist, polydimethylsiloxane, and other synthetic polymers).

[0051] To further enhance the density of the mucus layer, additional methods may be employed to create a high-density mucus layer on the surface of a mucus-producing cell monolayer. For example, in some embodiments, surface forces or mechanical stimuli may be applied to the surface of a cell monolayer containing mucus-producing cells to mimic (without diluting the mucus) the impact of surface tension forces that may be present during meniscus formation produced by ALI. Mechanisms for exerting shear forces in a non-dilutive manner may include, but are not limited to, a stirrer, mechanical rotation of the layer or material above the cell, or forward and backward movement parallel to the surface induced by a viscous layer overlaid on the cell surface (this may include providing rocking motion, for example, by a mechanical rocker). To facilitate mucus production and accumulation, mechanical stimuli may also be applied to the cell surface, for example, within the reservoir R of the luminal LS. Mechanical stimuli may include, but are not limited to, shear stress applied to the apical surface by fluid flow, mechanical scratching of the apical mucus layer, and / or periodic deformation of cells to mimic peristalsis, etc.

[0052] Any combination of the methods described herein may be used to facilitate the formation of a mucus layer above a cell monolayer containing mucus-producing cells. By adjusting the methods and combinations of methods described herein, mucus layers with different properties, including different thicknesses, different densities, and regions of different density / thickness, can be produced.

[0053] Accordingly, in some embodiments, the subject matter of this disclosure may provide a method for producing a living cell construct comprising a cell monolayer containing mucin-producing cells and a mucus layer, the method comprising: (a) culturing stem cells capable of differentiating into mucin-producing cells (e.g., intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, and the like) 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 covered by stem cells; and (b) further culturing the stem cells to produce a cell monolayer containing mucin-producing cells (e.g., goblet cells), the cell monolayer having a basal side and a luminal (apical) side, the mucin-producing cells of the cell monolayer establishing a mucus layer on the luminal side of the cell monolayer, and the mucus layer being substantially impermeable to microscopic objects, thereby producing a living cell construct comprising a cell monolayer containing mucin-producing cells and a mucus layer. In some embodiments, the mucus layer may have a thickness of about 1 micron to about 1 cm.

[0054] In some embodiments, the subject matter of the present disclosure may provide a method for producing a living cell construct comprising a cell monolayer containing mucinous cells and a mucus layer, the method comprising: (a) culturing stem cells capable of differentiating into mucinous cells (e.g., intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, and the like) on the upper surface of a cell support structure having an upper surface and a lower surface until at least a portion of the upper surface of the cell support structure is covered by stem cells; and (b) further culturing the stem cells to produce a cell monolayer containing mucinous cells (e.g., goblet cells), the cell monolayer having a basal side and a luminal (apical) side, the mucinous cells of the cell monolayer establishing a mucus layer on the luminal side of the cell monolayer, the mucus layer having a thickness of about 1 micron to 1 cm, thereby producing a living cell construct comprising a cell monolayer containing mucinous cells and a mucus layer. In some embodiments, the mucus layer may be impermeable or substantially impermeable to microscopic objects.

[0055] In some embodiments, the subject matter of this disclosure may provide a method for producing a living cell construct comprising a cell monolayer containing mucin-producing cells and a mucinous layer, the method being: (a) culturing stem cells capable of differentiating into mucin-producing cells (e.g., intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, and the like) 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 covered by stem cells; and (b) further culturing the stem cells to produce mucin-producing cells. The present invention comprises producing a cell monolayer containing cells (e.g., goblet cells), the cell monolayer having a basal side and a luminal (apical) side, the mucus-producing cells of the cell monolayer establishing a first mucus layer and a second mucus layer on the luminal side of the cell monolayer, the first mucus layer being above and adjacent to the second mucus layer, the second mucus layer being above and adjacent to the cell monolayer, the second mucus layer being impermeable or substantially impermeable to minute objects, thereby producing a living cell construct comprising a cell monolayer containing mucus-producing cells and a mucus layer. In some embodiments, the thickness of the second mucus layer may be about 1 micron to about 1 cm, as further described herein.

[0056] In some embodiments, a method is provided for producing a living cell construct comprising a cell monolayer containing mucin-producing cells and a mucin layer, the method being: (a) culturing stem cells capable of differentiating into mucin-producing cells (e.g., intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, and the like) 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 covered by stem cells; and (b) further culturing the stem cells to produce mucin-producing cells. The method includes producing a cell monolayer containing living cells (e.g., goblet cells), the cell monolayer having a basal side and a luminal (apical) side, the mucus-producing cells of the cell monolayer establishing a first mucus layer and a second mucus layer on the luminal side of the cell monolayer, the first mucus layer being above and adjacent to the second mucus layer, the second mucus layer being above and adjacent to the cell monolayer, the second mucus layer having a thickness of approximately 1 micron to 1 cm, thereby producing a living cell construct including a cell monolayer containing mucus-producing cells and a mucus layer. In some embodiments, the second mucus layer may be impermeable or substantially impermeable to minute objects.

[0057] In some embodiments, in the method of the subject matter of this disclosure, a basal reservoir may be defined below the basal side of the cell layer of mucinous cells, and a luminal reservoir may be defined above the luminal side of the cell monolayer containing mucinous cells, and the basal reservoir and the luminal reservoir may each contain a liquid medium; the method further includes: (a) removing the liquid medium from the luminal reservoir to create a gas-liquid interface on the luminal side of the cell layer containing mucinous cells; and / or (b) adjusting the volume of the liquid medium in the luminal (apical) reservoir to a depth of about 0.001 mm to about 10 mm, optionally, in the range of about 0.001 mm to about 1 mm, above the luminal side of the cell monolayer. In some embodiments, a mucus layer exists or develops between the liquid medium and the cell monolayer. Therefore, in some embodiments, the volume of liquid may be adjusted prior to, during, or after mucus production by the mucinous cells. Once mucus-producing cells are present in a living cell construct, at least a thin film of mucus can be constitutively produced by the mucus-producing cells (e.g., goblet cells). Subsequently, a thicker layer of mucus can accumulate after adjustment of the volume of fluid in the luminal reservoir. In some embodiments, the mucus layer may be continuous or discontinuous on the surface of the cell monolayer.

[0058] In some embodiments, stem cells useful to 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 cavity stem cells, reproductive organ 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.

[0059] In some embodiments, a living cell construct comprising a cell monolayer containing mucus-producing cells may include one or more different cell types (e.g., 1, 2, 3, 4, 5, or more) in addition to mucus-producing cells (e.g., goblet cells). As used herein, “cell type” means morphologically or phenotypic distinct cell morphologies of a certain kind. In some embodiments, the cells to be placed on the cell support structure may be from healthy, inflamed, or diseased human or animal tissue. In some embodiments, cells useful for making the living cell constructs of the subject of this disclosure may be from human or animal tissue having diseases including, but not limited to, inflammatory bowel disease, constipation, cystic fibrosis, irritable bowel syndrome, leaky gut syndrome, bacterial overgrowth syndrome, celiac disease, lactose intolerance, excess gas syndrome, diarrheal diseases, and / or polyps and appendicitis, including but not limited to mucus regulating disorders.

[0060] In some embodiments, the cell layer of the subject of this disclosure may be flat and two-dimensional, as illustrated, for example, in Figures 1A to 1E. In some embodiments, the cell monolayer of the subject of this disclosure may also be folded into a three-dimensional shape or structure, for example, to mimic the crypt structure or crypt-villous structure of the intestine in vivo.

[0061] In some embodiments, the subject matter of this 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 / above) a cell monolayer containing mucus-producing cells. In some embodiments, the passage of water may be controlled by controlling the movement of liquid / water or water vapor.

[0062] In some embodiments, an impermeable and / or partially permeable physical barrier may be positioned directly on the luminal side of a cell monolayer containing mucinous cells or directly on the mucus layer (if already present). In other words, the impermeable and / or partially permeable physical barrier may be positioned to be in direct contact with the mucinous cells and / or mucus layer. Alternatively, when a physical barrier is positioned, a liquid culture medium may be present between the physical barrier and the mucinous cells and / or mucus layer. In some embodiments, when present between the physical barrier and the mucinous cells and / or mucus layer, the liquid culture medium may be present above the luminal side of the cell monolayer (and / or above the luminal side of the mucus layer) and below the physical barrier, at a depth ranging from about 0.001 mm to about 10 mm, optionally from about 0.001 mm to about 1 mm.

[0063] In some embodiments, the volume of the liquid culture medium can be in depths ranging from about 0.001 mm to about 10 mm above the luminal side of the cell monolayer, either within the luminal (apical) reservoir (with or without a physical barrier) or on the luminal side of the cell (e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08) , 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5. 2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 10 mm, or any value or range within these ranges) (For example, approximately 0.005 mm to approximately 10 mm, 0.01 mm to approximately 10 mm, approximately 0.05 mm to approximately 10 mm, 0.1 mm to approximately 10 mm, approximately 0.5 mm to approximately 10 mm, 1 mm to approximately 10 (mm, approximately 5mm to approximately 10mm, approximately 0.001mm to approximately 1mm, approximately 0.005mm to approximately 1mm, approximately 0.01mm to approximately 1mm, approximately 0.05mm to approximately 1mm, approximately 0.1mm to approximately 1mm, approximately 0.5mm to approximately 1mm, approximately 0.001mm to approximately 0.1mm, approximately 0.005mm to approximately 0.1mm, approximately 0.01mm to approximately 0.1mm, approximately 0.05mm to approximately 0.1mm, approximately 0.1mm to approximately 0.1mm, approximately 0.5mm to approximately 0.1mm, or any of these values ​​or ranges).

[0064] As used herein, a “partially permeable physical barrier” is impermeable 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. That is, 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 to about 150 kDa. Mucin has a molecular weight of about 200 kDa to 200 MDa.

[0065] As used herein, the “impermeable physical barrier” is at least substantially, preferably completely, impermeable to liquid culture media (e.g., water) and mucin.

[0066] Therefore, in some embodiments, an impermeable or partially permeable physical barrier may be used to contain mucin on or near the surface of the mucin-producing cell monolayer. In some embodiments, an impermeable and / or partially permeable physical barrier may be used to prevent or reduce the dilution of mucin by the liquid medium as it is produced by the cell monolayer.

[0067] Examples of physical barriers, though not limited to them, include semi-liquid lumps (e.g., hydrogels), gas-impermeable membranes, gas-permeable membranes, and hygroscopic materials (honey, glycerin, sugars, 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 encompassing 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 (mucosity-impermeable) physical barriers may be used. Examples of vapor-permeable membranes useful for the subject matter of this disclosure, though not limited to them, include uncoated / filler-free polydimethylsiloxane (PDMS), some synthetic polymers, and / or meshes. Examples of impermeable membranes (impermeable to water and mucin) include, but are not limited to, solid floaters (e.g., wax, plastic, etc.), meshes (e.g., nylon, photoresist, polydimethylsiloxane, and other synthetic polymers), oils (e.g., mineral oil, perfluorocarbon, natural oil, etc.), and / or synthetic polymers.

[0068] In some embodiments, the thickness of the mucinous layer of the living cell construct of the subject of this disclosure may range from about 1 micron to about 1 cm (10,000 microns) (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245) ,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 5 55, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 71 0, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865 ,870,875,880,885,890,895,900,905,910,915,920,925,930,335,940,945,950,955,960,965,970,975,980,985,990,995,1000,1100,1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,(9500, 10,000 microns, or any value or range therein). Therefore, in some embodiments, the thickness of the mucus layer is approximately 2 microns to 1 cm, approximately 3 microns to 1 cm, approximately 4 microns to 1 cm, approximately 5 microns to 1 cm, approximately 6 microns to 1 cm, approximately 7 microns to 1 cm, approximately 8 microns to 1 cm, approximately 9 microns to 1 cm, approximately 10 microns to 1 cm, approximately 20 microns to 1 cm, approximately 30 microns to 1 cm, approximately 40 microns to 1 cm, approximately 50 microns to 1 cm, approximately 60 microns to 1 cm, approximately 70 microns to 1 cm, approximately 80 microns to 1 cm, Approximately 90 microns to approximately 1 cm, approximately 100 microns to approximately 1 cm, approximately 200 microns to approximately 1 cm, approximately 300 microns to approximately 1 cm, approximately 400 microns to approximately 1 cm, approximately 500 microns to approximately 1 cm, approximately 1000 microns to approximately 1 cm, approximately 1500 microns to approximately 1 cm, approximately 30 microns to approximately 7500 microns, approximately 40 microns to approximately 7500 microns, approximately 50 microns to approximately 7500 microns, approximately 60 microns to approximately 7500 microns, approximately 70 microns to approximately 7500 microns, approximately 80 microns to approximately 7500 microns, approximately 90 microns From approximately 7500 microns, from approximately 100 microns to approximately 7500 microns, from approximately 500 microns to approximately 7500 microns, from approximately 1000 microns to approximately 7500 microns, from approximately 30 microns to approximately 5000 microns, from approximately 40 microns to approximately 5000 microns, from approximately 50 microns to approximately 5000 microns, from approximately 60 microns to approximately 5000 microns, from approximately 70 microns to approximately 5000 microns, from approximately 80 microns to approximately 5000 microns, from approximately 90 microns to approximately 5000 microns, from approximately 100 microns to approximately 5000 microns, and from approximately 500 microns to approximately 50 00 microns, approximately 1000 to 5000 microns, approximately 30 to 2500 microns, approximately 40 to 2500 microns, approximately 50 to 2500 microns, approximately 60 to 2500 microns, approximately 70 to 2500 microns, approximately 80 to 2500 microns, approximately 90 to 2500 microns, approximately 100 to 2500 microns, approximately 200 to 2500 microns, approximately 300 to 2500 microns, approximately 500 to 2500 microns,Approximately 10 microns to approximately 1000 microns, approximately 20 microns to approximately 1000 microns, approximately 30 microns to approximately 1000 microns, approximately 40 microns to approximately 1000 microns, approximately 50 microns to approximately 1000 microns, approximately 60 microns to approximately 1000 microns, approximately 70 microns to approximately 1000 microns, approximately 80 microns to approximately 1000 microns, approximately 90 microns to approximately 1000 microns, approximately 100 microns to approximately 1000 microns, approximately 200 microns to approximately 1000 microns, approximately 3 00 microns to approximately 1000 microns, approximately 500 microns to approximately 1000 microns, approximately 30 microns to approximately 500 microns, approximately 40 microns to approximately 500 microns, approximately 50 microns to approximately 500 microns, approximately 60 microns to approximately 500 microns, approximately 70 microns to approximately 500 microns, approximately 80 microns to approximately 500 microns, approximately 90 microns to approximately 500 microns, approximately 100 microns to approximately 500 microns, approximately 200 microns to approximately 500 microns, approximately 30 microns to approximately 40 0 microns, approximately 50 to 400 microns, approximately 70 to 400 microns, approximately 100 to 400 microns, approximately 50 to 350 microns, approximately 70 to 350 microns, approximately 100 to 350 microns, approximately 50 to 300 microns, approximately 70 to 300 microns, approximately 100 to 300 microns, approximately 30 to 250 microns, approximately 50 to 250 microns, approximately 30 microns The possible ranges are approximately 200 microns from 1 micron, 50 microns to 200 microns, 100 microns to 200 microns, 30 microns to 150 microns, 50 microns to 150 microns, 1 micron to 100 microns, 5 microns to 100 microns, 10 microns to 100 microns, 20 microns to 100 microns, 30 microns to 100 microns, 50 microns to 100 microns, or any of these ranges or values.

[0069] In some embodiments, the mucus layer produced by the mucus-producing cells of the living cell construct of the subject of this disclosure is composed of microscopic objects (collections of objects) (e.g., from about 0.1 microns to about 100 microns (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 22, 23, 24, 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, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, Microorganisms or microparticles (e.g., beads) in the size range of 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 microns (μm)) may be impermeable or substantially impermeable (e.g., about 0.1 to about 5 μm, about 0.1 to about 10 μm, about 0.1 to about 2 μm, about 0.1 to about 30 μm, approximately 0.1 to approximately 40 μm, approximately 0.1 to approximately 50 μm, approximately 0.1 to approximately 60 μm, approximately 0.1 to approximately 70 μm, approximately 0.5 to approximately 5 μm, approximately 0.5 to approximately 10 μm, approximately 0.5 to approximately 2 μm, approximately 0.5 to approximately 30 μm, approximately 0.5 to approximately 40 μm, approximately 0.5 to approximately 50 μm, approximately 0.5 to approximately 60 μm, approximately 0.5 to approximately 70 μm, approximately 0.5 to approximately 80 μm, approximately 0.5 to approximately 90 μm, approximately 0.5 to approximately 100 μm, approximately 1 to approximately 5 μm, approximately 1 to approximately 10 μm, approximately 1 to approximately 2 μm, approximately 1 to approximately 30 μm, approximately 1 to approximately 40 μm, approximately 1 to approximately 50 μm, approximately 1 to approximately 60 μm, approximately 1 to approximately 70 μm, approximately 1 to approximately 80 μm, approximately 1 to approximately 90 μm, approximately 1 to approximately 100 μm, approximately 2 to approximately 5 μm, approximately 2 to approximately 10 μm, approximately 2 to approximately 20 μm, approximately 2 to approximately 50 μm, approximately 2 to approximately 70 μm, approximately 2 to approximately (80 μm, approximately 2 to approximately 90 μm, approximately 2 to approximately 100 μm, approximately 5 to approximately 20 μm, approximately 5 to approximately 50 μm, approximately 5 to approximately 70 μm, approximately 5 to approximately 80 μm, approximately 5 to approximately 90 μm, approximately 5 to approximately 100 μm, approximately 10 to approximately 20 μm, approximately 10 to approximately 50 μm, approximately 10 to approximately 70 μm, approximately 10 to approximately 80 μm, approximately 10 to approximately 90 μm, approximately 10 to approximately 100 μm, or any amount or range within these ranges). Microorganisms useful for measuring mucus permeability may be, for example, bacteria. Examples of bacteria known in this field that can be used to determine the permeability of the mucus layer include, but are not limited to, Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, and Bifidobacterium. (In some embodiments, the bacteria may belong to the genera Escherichia spp. or Lactobacillus spp.) In addition, microobjectives useful for determining the impenetrability of the mucus layer may be microparticles (e.g., beads) having a size of approximately 0.1 to 100 microns. Generally, microparticles useful for measuring permeability are spherical and composed of one or more polymers (e.g., beads).

[0070] As used herein, “substantially impenetrable” means that more than approximately 70% of a population of microparticles are unable to penetrate the mucus layer (for example, more than approximately 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% of a population of microparticles, and any range or value within these; for example, about 70 The ranges or values ​​from % to approximately 99%, approximately 75% to approximately 99%, approximately 80% to approximately 99%, approximately 85% to approximately 99%, approximately 90% to approximately 99%, approximately 95% to approximately 99%, and any of these ranges or values ​​are not capable of penetrating the mucus layer, and / or permeating minute objects can travel a distance of less than approximately 30% into the thickness of the mucus layer (or less than 30% of the distance from the lumen side of the mucus layer to the base side of the mucus layer) (e.g., approximately 0.5, 0.6, 0.7, 0. 8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30%; for example, approximately 0.5% to approximately 30%, approximately 1% to approximately 30%, approximately 5% to approximately 30%, approximately 10% to approximately 30%, approximately 15% to approximately 30%, approximately 20% to approximately 30%, approximately 0.5% to approximately 20%, approximately 1% to approximately 20%, approximately 5% This means that the percentages are approximately 20%, 10% to 20%, 15% to 20%, 20% to 25%, 0.5% to 15%, 1% to 15%, 5% to 15%, 10% to 15%, 0.5% to 10%, 1% to 10%, 5% to 10%, 0.5% to 5%, 1% to 5%, 2.5% to 5%, 0.5% to 1%, and any range or value therein. In some embodiments, a substantially impermeable mucus layer may be impermeable to more than 90% of a population of minute objects (e.g., approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and any range or value therein; e.g., approximately 90% to approximately 99%). In some embodiments, a substantially impermeable mucus layer may be impermeable to more than 90%, and permeable minute objects travel less than 10% of the distance from the luminal side to the basal side of the mucus layer (e.g., less than 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%).

[0071] As used herein, “impermeable” means that more than approximately 99% of a population of microparticles (e.g., approximately 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 100%, and any range or value therein; e.g., 99–100%) are unable to penetrate the mucus layer, and / or the penetrating microparticles travel a distance of less than approximately 10% into the thickness of the mucus layer (e.g., less than 10% of the distance from the luminal side of the mucus layer to the basal side of the mucus layer) (e.g., less than 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10%).

[0072] As used herein, "cell support structure" may be any structure on which one or more cells and / or tissues can be placed, and may be, for example, an organic, inorganic, or similar composite encompassing any porous or mesh membrane.

[0073] In some embodiments, the cell support structure may include an organic polymer such as collagen, typically in combination with other components discussed below. In some embodiments, the support is porous. The support may be provided or mounted on a porous carrier (e.g., a porous membrane, mesh, inorganic grid, hydrogel, or a combination thereof) to give it structural support, as also discussed below. The support may be any preferred shape or configuration, including flat, tubular, curved, spherical, ellipsoidal, etc. (for example, to mimic macroscopic anatomical structures), and may include such composites.

[0074] Therefore, cell support structures useful for the subject matter of this disclosure may include, but are not limited to, membranes, ECM (extracellular matrix), hydrogels, natural or synthetic polymers, and / or two- or three-dimensional scaffolds, and / or any combination thereof. In some embodiments, for example, the lower wall of a luminal 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, 200, or 300 microns, a maximum depth of 800 or 1000 microns or more, and / or a width of about 10 or 50 microns, a maximum width of 100 or 200 microns or more; e.g., microwells, posts, and / or grooves)). In some embodiments, the cell support structure may be constructed from, 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.

[0075] In some embodiments, materials useful as cell support structures of the subject matter of this disclosure that are not naturally porous can be made porous by methods including, but not limited to, sintering, etching, leaching, lithography, laser micromachining, etc. For example, porous meshes of silicon and gold can be fabricated by lithography / etching. In some embodiments, photoreactive polymers such as photoresists fabricated into films or micro- or nanomeshes having micro or nanopores by photolithography can be used as cell support structures. In some embodiments, elastomer films such as polydimethylsiloxane (PDMS) or EcoFlex fabricated into porous films or micro / nanomeshes by soft lithography or mold 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.

[0076] Cells and / or tissues may be placed on a cell support structure or scaffold 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 such as collagen, gelatin, laminin, elastin, fibronectin, vitronectin, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, a mixture of gelatinous proteins secreted by Engelbres-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, hydrogels from natural polymers, synthetic polymers, and hybrid hydrogels may be used to construct the scaffold in two or three dimensions. Examples of natural and synthetic polymers include, but are not limited to, chitosan, agarose, alginic acid (e.g., AlgiMatrix®), fibrin, silk, polyvinyl alcohol, sodium polyacrylate, acrylic acid polymers, polyethylene glycol (PEG), synthetic peptides, poly-N-isopropylacrylamide, and / or polyacrylamide, and / or any combination thereof. In some embodiments, the scaffold surface may be artificially created to promote cell adhesion by any one or a combination of ECM molecules, natural or synthetic polymers, or synthetic peptides, including but not limited to poly-l-lysine, RGD peptide, and other integrin-recognizing peptide segments. In some embodiments, cell support structures useful for the subject matter of this disclosure may 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 support structure may include two- or three-dimensional micropatterns or microstructures.

[0077] In some embodiments, cells of a living cell construct may be cultured in a liquid medium containing, for example, additives, compounds, and / or solutions that contribute to water equilibrium through the cell layer and thereby assist in the formation of the mucus layer. In some embodiments, the 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, the additives, compounds, and / or solutions may be present in / introduced into the luminal reservoir (the luminal side of the cell monolayer) and / or the basal reservoir (the basal side of the cell monolayer). For example, hormones that stimulate the secretion of water and electrolytes into the intestinal lumen may be added to the basal side of the cell monolayer (basal reservoir) to assist in the equilibrium of fluid movement through the cell monolayer. In contrast, food additives and bacterial metabolites may be added to the luminal side of the cell monolayer (luminal reservoir).

[0078] In some embodiments, hormones useful to the subject matter of this disclosure may include, but are not limited to, vasoactive intestinal peptides (VIPs), 5-hydroxytryptamine (serotonin, 5-HT), substance P, bone morphogenetic proteins (BMPs), gastrin, cholecystokinin, secretin, ghrelin, motilin, gastric inhibitory polypeptides, leptin, glucagon-like peptides, somatostatin, and / or neurotensin.

[0079] Useful chemical additives, though not limited to these, include butyric acid, dibenzazepine, gamma-secretase inhibitors (DAPT, LY411575), forskolin, guaifenesin, carbachol, prostaglandins, phorbal ester (phorbal 12-myristo 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).

[0080] Examples of food additives include N-nitrosoanabasin, matairesimol, and / or caffeine.

[0081] In some embodiments, bacterial metabolites may include, but are not limited to, short-chain fatty acids.

[0082] In some embodiments, the salts in the hypertonic salt solution useful for the subject matter of this disclosure may include, but are not limited to, sodium, chlorine, potassium, magnesium, phosphoric acid, carbonic acid, and / or lithium. In some embodiments, the concentration of the salt in the hypertonic solution may range from about 1 mM to about 1000 mM (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 4 95, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 76 0, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 335, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000 mM, and any value or range within these ranges).Therefore, in some embodiments, the concentration of salt in the 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, and approximately 200 mM to 500 mM. M can be approximately 200mM to 1000mM, approximately 300mM to 500mM, approximately 300mM to 800mM, approximately 300mM to 1000mM, approximately 400mM to 500mM, approximately 400mM to 800mM, approximately 400mM to 1000mM, approximately 500mM to 750mM, approximately 500mM to 1000mM, approximately 600mM to 1000mM, approximately 700mM to 1000mM, approximately 800mM to 1000mM, or any value or range within these ranges.

[0083] Any substance useful for the growth / maintenance of cells and / or tissues may be introduced into the basal reservoir or the luminal reservoir. In some embodiments, the substance may include, but is not limited to, fibronectin; laminin; epidermal growth factor (EGF); R-spondin; 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-associated integration sites) (e.g., Wnt3, Wnt3A, and other Wnt); Notch signaling factors (Notch receptors); Dll1 / 4; noggin; Grem1; Grem2; acetate; butyric acid; propionic acid (proprionate), desaminotyrosine, catecholamines (e.g., dopamine, norepinephrine), cytokines, and / or short-chain fatty acids.

[0084] In some embodiments, forces may be applied, for example, parallel to the surface of the cell layer, to assist in establishing a mucus layer. In some embodiments, the force may be, for example, a surface tension force (e.g., a gas-liquid interface (ALI)), or it may be by the application of mechanical forces. Not limited examples of the application of mechanical forces include motions created by stirring bars, moving semi-solid materials (e.g., hydrogels) parallel to the cell surface, and / or circulating a slurry above the cell surface.

[0085] The subject matter of this disclosure further provides a living cell construct comprising a cell monolayer containing mucin-producing cells and a mucinous layer, wherein the mucinous layer is impermeable or substantially impermeable to microscopic objects (e.g., microorganisms (e.g., bacteria) or microparticles (e.g., beads)). In some embodiments, the mucinous layer may be impermeable or substantially impermeable to microscopic objects in a size range of about 0.1 to about 100 microns. In some embodiments, the mucinous layer may have a thickness (depth) of about 1 micron to about 1 cm. In some embodiments, the mucinous layer of the living cell construct of the subject matter of this disclosure may comprise a basal side and a luminal side, the basal side being below the mucinous layer and adjacent to (directly above) the mucin-producing cells, and the luminal side being above the mucus and mucin-producing cells and adjacent to (directly below) the liquid culture medium or air in the luminal reservoir.

[0086] The subject matter of this disclosure further provides methods using the living cell construct of the subject matter of this disclosure to study (a) the ability of organisms, drugs, or particles to cross (permeate) the mucus layer of cells; (b) the immunological response of cells including the mucus layer to biological invasion or contact with particles and / or chemicals / compounds; (c) the ability of organisms to infect cells including the mucus layer; (d) the effectiveness of drugs to prevent biological infection or reduce the ability of infecting organisms; and (e) the study of mucus dysregulation in diseases including, but not limited to, inflammatory bowel disease, constipation, cystic fibrosis, irritable bowel syndrome, leaky gut syndrome, bacterial overgrowth syndrome, celiac disease, lactose intolerance, excess gas syndrome, diarrheal diseases, and / or polyps and appendicitis.

[0087] Therefore, in some embodiments, the subject matter of the present disclosure provides a method for determining the ability of an organism, drug, or particle to traverse (permeate) the mucus layer of a cell monolayer: bringing the luminal side of the mucus layer of a living cell construct of the subject matter of the present disclosure into contact with the organism, drug, or particle; and measuring the distance the organism, drug, or particle moves into / through the mucus layer (e.g., from the luminal side to the basal side of the mucus layer), thereby determining the ability of the organism, drug, or particle to traverse (permeate) the mucus layer of a cell monolayer of a living cell construct. In some embodiments, the distance the organism, drug, or particle moves into the mucus layer may be measured over time, thereby determining the speed of movement of the organism, drug, or particle into / through the mucus layer of a cell monolayer of a living cell construct.

[0088] In some embodiments, methods are provided for studying and evaluating the ability of an organism to infect a cell monolayer including a mucus layer, the method comprising: bringing the luminal side of the mucus layer of a living cell construct of the subject of this disclosure into contact with the organism; and determining whether the organism traverses the mucus layer and comes into contact with the cell monolayer of the living cell construct. When it is determined that the organism traverses the mucus layer and comes into contact with the cell monolayer, the organism is determined to have the ability to infect a cell monolayer including a mucus layer.

[0089] In some embodiments, a method is provided for evaluating the effectiveness of a drug in preventing infection by an organism or reducing the ability of an infectious organism to infect: comprising: bringing the luminal side of the mucus layer of a living cell construct of the subject of this disclosure into contact with an organism; bringing the luminal side of the mucus layer of the living cell construct into contact with a drug; and determining whether the organism permeates the mucus layer and / or infects one or more cells of the cell monolayer of the living cell construct. If the organism does not permeate the mucus layer and / or infect one or more cells of the cell monolayer of the living cell construct compared to a control (i.e., brought into contact with the organism but not with the drug), the drug is determined to be effective in preventing infection or reducing the ability of an infectious organism to infect. In some embodiments, bringing the luminal side of the mucus layer of the living cell construct into contact with an organism precedes, simultaneously with, or after bringing the luminal side of the mucus layer of the living cell construct into contact with the drug. In some embodiments, the drug may be determined to be effective when approximately 25% to approximately 100% of the organisms are prevented from penetrating the mucus layer and / or killed (e.g., 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, 63, 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 range or value therein is prevented from penetrating the mucus layer and / or killed.

[0090] In some embodiments, the subject matter of the present disclosure provides a method for evaluating the immunological response of a cell monolayer, including a mucous layer, to biological invasion, contact by particles, and / or contact by chemicals / compounds: comprising: contacting the luminal side of the mucous layer of a living cell construct of the subject matter of the present disclosure with a biological organism, particles, and / or chemicals / compounds; 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 by biological organisms, particles, and / or chemicals / compounds.

[0091] In some embodiments, the chemical substances and / or compounds may include, but are not limited to, food metabolites and / or bacterial metabolites, such as vitamins or short-chain fatty acids.

[0092] The organisms that can be studied using the methods and living cell constructs of the subject matter of this disclosure may be any organism, including, for example, bacteria, viruses, fungi, protozoa, and / or helminths. Therefore, any bacteria, virus, fungi, protozoa, or helminth may be studied for its ability to penetrate the mucus layer of cells, for example, to evaluate the effectiveness of drugs that prevent infection by organisms / reduce the ability of infectious organisms and / or to evaluate the immunological response of cells in living cell constructs in response to contact by organisms.

[0093] In some embodiments, the organism may be a bacterium. Examples of bacteria that are not limited to these include those from the genera Escherichia spp., Yersinia spp., Salmonella spp., Campylobacter spp., Clostridium spp., Helicobacter spp., Bacteroides spp., Peptostreptococcus spp., Vibrio spp., Sigella spp., Salmonella spp., Listeria spp., and Staphylococcus spp.In some embodiments, the bacteria include Acinetobacter baumannii, Actinomyces israeri, Bacillus anthrasis, Bacteroides fragilis, Bartonella henselae, Bordetella partsis, Borrelia burgdorferi, Borrelia galini, Borrelia afzeril, Borrelia licarensis, Brucella avoltus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia pseudomalei, Campylobacter jejuni, Chlamydia pneumoniae, and Chlamydia. Midia trachomatis, Chlamydophila sittasi, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium amicolatum, Corynebacterium diphtheriae, Coxiella barnetii, Ehrlichia canis, Ehrlichia shafensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterotoxigenic Escherichia coli, Enteropathogenic Escherichia coli, Enteroinvasive Escherichia coli, Enterohemorrhagic Escherichia coli Bacteria, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorea, Neisseria meningitidis, Parachlamydia, Pseudomonas erginosa, Nocardia asteroides, Rickettsia rickettsia This may include, but is not limited to, *Streptococcus tschii*, *Salmonella bongoli*, *Salmonella enterica*, *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus saprophyticus*, *Streptococcus agalactia*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Streptococcus viridans*, *Treponema pallidum*, *Vibrio cholera*, *Vibrio vulnificus*, *Vibrio parahemorrhagicus*, and / or *Yersinia pestis*.

[0094] In some embodiments, the organism may be a protozoan. Examples of protozoans, though not limited to them, include those from the phylum Amoebozoa, Excavata, and / or Chromalveolata. In some embodiments, the protozoan may include, but is not limited to, those from the genera Amoeba spp., Entomoeba spp., Plasmodium spp., Giardia spp., and / or Trypanosoma spp. In some embodiments, the protozoan may include, but is not limited to, Entomoeba historica, Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora caetanensis, and / or Giardia lambria.

[0095] In some embodiments, the organism may be a virus. Examples of viruses, though not limited to them, include: simplex virus, varicerovirus, cytomegalovirus, roseolovirus, lymphocryptovirus, radinovirus, adenovirus, astrovirus, calicivirus, mastadenovirus, alphapapillomavirus, betapapillomavirus, gammapapillomavirus, mupapillomavirus, neopapillomavirus, polyomavirus, molucipoxvirus, orthopoxvirus, parapoxvirus, alphatork virus, betatork virus, gammatork virus, gemiscircularvirus, erythrovirus, dipendovirus, bocavirus, cortivirus, rotavirus, cidernavirus, hepevirus, alphacoronavirus, betacoronavirus, torovirus, mumastrovirus, norovirus, sapovirus, flavivirus, and hepasiwi. This includes Russ, Pegivirus, Cardiovirus, Cosavirus, Enterovirus, Hepatovirus (e.g., Hepatitis A), Cobbvirus, Parechovirus, Rosavirus, Sarivirus, Alphavirus, Rubivirus, Deltavirus, Lyssavirus, Vesiculovirus, Filoviridae, Ebolavirus, Marburgvirus, Paramyxoviridae, Henipavirus, Morbillivirus, Respirovirus, Rubravirus, Metapneumovirus, Pneumovirus, Arenavirus, Peribunyaviridae, Orthobunyavirus, Hantavirus, Nairovirus, Phenuiviridae, Phlebovirus, Influenza A virus, Influenza B virus, Influenza C virus, Togotovirus, Gammaretrovirus, Deltaretrovirus, Lentivirus, Supumavirus, and / or Orthohednavirus.

[0096] In some embodiments, the organism may be helminths, including but not limited to enteric flukes, roundworms, pinworms, and / or tapeworms. In some embodiments, helminths may include, but not limited to, helminths from the genera Ascaris spp., Ancillostoma spp., Trichris spp., Stronyloides spp., Necator spp., Schistosoma spp., and / or Trichinella spp. Further non-limiting examples of helminths include Ascaris lambricoides (roundworm), Ancillostoma duodenale (hookworm), Necator americanus (hookworm), Stronyloides stercolalis, Trichinella spiralis, and / or Trichris trichiura (whipworm).

[0097] In some embodiments, the organism may be a fungus. Examples of fungi that are not limited to the following include those from the genera Candida spp., Aspergillus spp., Mucor spp., Fusarium spp., Blastomyces spp., Coccidioides spp., Cryptococcus spp., Histoplasma spp., Rhizopus spp., Licthimia spp., Pneumocystis spp., Sporospirix spp., and / or Canninghamella spp. Further examples of fungi that are not limited to the following include Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Cryptococcus gattii, Pneumocystis irovechii, and / or Torlopsis glabrata.

[0098] The subject matter of this disclosure further provides methods for evaluating mucin dysregulation in an in vitro cell system. In some embodiments, the method for evaluating mucin dysregulation in an in vitro cell system includes creating a living cell construct of the subject matter from stem cells of a subject having a disease associated with mucin dysregulation. In some embodiments, the living cell construct of the subject matter of this disclosure may be created from stem cells of a healthy subject that has been modified by genome editing (e.g., CRISPR-Cas9, TALEN, meganuclease) to generalize cells from a disease associated with mucin dysregulation. The mucin layer thus created from the in vitro cell system can then be studied for properties including, but not limited to, thickness, composition, viscosity, permeability by microorganisms, ability to infect microorganisms, and / or responsiveness to drugs, as described herein. Disorders associated with mucus dysregulation include, but are not limited to, inflammatory bowel disease, constipation, cystic fibrosis, irritable bowel syndrome, leaky gut syndrome, bacterial overgrowth syndrome, celiac disease, lactose intolerance, excess gas syndrome, diarrheal disorders, and / or polyps and appendicitis.

[0099] Herein, the subject matter of this disclosure is described by reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims to the subject matter of this disclosure, but rather to illustrate certain embodiments. Any variation of the illustrated methods that can be imagined by those skilled in the art is intended to fall within the scope of the subject matter of this disclosure. [Examples]

[0100] The following examples are included to further illustrate various embodiments of the subject matter of this disclosure. However, in light of this disclosure, those skilled in the art will 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 will still be able to obtain similar or equivalent results.

[0101] Materials and methods for Examples 1-4 In vitro expansion culture of human colon epithelial stem cells on collagen hydrogel. To expand the culture of human-derived intestinal epithelial stem cells, monolayer culture technology was used according to a previously published protocol. 27,30 In short, colonic crypts were isolated from a transverse colon tissue specimen of a cadaveric donor (male, 23 years old), directly plated onto collagen hydrogel at a well density of 1,000 crypts / standard 6-well culture plate (containing 1 mm thick collagen hydrogel), and layered with 4 mL of stem cell medium (SM-Table 1). The medium was changed every 48 hours. When cell confluence reached ≥80% (typically 5-7 days), the monolayer was passaged and subcultured on fresh collagen hydrogel at a passage ratio of 1:3. 27,30 The cells were analyzed for karyotype at P11, and all 10 out of 10 smears showed a normal karyotype. All experiments in this specification used cells at passages less than P15.

[0102] Generation of a mucus layer on a monolayer of human colon epithelium. Transwell inserts containing a porous membrane (0.4 μm pore size, Corning Incorporated, #3460) were coated with 1 vol% Matrigel in phosphate-buffered saline (PBS) overnight at 37°C. Prior to cell plating, the inserts were rinsed with PBS × 1. Intestinal epithelial cells were passaged according to the procedure described above. However, the cells were suspended in expanded culture medium (EM-Table 1) and plated directly into the upper compartment of the Transwell insert. Cells from one well of a 6-well plate were dispersed into six separate 12-well Transwell inserts (1 mL in the upper [apical] reservoir and 2 mL in the lower [basal] reservoir). The medium was changed every 48 hours. To induce cell differentiation and mucus production, the medium was switched to differentiation medium after 5 days (DM-Table 1). For immersion culture, 1 mL of DM was added to the apical reservoir and 2 mL to the basal reservoir. The medium was then changed every 48 hours. For ALI culture, the medium in the apical reservoir was completely aspirated, and 1 mL of DM or DM-VIP (DM containing 330 ng / mL of VIP [AnaSpec, Inc., #AS-22872]) was added to the basal reservoir. The medium was then changed every 24 hours. By day 10, the system was suitable for characterization and cytokine assays.

[0103] [Table 1]

[0104] Characterization of human colon epithelium and mucus layer. Cells and mucus layer were fixed with Carnoy's solution (ethanol 6: acetic acid 3: chloroform 1, v / v / v) at 4°C for 2 hours. The tissue was dehydrated with ethanol and embedded in paraffin for sectioning. Sections were stained with anti-Muc2 antibody (Santa Cruz Biotechnology, #sc-15334) to reveal the mucus layer (Muc2 is a major structural component of the colonic mucus layer), and DNA in the cell nuclei was stained with Hoechst 33342 (Thermo Fisher Scientific, #62249). 30 To clarify the characteristics of the mucus layer and cell apex, the tissues were fixed at 4°C for 2 hours with either Carnoy's solution (in immersion and ALI cultures) or glyoxal (in ALI cultures with DM-VIP), dehydrated stepwise with ethanol (25%, 50%, 75%, and 100%), dried using a critical point dryer (Tousimis Research Corporation Semidri-PVT3), coated with 10 nm metal using a sputter coater (Cressington Scientific Instruments 108), and examined by SEM (FEI-Quanta200ESEM, FEI Company). To demonstrate the bacterial isolation ability of the mucus layer, 1 mL of medium containing 2 μg / mL of Hoechst 33342 is added to the basal reservoir over 1 hour to first stain living epithelial cells with Hoechst 33342, and then stain them with GFP-EC (ATCC, #25922GFP) or 10¹⁰ GFP at a density of 200 million colony-forming units (cfu) / mL. 8A 0.5 mL suspension of 1 μm red fluorescent beads (Thermo Fisher Scientific, #F13083) at a density of beads / mL was added to the apical reservoir. After seeding for 20 mins, the Transwell insert was placed on a coverslip, and the tissue was imaged using an Olympus FluoView-FV3000 confocal laser scanning microscope.

[0105] Toxin A Experiment: 20 μL of a mixture of 0 or 12 μg / mL of natural C. difficile A toxin protein (Abcam PLC, #ab123999) and 5 mg / mL of FITC-dextran (Sigma-Aldrich Co. LLC, #FD40S) was added to the apical side of a human colon epithelial monolayer (± mucus). This spread to form a 180 μm liquid layer. 1 mL of Hanks equilibrium salt solution (with calcium / magnesium, supplemented with 10% fetal bovine serum and 10 mM HEPES) was added to the basal compartment. 150 μL of sample was collected from the basal compartment at 2, 4, 8, and 24 h. The fluorescence intensity of the collected samples was measured using a microplate reader, and the percentage of permeability was calculated as % permeability = 100 × (sample - blank) / (transwell - blank). In the formula, "Transwell" refers to the liquid collected from the cell-free insert. Cells were fixed with ethanol and stained with phalloidin (Thermo Fisher Scientific, #R37110) and ZO-1 antibody (Proteintech Group, Inc., #21773-1-AP) to reveal F-actin and tight junctions.

[0106] Co-culture of E. coli and PBMCs. Fresh, normal human PBMCs were purchased from Physician's Plasma Alliance (Johnson City, TN). PBMCs were suspended at 2.86 million cells / mL in RPMI medium containing 10% fetal bovine serum (FBS) and 100 μg / mL gentamicin. GFP-EC was cultured in nutrient broth containing 100 μg / mL ampicillin. A suspension of GFP-EC with a density of 200 million cfu / mL was prepared in 10 mL of phosphate-buffered saline (PBS), centrifuged at 2300 g, and washed twice with PBS. GFP-EC was resuspended in 0.2 mL of RPMI medium containing 10% FBS. Gentamicin (100 μg / mL) was added to the medium to avoid uncontrolled bacterial growth. 54 For monolayer colon cultures immersed in DM for 5 days, the culture medium was aspirated from both the upper and lower reservoirs. For ALI cultures immersed in DM-VIP for 5 days, the culture medium was aspirated only from the lower reservoir. 20 μL of GFP-EC suspension (20 million cfu) was added to the apical side of the epithelium. 500 μL of PBMC suspension (1.43 million) was added to the basal reservoir. After 24 hours of co-culture, the culture medium was collected from the basal reservoir, centrifuged at 5000 rpm for 6 minutes, divided into smaller portions, and stored at -20°C.

[0107] Cytokine Quantification. The concentrations of cytokines (IL-8, IL-6, IL-1β, and TNF-α) were determined using an ELISA kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Samples were diluted 40× (for IL-8), 10× (for IL-6), or 5× (for IL-1β and TNF-α) so that the measurements fell within the linear range of the given kit. Three samples were used for each condition. Changes in cytokine concentrations compared to the untreated control were statistically analyzed by an unpaired two-sided t-test. In all figures, "**" indicates p<0.005, "*" indicates p<0.05, and "#" indicates not statistically significant.

[0108] Example 1 Air-liquid interface (ALI) culture allows for the accumulation of a compact mucus layer on the apical colon epithelium. First, to aid in the formation of a mucus layer, we tested gas-liquid interface (ALI) culture. This was based on the hypothesis that in standard immersion culture, the layer of culture medium on top would dilute the mucus as it was secreted, preventing the formation of a dense mucus layer. Human epithelial stem cells obtained from the transverse colon were plated onto Matrigel-coated Transwell (porous membrane) and cultured in expanded culture medium (EM) for 5 days to allow the cells to proliferate and form a confluent monolayer. On the 5th day, the medium was switched to differentiation medium (DM), and the cells were cultured for an additional 5 days either as immersion culture (medication in both apical and basal reservoirs) or as ALI culture (medication in the basal reservoir only) (Figure 2A). Due to the absence of growth factors in DM, the cells lost their proliferative capacity and spontaneously differentiated into a mature cell lineage consisting of a mixture of colon cells, goblet cells, and enteroendocrine cells. 29 On day 10 of culture, the mucinous layer was absent in the immersion culture (Figure 2B). As shown in Figure 2C, when viewed by scanning electron microscopy (SEM), the mucinous layer was not visible on these monolayers, and the epithelium and its apical features (goblet cell secretory granules, arrows) were easily discernible (Figure 1C, upper panel). As previously described, lysed mucin was detectable in the luminal medium by enzyme-linked immunosorbent assay (ELISA). 30 Therefore, the mucin secreted by goblet cells was rapidly diluted by the culture medium, and thus lacked the ability to form a dense physiological hydrogel.

[0109] In ALI culture, a continuous mucus layer (Muc2 +) was observed across the entire apical surface of the epithelium by immunofluorescence (Figure 2B). The thickness of this mucus layer was heterogeneous, ranging from 76 to 154 μm. The mucus was sometimes associated with goblet cells (indicated by the arrows in Figure 2B). When viewed by SEM, the mucus demonstrated continuous coverage of the epithelium (Figure 2C, lower panel) and had scattered fissures due to the dehydration process required for SEM imaging. Cells beneath the dense mucus layer could not be visualized (high-magnification image in the right panel). As seen here, in addition to the potential for dilution, various mechanisms that affect mucus production may be at play to enhance mucus secretion. Although ALI culture has been shown to increase mucus production in several tissue-cultured tumor cell lines derived from the respiratory system, as well as the stomach and intestines, 31-37 This effect has not been previously demonstrated using primary intestinal tissue. Mechanical forces in combination with ALI have been shown to enhance mucus production by the tumor cell matrix, and therefore, the surface tension force exerted by a thin fluid layer may be a contributing factor. 37 Evaporation can also provide additional stimulation due to the increased osmorality of the remaining thin film of water, because high-osmolarity solutions have been shown to induce mucus hypersecretion in human bronchial epithelial cells. 38,39 Finally, it is known that changes in osmorality also stimulate the production of trefoil factor 3 by colonic epithelial cells, which acts to improve the quality and density of mucus. 8,40 .

[0110] To determine whether a compact mucus layer acts as a barrier for effectively separating microbeads or bacteria from epithelium, 1 μm red fluorescent beads (Figure 2D) or a suspension of green fluorescent protein (GFP)-expressing Escherichia coli (GFP-EC) (Figure 2E) were added to the luminal reservoir of epithelium stained with Hoechst 33342 after ALI culture. The thickness of the mucus layer was then evaluated by confocal microscopy (Figures 2D and 2E). The distances of the beads and bacteria from the epithelial cells ranged from 71 to 381 μm in the representative images shown in Figures 2D and 2E, with an average of 138 ± 62 μm (N = 3 locations). This mucus layer acted effectively as a barrier for separating microbeads or bacteria, but it formed mucus aggregates with heterogeneous thickness.

[0111] Example 2 By promoting water secretion within the luminal cavity in modified ALI cultures, a thick, hydrated mucus layer can be produced. Gases originating from bacterial metabolism are present in the rumen of the large intestine; however, the in vivo luminal surface is primarily in contact with non-digestible material and waste products that have a high water content. Therefore, in some embodiments, due to the absence of water on the apical surface, the ALI cultures described above do not accurately reflect the in vivo intestinal lumen environment. In fact, the water and electrolyte homeostasis of the colonic mucosa is equilibrated by the movement of water in and out of the rumen. In healthy adults, the rate of water movement from the rumen out is 17.8 mL / min, and the rate of water movement into the rumen is 16 mL / min, resulting in a net water outflow of 1.85 mL / min, which causes fecal solidification. 41 Intestinal hormones, such as 5-hydroxytryptamine (serotonin; 5-HT), vasoactive intestinal peptides (VIPs), and substance P, play a major role in regulating the fluid balance or water content of the lumen contents of the large intestine. 42To develop a strategy for producing a thick, highly hydrated, and uninterrupted mucus layer of uniform thickness, we assisted in balancing fluid movement through the epithelium by exposing the basal epithelial surface to the hormone VIP using VIP-containing DM (DM-VIP) (Figure 3A). VIP is an endogenous hormone, and its plasma concentration in healthy adults ranges from 14 to 76 pg / mL. 43 Its role in the intestines is to stimulate the secretion of water and electrolytes into the intestinal rumen. 44 VIP may also act to increase mucus secretion and production by goblet cells and to strengthen lineage distribution to goblet cells. 45 .

[0112] To investigate the function of VIP in stimulating water secretion, human colon stem cells were cultured in EM for 5 days. On the 5th day, the basal medium was switched to DM with fluctuating VIP concentrations, and the cells were placed under ALI culture conditions. After 24 hours, the water accumulated in the apical reservoir was collected and weighed using an analytical balance. In the absence of VIP, water accumulation in the apical reservoir was not measurable. In the presence of VIP, water secretion was VIP concentration-dependent, with an effective dose of 210 pg / mL being 50 ED. 50 (Figure 3B) A plateau was reached when the VIP concentration was >1 ng / mL. At this saturated concentration, the concentration was approximately 42 mg / cm³, corresponding to the depth of water in a 420 μm reservoir. 2 The water accumulated after a 24-hour incubation with the VIP.

[0113] In ALI culture using DM-VIP (DM containing saturated VIP), a liquid layer accumulated apex-side within 24 hours, and this liquid layer was maintained during the 5-6 day differentiation culture conditions. The apical liquid layer gradually became viscous, and by day 5, a slippery mucohydrogel was formed. This could be lifted from the epithelium with tweezers for visualization (Figure 3C). Under these conditions, the mucus thickness was dependent on the duration of mucus accumulation (i.e., the time under ALI plus DM-VIP). To demonstrate the accumulation of the mucus layer over time, human colon epithelial cells were maintained under the ALI conditions described above by immersion culture with EM for 5 days, followed by culture with DM-VIP for 6 days. Confocal imaging was performed at days 0, 2, 4, and 6. Monolayer cell nuclei were stained with Hoechst 33342. The GFP-EC suspension was overlaid on the mucus layer as described, and the distance between bacteria and epithelial cells was measured as described for the microbeads. The distance between E. coli (labeled by arrows) and nuclei (labeled by arrows) indicates that the mucus layer component effectively separated the bacteria from the epithelium over time (Figure 3D). On days 0 and 2, the bacteria were in contact with the apical surface of the epithelium. After 4 days, the mucus layer separating the bacteria was 142 ± 21 μm thick (N=3 samples). After 6 days, the thickness of the mucus layer had increased to 302 ± 28 μm, as shown by the separation between bacteria and epithelial monolayers. See Figure 3D. These results demonstrate that mucus thickness can be easily adjusted depending on the duration of ALI (Figure 3E). Possibly, due to its ability to flow and redistribute as a result of its water content, the mucus thickness remained constant across the surface (302 ± 28 μm). Similar results were observed by overlaying 1 μm red fluorescent beads onto the mucus layer (Figure 3F). This in vitro-produced mucus layer resembles the properties of the inner mucus layer of the colon in vivo by forming an effective barrier to isolate the epithelium from luminal microorganisms and microbeads. 46To visualize both the mucus and epithelium, the sample was dehydrated and the mucus was partially removed from the epithelium (the dotted line in Figure 3G indicates the boundary). E. coli was found only on the surface of the mucus layer and not in contact with the epithelium. Again, this result demonstrates that the hydrated mucus layer formed a barrier to isolate the epithelium and microorganisms (Figure 3G).

[0114] ALI culture method uses bronchial epithelial cells 47 keratinocyte 48 , adenocarcinoma intestinal cell line 37 , and intestinal organoids 49 ALI is used for culturing primary intestinal epithelial cells on porous membrane inserts using commercially available products (MatTek Corporation's 3D tissue model from MatTek Corporation). However, MatTek's 3D tissue model has not been shown to support a dense, continuous mucus layer for separating microbeads or bacteria with a controllable thickness. The applicant tested primary human small intestinal (jejunal) epithelial cells using the above ALI strategy (DM or DM-VIP), but a dense and thick mucus layer was not produced. The method of the present disclosure is applicable to primary human colon cells, and these data represent the first demonstration of creating an in vitro colonic mucus system with in vivo-like mucus properties.

[0115] Example 3 The mucous layer interferes with the effects of C. difficile toxin on primary human colon epithelium. To demonstrate that the mucus layer improves the physiological relevance of an in vitro colon epithelial model, primary human colon epithelium (GC and / or OC) was exposed to C. difficile toxin in the absence or presence of the mucus layer (Figure 4A). C. difficile produces two potent toxins, A and B, which can inactivate host GTPases (including Rho, Rac, and Cdc42), leading to alteration of the epithelial barrier, damage to the human intestinal mucosa, and inflammation of the colon. 50In immersion culture, in the absence of a mucus layer, toxin A rapidly induced cell damage, with the earliest changes observed within 2 hours of toxin incubation. At 4 hours, intercellular permeability was significantly increased in the toxin-treated epithelial monolayer (Figure 4B), and IL-8 secretion was also significantly enhanced (Figure 4C). Both apical F-actin structure and ZO-1 tight junctions were significantly altered by toxin treatment. While the control epithelial monolayer exhibited an ordered F-actin and continuous "chicken wire" pattern of ZO-1 at the apical brush border, the toxin-treated monolayer showed cellular globulation, disruption and destruction of normal F-actin, and degradation of the ZO-1 architecture (Figure 4D). This trend was observed at 8 hours (Figures 4E and 4F) and 24 hours. These results demonstrate that toxin A disrupts epithelial barrier function and induces an immunological response. This is consistent with previous studies on other intestinal epithelial models. 15,51,52 .

[0116] In the presence of the mucus layer produced by ALI plus DM-VIP, no cellular response to toxin treatment was observed at 4h in terms of changes in permeability and IL-8 production (Figures 4B and 4C), as well as in cell morphology, F-actin, and ZO-1 architecture (Figure 4D). However, changes in permeability and IL-8 production were observed at 8h (Figures 4E and 4F). These results suggest that the mucus layer acted as a physical barrier or trap for the toxins, and as a result, they reached the epithelium by pure diffusion (regardless of the presence or absence of mucus binding sites), and convective mixing no longer had the ability to accelerate toxin transport to the epithelium. These findings suggest that our intestinal mucus-epithelium is an improved physiologically appropriate model for studying host and pathogen factors in C. difficile infection.

[0117] Example 4 The mucus layer acts as a physical barrier, eliminating the immune response induced by bacteria. In vivo, the mucus layer acts as an essential component of the mucosal immune system by serving as a physical barrier that isolates commensal microorganisms from the host epithelium. This physical isolation allows the commensal microorganisms to coexist with the epithelium without initiating a host inflammatory response. 6 To demonstrate that the in vitro mucus layer can provide the same protective function, epithelial monolayers were co-cultured for 24 hours with Escherichia coli (luminal side) and peripheral blood mononuclear cells (PBMCs, basal side) in the absence of the mucus layer (cultured by immersion method) or in the presence of the mucus layer (created by ALI plus DM-VIP method) (Figure 5A). Inflammatory cytokines secreted from the basal epithelium were quantified (Figures 5B to 5E). Without exposure to Escherichia coli, PBMCs produced relatively low levels of cytokines. Co-culture of PBMCs and epithelium produced similar levels of cytokines. However, after epithelium was exposed to bacteria in the absence of the mucus layer, a significant relative increase in cytokine production was observed compared to the control without bacteria. These results are similar to those obtained when non-mucus-producing Caco-2 cells are grown in the presence of non-pathogenic Escherichia coli. 53,54 .

[0118] In contrast, in the presence of a mucus layer, the cytokine response to co-cultured E. coli was eliminated. Without E. coli attack, co-culture of PBMCs, epithelium, and mucus layer produced relatively low levels of cytokines. After 24 hours of co-culture with E. coli, cytokine production was statistically no different from that without bacteria. These data demonstrate that the mucus layer successfully functionally isolates microorganisms from the epithelium, thus eliminating the immune response and mimicking in vivo conditions.

[0119] It will be understood that various details of the subject matter of this disclosure may be altered without departing from the scope of the subject matter. Furthermore, the foregoing statements are for illustrative purposes only and not for limiting purposes.

[0120] reference All references listed herein, including but not limited to all patents, patent applications, and similar publications, scientific journal articles, and database entries (e.g., GENBANK® database entries and all available annotations therein), are incorporated herein by reference in their entirety to the extent that they supplement, explain, provide background to, or teach the methodologies, techniques, and / or compositions employed herein. 1. Allen A., Carroll NJH, "Adherent and Soluble Mucus in the Stomach and Duodenum," Digestive Diseases and Sciences, 1985; Vol. 30: p. 55S. 2. Pelaceyed T, Bergstrom JH, Gustafsson JK, Ermund A, Birchenough GMH, Schutte A, van der Post S, Svensson F, Rodriguez-Pineiro AM, Nystrom EEL, Wizing C, Johansson MEV, Hansson GC, "The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system," Immunological Reviews. (reviews), 2014; Vol. 260: pp. 8-20 3. Murgia X, Loretz B, Hartwig O, Hittinger M, Lehr CM, "The role of mucus on drug transport and its potential to affect therapeutic outcomes," Advanced Drug Delivery Reviews, 2018; Vol. 124: pp. 82-97. 4. Lehr (CM), Poelma (FGJ), Junginger (HE), and Tukker (JJ), "An estimate of turnover time of intestinal mucus gel layer in the rat in situ loop," International Journal of Pharmaceutics, 1991; Vol. 70: pp. 235-240. 5. Wei XC, Yang Z, Rey FE, Ridaura VK, Davidson NO, Gordon JI, Semenkovich CF, "Fatty Acid Synthase Modulates Intestinal Barrier Function through Palmitoylation of Mucin 2," Cell Host & Microbe, 2012; Vol. 11: pp. 140-152. 6. Johansson (MEV), Phillipson (M), Peterson (J), Velcich (A), Holm (L), Hansson (GC), "The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria," Proceedings of the National Academy of Sciences, 2008; Vol. 105: pp. 15064-15069. 7. Hansson, GC., "Role of mucus layers in gut infection and inflammation," Current Opinion in Microbiology, 2012; Vol. 15: pp. 57-62. 8. Carlson (TL), Lock (JY), Carrier (RL), "Engineering the Mucus Barrier," Annual Review of Biomedical Engineering, 2018; Vol. 20: pp. 197-220. 9. Werlang (C), Carcarmo-Oyarce (G), and Ribbeck (K), "Engineering mucus to study and influence the microbiome," Nature Reviews Materials, 2019; Vol. 4: pp. 134-145. 10. Rogier (E.W.), Frantz (AL), Bruno (MEC), Kaetzel (CS), "Secretory IgA is Concentrated in the Outer Layer of Colonic Mucus along with Gut Bacteria," Pathogens, 2014; Vol. 3: pp. 390-403. 11. Gunasekara (DB), Speer (J), Wang (Y), Nguyen (DL), Reed (MI), Smiddy (NM), Parker (JS), Fallon (JK), Smith (PC), Sims (CE), Magness (ST), Allbritton (NL), "A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies," Analytical Chemistry, 2018; Vol. 90: pp. 13331-13340. 12. Quigley, EMM., "Gut bacteria in health and disease," Gastroenterology & Hepatology, 2013; Vol. 9: pp. 560-569. 13. Gagnon M, Berner A., ​​Chervet N, Chassard C, Lacroix C, "Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion," Journal of Microbiological Methods, 2013; Vol. 94: pp. 274-279. 14. Lesuffleur (T), Barbat (A), Dussaulx (E), Zweibaum (A), "Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells," Cancer Research, 1990; Vol. 50: pp. 6334-6343. 15. Nusrat A, von Eichel-Streiber C, Turner JR, Verkade P, Madara JL, and Parkos CA, "Clostridium difficile Toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins," Infection and Immunity, 2001; Vol. 69: pp. 1329-1336. 16. Sato (T), Vries (RG), Snippert (HJ), van de Wetering (M), Barker (N), Stange (DE), van Es (JH), Abo (A), Kujala (P), Peters (PJ), Clevers (H), "Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche," Nature, 2009; Vol. 459: p. 262-U147. 17. Date (S.) and Sato (T.), "Mini-Gut Organoids: Reconstitution of Stem Cell Niche," in Schekman (R.), *Annual Review of Cell and Developmental Biology*, Vol. 31, 2015: pp. 269-289. 18. Sato T, Stange D., Ferrante M, Vries R.G.J., van Es J.H., van den Brink S., van Houdt W.J., Pronk A., van Gorp J., Siersema PD., Clevers H., "Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium," Gastroenterology, 2011; Vol. 141: pp. 1762-1772. 19. Yin (XL), Farin (HF), van Es (JH), Clevers (H), Langer (R), Karp (JM), "Niche-independent high-purity cultures of Lgr5(+) intestinal stem cells and their progeny," Nature Methods, 2014; Vol. 11: pp. 106-112. 20. Fatehullah A, Appleton P, and Nathke I, "Cell and tissue polarity in the intestinal tract during tumourigenesis: cells still know the right way up, but tissue organization is lost," Philosophical Transactions of the Royal Society B - Biological Sciences, 2013; Vol. 368: p. 20130014. 21. Moon (C), VanDussen (KL), Miyoshi (H), and Stappenbeck (TS), "Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis," Mucosal Immunology, 2014; Vol. 7: pp. 818-828. 22. VanDussen (KL), Marinshaw (JM), Shaikh (N), Miyoshi (H), Moon (C), Tarr (PI), Ciorba (MA), and Stappenbeck (TS), "Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays," Gut, 2015; Vol. 64: pp. 911-920. 23. In (J), Foulke-Abel (J), Zachos (NC), Hansen (AM), Kaper (JB), Bernstein (HD), Halushka (M), Blutt (S), Estes (MK), Donowitz (M), Kovbasnjuk (O), "Enterohemorrhagic Escherichia coli Reduces Mucus and Intermicrovillar Bridges in Human Stem Cell-Derived Colonoids," Cellular and Molecular Gastroenterology and Hepatology, 2016; Vol. 2: pp. 48-62. e3 24. Kozuka (K), He (Y), Koo-McCoy (S), Kumaraswamy (P), Nie (B), Shaw (K), Chan (P), Leadbetter (M), He (L), Lewis (JG), Zhong (Z), Charmot (D), Balaa (M), King (AJ), Caldwell (JS), Siegel (M), "Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform," Stem Cell Reports, 2017; Vol. 9: pp. 1976-1990. 25. Noel G, Baetz NW, Staab JF, Donowitz M, Kovbasnjuk O, Pasetti MF, Zachos NC, "A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions," Scientific Reports, 2017; Vol. 7: p. 45270. 26. Puzan (M), Hosic (S), Ghio (C), Koppes (A), "Enteric Nervous System Regulation of Intestinal Stem Cell Differentiation and Epithelial Monolayer Function," Scientific Reports, 2018; Vol. 8: p. 6313. 27. Wang (YL), DiSalvo (M), Gunasekara (DB), Dutton (J), Proctor (A), Lebhar (MS), Williamson (IA), Speer (J), Howard (RL), Smiddy (NM), Bultman (SJ), Sims (CE), Magness (ST), Allbritton (NL), "Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells," Cellular and Molecular Gastroenterology and Hepatology, 2017; Vol. 4: p. 165-+ 28. Madden (LR), Nguyen (TV), Garcia-Mojica (S), Shah (V), Le (AV), Peier (A), Visconti (R), Parker (EM), Presnell (SC), Nguyen (DG), Retting (KN), "Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME / Tox Functions," iScience, 2018; Vol. 2: pp. 156-167. 29. Wang Y, Kim R, Hwang S-HJ, Dutton J, Sims CE, Allbritton NL, "Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform," Analytical Chemistry, 2018; Vol. 90: pp. 11523-11530. 30. Wang (YL), Kim (R), Gunasekara (DB), Reed (MI), DiSalvo (M), Nguyen (DL), Bultman (SJ), Sims (CE), Magness (ST), Allbritton (NL), "Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer," Cellular and Molecular Gastroenterology and Hepatology, 2018; Vol. 5: pp. 113-130. 31. Whitcutt (MJ), Adler (KB), and Wu (R), "A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells," *In Vitro Cellular & Developmental Biology*, 1988; Vol. 24: pp. 420-428. 32. Gray (TE), Guzman (K), Davis (CW), Abdullah (LH), Nettesheim (P), "Mucociliary differentiation of serially passed normal human tracheobronchial epithelial cells," American Journal of Respiratory Cell and Molecular Biology, 1996; Vol. 14: pp. 104-112. 33. Raredon (MSB), Ghaedi (M), Calle (EA), Niklason (LE), "A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium," Cell Medicine, 2015; Vol. 7: pp. 109-121. 34. O'Boyle (N), Sutherland (E), Berry (CC), Davies (RL), "Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface," PLOS One, 2017; Vol. 12. 35. Ootani (A), Toda (S), Fujimoto (K), Sugihara (H), "An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture," Biochemical and Biophysical Research Communications, 2000; Vol. 271: pp. 741-746. 36. Yokoyama F, Sakata Y, Ootani A, Fujise T, Kakimoto T, Amemori S, Shiraishi R, Kuroki T, Tsunada S, Iwakiri R, Fujimoto K, "Differentiation of gastric surface mucous cells (GSM06) induced by air-liquid interface is regulated partly through mitogen-activated protein kinase pathway," Journal of Gastroenterology and Hepatology Hepatology, 2007; Vol. 22: pp. 2310-2315 37. Navabi (N), McGuckin (MA), Linden (SK), "Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation," PLOS One, 2013; Vol. 8. 38. Elkins (MR) and Bye (PTP), "Mechanisms and applications of hypertonic saline," Journal of the Royal Society of Medicine, 2011; Vol. 104: pp. S2-S5. 39. JT, JMP, VPK, XDZ, "Effect of osmotic response element binding protein on mucus secretion with hypertonicity in human airway epithelial cells," Chinese Medical Journal (Zhonghua Yi Xue Za Zhi), 2011; Vol. 91: pp. 549-553. 40. Ludeking (A), Fegert (P), Blin (N), Gott (P), "Osmotic changes and ethanol modify TFF gene expression in gastrointestinal cell lines," Febs Letters, 1998; Vol. 439: pp. 180-184. 41. Shields R, Miles J.B., Gilbertson C., "Absorption and secretion of water and electrolytes by the intact colon in a patient with primary aldosteronism," British Medical Journal, 1968; Vol. 1: pp. 93-96. 42. Wapnir (RA) and Teichberg (S), "Regulation mechanisms of intestinal secretion: implications in nutrient absorption," The Journal of Nutritional Biochemistry, 2002; Vol. 13: pp. 190-199. 43. Koch (TR), Michener (SR), Go (VLW), "Determination of Plasma Vasoactive Intestinal Polypeptide Concentration in Patients with Diarrhea," Gastroenterology, 1991; Vol. 100: pp. 99-106. 44. Schwartz (C.J.), Kimberg (DV), Sheerin (HE), Field (M.), and Said (SI), "Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa," Journal of Clinical Investigation, 1974; Vol. 54: pp. 536-544. 45. Wu X, Conlin VS, Morampudi V, Ryz NR, Nasser Y, Bhinder G, Bergstrom KS, Yu HB, Waterhouse CCM, Buchan AMJ, Popescu OE, Gibson WT, Waschek JA, Vallance BA, Jacobson K, "Vasoactive Intestinal Polypeptide Promotes Intestinal Barrier Homeostasis and Protection Against Colitis in Mice," PLOS One, 2015; Vol. 10: p.e0125225 46. ​​Johansson (MEV), Gustafsson (JK), Holmen-Larsson (J), Jabbar (KS), Xia (L), Xu (H), Ghishan (FK), Carvalho (FA), Gewirtz (AT), Sjovall (H), Hansson (GC), "Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis," Gut, 2014; Vol. 63: pp. 281-291. 47. Lin HX, Li H, Cho HJ, Bian S, Roh HJ, Lee MK, Kim JS, Chung SJ, Shim CK, Kim DD, "Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studies," Journal of Pharmaceutical Sciences, 2007; Vol. 96: pp. 341-350. 48. Bernstam (LI), Vaughan (FL), Bernstein (IA), "Keratinocytes grown at the air-liquid interface," In Vitro Cellular & Developmental Biology, 1986; Vol. 22: pp. 695-705. 49. Ootani A, Li XN, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugihara H, Fujimoto K, Weissman IL, Capecchi MR, Kuo CJ, "Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche," Nature Medicine, 2009; Vol. 15: p.1-U140. 50. Voth (DE) and Ballard (JD), "Clostridium difficile toxins: mechanism of action and role in disease," Clinical Microbiology Reviews, 2005; Vol. 18: pp. 247-263. 51. He (D), Sougioultzis (S), Hagen (S), Liu (J), Keates (S), Keates (AC), Pothoulakis (C), LaMont (JT), "Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation," Gastroenterology, 2002; Vol. 122: pp. 1048-1057. 52. Mahida (YR), Makh (S), Hyde (S), Gray (T), Borriello (SP), "Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment," Gut, 1996; Vol. 38: pp. 337-347. 53. Haller D, Bode C, Hammes W., Pfeifer A., ​​Schiffrin E., Blum S., "Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell / leukocyte co-cultures," Gut, 2000; Vol. 47: pp. 79-87. 54. Parlesak (A), Haller (D), Brinz (S), Baeuerlein (A), Bode (C), "Modulation of Cytokine Release by Differentiated CACO-2 Cells in a Compartmentalized Coculture Model with Mononuclear Leucocytes and Nonpathogenic Bacteria," Scandinavian Journal of Immunology, 2004; Vol. 60: pp. 477-485. [Explanation of symbols]

[0121] A Apical side B Basal side BS basal side CML concentrated mucus layer DM differentiation medium EM Expanded Culture Medium GC goblet cell LS lumen side M Mucin OC Other cell types PM Porous Matrix R Reservoir SC stem cells

Claims

1. Method: The method involves culturing stem cells capable of differentiating into mucin-producing cells 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 covered by stem cells; Further culturing of stem cells produces a cell monolayer containing mucus-producing cells, and the cell monolayer has a basal side and a luminal side. Includes, A monolayer of mucus-producing cells establishes a mucus layer on the luminal side of the monolayer, thereby creating a living cell construct that includes both the monolayer containing mucus-producing cells and the mucus layer. A method for producing a living cell construct comprising a cell monolayer containing mucus-producing cells and a mucus layer.

2. The method according to claim 1, wherein the mucus layer is substantially impermeable to minute objects.

3. The method according to claim 1, wherein the thickness of the mucus layer is approximately 1 micron to approximately 1 cm.

4. The method according to claim 3, wherein the thickness of the mucus layer is approximately 30 microns to approximately 1 cm.

5. The basal reservoir is located below the basal side of the cell monolayer containing mucin-producing cells, and the luminal reservoir is located above the luminal side of the cell monolayer containing mucin-producing cells; both the basal reservoir and the luminal reservoir each contain a liquid culture medium; Method: Furthermore, Removing the liquid culture medium from the luminal reservoir to create a gas-liquid interface on the luminal side of a cell monolayer containing mucus-producing cells; and / or The volume of the liquid culture medium in the luminal reservoir is adjusted to a depth of approximately 0.001 mm to approximately 10 mm, or arbitrarily, between approximately 0.001 mm and approximately 1 mm, above the luminal side of the cell monolayer. including, The method according to claim 1.

6. The method according to any one of claims 1 to 5, further comprising placing an impermeable physical barrier and / or a partially permeable physical barrier on the luminal side or above the cell monolayer containing mucus-producing cells.

7. The method according to claim 6, wherein an impermeable physical barrier and / or a partially permeable physical barrier is in direct contact with the luminal side of a cell monolayer containing mucus-producing cells and / or a mucus layer produced by mucus-producing cells in a cell monolayer.

8. The method according to claim 6, wherein the liquid culture medium is located between an impermeable physical barrier and / or a partially permeable physical barrier and the luminal side of a cell monolayer and / or mucus layer containing mucus-producing cells, and the depth of the liquid culture medium is in the range of about 0.001 mm to about 10 mm, and optionally about 0.001 mm to about 1 mm.

9. The method according to claim 5, wherein the liquid culture medium comprises hormones, chemical additives, food additives, bacterial metabolites, and / or hypertonic salt solutions (hormones, chemical additives, food additives, bacterial metabolites, and / or hypertonic salt solutions).

10. The method according to any one of claims 1 to 5, wherein the stem cells are epithelial stem cells, intestinal epithelial stem cells, basal stem cells, induced pluripotent stem cells, respiratory stem cells, gastric stem cells, nasal cavity stem cells, reproductive organ cells (cervix, vagina, uterus), urethral cells, olfactory cells, oral cells, tongue cells, and / or conjunctival cells.

11. The method according to claim 1, wherein the stem cells are intestinal epithelial stem cells.

12. The method according to claim 1, wherein the mucus layer is substantially impermeable to beads or microorganisms in a size range of about 1 to about 100 microns.

13. The method according to any one of claims 1 to 12, wherein the force is applied parallel to the surface of a cell monolayer.

14. The method according to claim 13, wherein the force includes the application of surface tension force or mechanical force.

15. The method according to claim 14, wherein the mechanical force is the circulation of a stirring bar, a semi-solid material moving parallel to the cell surface, and / or a slurry above the cell surface.

16. A living cell construct comprising a cell monolayer containing mucus-producing cells and a mucus layer, produced by the method according to any one of claims 1 to 15.

17. The living cell construct according to claim 16, wherein the mucus layer includes a basal side and a luminal side, and the basal side is adjacent to and below the mucus-producing cells.

18. A living cell construct comprising a cell monolayer containing mucus-producing cells and a mucus layer, wherein the mucus layer is substantially impermeable to micro-objectives.

19. The living cell construct according to claim 18, wherein the mucus layer includes a thickness of approximately 1 micron to approximately 1 cm.

20. The living cell construct according to claim 18, wherein the mucus layer is impermeable to minute objects in a size range of approximately 1 micron to approximately 100 microns.

21. The living cell construct according to claim 18, wherein the mucus layer includes a basal side and a luminal side, and the basal side is adjacent to and below the mucus-producing cells.

22. Method: The luminal side of the mucus layer of the living cell construct according to claim 16 is brought into contact with a living organism, drug, or particle; To measure the distance that organisms, drugs, or particles move through the mucus layer, and thereby determine the ability of organisms, drugs, or particles to traverse the mucus layer of the cell monolayer of a living cell construct, including, A method for determining the ability of a biological organism, drug, or particle to traverse a mucous layer of a single cell.

23. The method according to claim 22, wherein the organism is a bacterium, virus, fungus, protozoan, and / or helminth.

Images