Formation of arrays of planar intestinal crypts with stem cell / proliferative cell compartments and differentiated cell zones

Abstract

To provide a method for creating a tissue construct including two or more individual regions each of which includes a different cell population or cell line.SOLUTION: A method includes: a step of providing a support base material or a substrate assembly including two or more physically separate regions, in which two or more physically separate regions of the support base material or the substrate assembly are different from each other; and a step of depositing / arranging one or more cells on the support base material or the substrate assembly. The cells have at least two zones (e.g. proliferation zone and non-proliferation zone), and can form a continuous monolayer capable of operating as an in-vitro intestinal model. The model is two-dimensional, and therefore, facilitates quick and light imaging. A system including a tissue construct, and a method for measuring the effects of a medicine, a nutritional supplementary food and a metabolite for enterocytes by using a tissue construct are also provided.SELECTED DRAWING: Figure 1C

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 676,418, filed May 25, 2018, the disclosure of which is incorporated herein by reference in its entirety. Statement of Federal Support

[0002] This invention was made with government support under Grant No. DK109559 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] The presently disclosed subject matter relates to methods for generating tissue constructs that include two or more physically distinct regions containing different cell populations or lineages. The presently disclosed subject matter further relates to methods and devices for generating the tissue constructs, as well as methods of using the tissue constructs. [Background technology]

[0004] The development of in vitro culture systems (e.g., intestinal organoid cultures, self-renewing intestinal monolayers, gut-on-a-chip devices, etc.) provides useful in vitro platforms that offer advantages over the use of in vivo animal models. However, currently available in vitro platforms either fail to recapitulate the in vivo microenvironment and / or cellular compartmentalization of intestinal structures or are difficult to assay or image (especially in high-throughput assays) due to their three-dimensional nature. Thus, there is a continuing need for new methods for generating tissue constructs of interest on a flat surface, particularly tissue constructs that may contain two or more distinct cell populations (eg, intestinal tissue). Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made to solve the above-mentioned problems of the conventional technology. [Means for solving the problem]

[0006] This Summary lists several embodiments of the presently disclosed subject matter and, in many cases, lists variations and permutations of these embodiments. This Summary is merely illustrative of various embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such embodiments may typically exist with or without the mentioned feature, and similarly, such features may apply to other embodiments of the presently disclosed subject matter, whether or not they are listed in this Summary. To avoid excessive repetition, this Summary does not list or suggest every possible combination of such features.

[0007] In some embodiments, methods are provided for generating tissue constructs comprising two or more distinct regions, each region containing a different cell population or cell lineage. Such methods may, in some aspects, include: (a) providing a support substrate comprising two or more physically distinct regions, wherein the two or more physically distinct regions of the support substrate are distinct from one another; and depositing / disposing one or more cells on the support substrate, wherein the one or more cells anchor or adhere to the support substrate and grow on the support substrate. The one or more cells are capable of converting into different cell populations or cell lineages on the two or more physically distinct regions of the support substrate.

[0008] In some aspects, the two or more physically distinct regions of the support substrate comprise different physical properties, and one or more cells transform into different cell populations or cell lineages on the two or more physically distinct regions of the support substrate in response to the different physical properties of the two or more physically distinct regions of the support substrate. In some embodiments, the different physical properties are porosity, permeability, stiffness modulus, or a combination thereof. In some embodiments, the one or more cells comprise primary cells, and optionally, the one or more cells comprise primary epithelial cells.

[0009] Such methods may further include exposing one or more of the two or more physically distinct regions to one or more stimuli, each of the one or more stimuli selected from the group consisting of a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial-derived compound. In some embodiments, the disclosed methods may further include detecting or measuring an effect of the one or more stimuli, optionally wherein the detecting or measuring includes comparing one or both of cell differentiation and cell proliferation after exposure to the one or more stimuli with one or both of cell differentiation and cell proliferation before exposure to the one or more stimuli and / or one or both of cell differentiation and cell proliferation in a comparable tissue construct that has not been exposed to the one or more stimuli.

[0010] Disclosed herein is an apparatus for generating a tissue construct comprising two or more distinct regions, the apparatus comprising: a luminal container comprising a bottom wall, at least one side wall extending upwardly from the bottom wall, and an upper opening defined by the at least one side wall; and a cell support substrate on or comprising the bottom wall, the cell support substrate comprising two or more physically distinct regions, the two or more physically distinct regions of the cell support substrate being different from one another, and the two or more physically distinct regions of the cell support substrate comprising different physical properties. In some embodiments, the cell support substrate comprises a single layer of material comprising two or more physically distinct regions comprising different physical properties. In some embodiments, the cell support substrate comprises a first layer of material and a second layer of material, the first layer overlying the second layer, the first layer and the second layer having different physical properties, one of the first layer and the second layer comprising one or more openings extending from one surface of the first layer or the second layer to the opposite surface of the first layer or the second layer, and optionally, the one or more openings being micropores. In some embodiments, the first layer comprises a porous material and the second layer comprises a non-porous material, the second layer comprising one or more micropores, and optionally, the second layer is the bottom wall of the luminal container. In some embodiments, the first layer comprises a hydrogel, and optionally, the first layer comprises collagen.

[0011] In some embodiments, the device further includes a base container including a bottom wall and at least one side wall extending upward from the bottom wall, the bottom wall and the at least one side wall defining a well, the luminal container being held within the well of the base container, the bottom wall of the base container being spaced apart from the bottom wall of the luminal container, and the base container being defined between the bottom wall of the base container and the bottom wall of the luminal container, and / or between at least one side wall of the base container and at least one side wall of the luminal container.

[0012] Also provided herein are methods for preparing a two-dimensional live cell culture model of intestinal or colonic crypts, the method comprising: providing a device disclosed herein; and depositing / disposing one or more epithelial cells on a cell support substrate, wherein the one or more cells anchor or adhere to the cell support and proliferate on the cell support substrate or substrate assembly, and the one or more cells transform into different cell populations or cell lineages on two or more distinct regions of the support substrate or substrate assembly depending on the different physical properties of the physically distinct regions of the support substrate or substrate assembly. In some embodiments, the device includes a base reservoir defined between a bottom wall of the base reservoir and a bottom wall of a luminal reservoir, and / or between at least one side wall of the base reservoir and at least one side wall of the luminal reservoir, the method comprising providing a first growth medium to the base reservoir and a second growth medium to the luminal reservoir, wherein the first growth medium and the second growth medium can be the same or different. In some embodiments, the first and second growth media are the same for at least a first period of time after the one or more cells are deposited / placed, hi some embodiments, the first and second growth media comprise growth factors that support the proliferation of stem cells.

[0013] In some embodiments, the method includes replacing the first or second growth medium with a third growth medium after the first period of time, the third growth medium being different from the first and second growth media, and optionally the third growth medium being devoid of growth factors that support stem cell proliferation. In some embodiments, the third growth medium includes one or more stimuli, each selected from a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial-derived compound. In some embodiments, one or both of the first and second growth media includes one or more stimuli, each selected from a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial-derived compound. In some embodiments, the method further comprises detecting or measuring the effect of the one or more stimuli on one or more of the different cell populations or cell lineages, and optionally, the detecting or measuring comprises comparing one or both of cell differentiation and cell proliferation in the one or more different cell populations or cell lineages after exposure to the one or more stimuli with one or both of cell differentiation and cell proliferation in the one or more different cell populations or cell lineages before exposure to the one or more stimuli and / or with one or both of cell differentiation and cell proliferation in the one or more cell populations or cell lineages of a comparable cell culture model that has not been exposed to the one or more stimuli.

[0014] These and other objects and aspects of the present disclosure are explained in detail hereinafter.

[0015] While the embodiments of the presently disclosed subject matter described above may be achieved in whole or in part by the presently disclosed subject matter, other embodiments will become apparent as the description proceeds, along with the accompanying examples detailed below.

[0016] The presently disclosed subject matter can be better understood by reference to the following drawings. The components in the drawings are not necessarily drawn to scale, emphasis instead being placed upon illustrating, often in a schematic manner, the principles of the presently disclosed subject matter. In the drawings, like reference numerals represent corresponding elements among the different drawings. A further understanding of the presently disclosed subject matter can be obtained by reference to the embodiments illustrated in the accompanying drawings. While the illustrated embodiments are merely exemplary of systems embodying the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter generally, together with further objects and advantages thereof, will be more readily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of the presently disclosed subject matter, which is particularly set forth in the appended or later amended claims, but merely to clarify and illustrate the presently disclosed subject matter.

[0017] For a more complete understanding of the presently disclosed subject matter, reference is now made to the following drawings, in which: [Brief explanation of the drawings]

[0018] [Figure 1A] 1 is a schematic diagram showing a cross-sectional side view of an exemplary device of the presently disclosed subject matter, including a cell support substrate and a luminal reservoir. The cell support substrate comprises a layer of porous material overlying a layer of non-porous material containing micropatterned holes. The cell support substrate comprises two distinct regions: in one region, the porous material overlies the non-porous material, and in another region, the porous material is positioned over the holes in the non-porous material. [Figure 1B] 1B is a schematic diagram showing a cross-sectional side view of an exemplary device of the presently disclosed subject matter, with the device of FIG. 1A inserted into a base container. [Figure 1C]1C is a schematic diagram showing a cross-sectional side view of the exemplary device of FIG. 1B after cells have been deposited and grown, where two different regions of the cell support substrate are exposed to cell culture media (optionally different cell culture media) and the cells form two different populations: a first cell population (cells 1, e.g., stem cells, drug-treated cells, etc.) grows on the top surface of the porous material layer in the region above the holes, while a second cell population (cells 2, e.g., differentiated cells, untreated cells, etc.) grows on the top surface of the porous material layer in the region not directly above the holes. [Figure 2A] 1 is a schematic diagram showing a side view of an exemplary cell support substrate of the presently disclosed subject matter, comprising two regions of different porosity / permeability: the outer portions of the support comprise a non-porous support material (NPM), while the central portion comprises a porous support material (PM), thereby achieving a monolayer substrate with materials having different physical properties. [Figure 2B] 1 is a schematic diagram showing a side view of one exemplary cell support substrate of the presently disclosed subject matter, comprising two regions of different porosity / permeability: a central portion comprising a porous support material (PM), while an outer portion comprises a less porous material (RPM) (e.g., compared to the central portion PM). [Figure 2C] 1 is a schematic diagram showing a side view of one exemplary cell support substrate of the presently disclosed subject matter, comprising two regions of different porosity / permeability: a layer of non-porous support material (NPM) containing micropatterned holes overlaid with a layer of porous membrane (PM). [Figure 2D] 1 is a schematic diagram showing a side view of an exemplary cell support substrate of the presently disclosed subject matter, comprising two regions of different porosity / permeability: a layer of low-porosity porous material (RPM) containing micropatterned holes, overlaid with a layer of porous membrane (PM). [Figure 2E] 1 is a schematic diagram showing a side view of one exemplary cell support substrate of the presently disclosed subject matter, comprising two regions of different porosity / permeability: a layer of porous material (PM) overlaid with a layer of non-porous material (NPM) containing micropatterned holes. [Figure 2F]1 is a schematic diagram showing a side view of an exemplary cell support substrate of the presently disclosed subject matter, comprising regions of two different porosity / permeability: a layer of porous material (PM) overlaid with a layer of low-porosity porous material (RPM) containing micropatterned holes. [Figure 3] Schematic diagram showing an exemplary process for fabricating a cell support substrate having multiple regions with different material properties within a single support. A layer of porous photoresist material is provided (left), which is then covered with a mask that blocks a portion of the top surface of the material. The unmasked portions of the material are exposed to a chemical reaction or light, which converts the unmasked portions of the porous material into a material with low porosity (center). When the mask is removed, a support is obtained with regions of high porosity and regions of low porosity (right). [Figure 4A] Schematic diagram showing an exemplary process for preparing a device containing a cell support substrate with multiple regions of different material properties that can be used to grow planar crypt arrays. The top left panel shows a side view of a photoresist (1002F) film on the surface of a glass slide with an array of microholes patterned therein. After the photoresist film is removed from the slide (top center panel), a layer of collagen is applied over the photoresist film (top right panel) and dehydrated, leaving behind a condensed collagen layer (second-to-top right panel). After rehydration (second-to-bottom right panel), cells are deposited on top of the collagen, and culture medium is added to the basal and luminal containers / reservoirs of the device. [Figure 4B] 4A (a) is a schematic diagram showing the top view of the photoresist film shown in the top left panel of FIG. 4A, illustrating the pattern dimensions of the microhole array. A unit crypt is defined as a 350 micrometer (μm) × 350 μm square with a microhole in its center. Each hole is 50 μm in diameter, with a 350 μm distance between the center of one hole and the center of each adjacent hole. [Figure 4C]A pair of scanning electron microscope (SEM) images of the top surface of a collagen matrix overlaid with a photoresist film. The top image is before rehydration (i.e., the film in the second panel from the top on the right side of Figure 4A) and the bottom image is after rehydration (i.e., the film in the second panel from the bottom on the right side of Figure 4A). Salt crystals are visible on the surface of the dehydrated collagen matrix, as indicated by the arrows. The black bar in the top image represents 200 μm, and the white bar in the bottom image represents 2 μm. [Figure 4D] A pair of panels show a model of the diffusion of a 40 kilodalton (kDa) molecule from the basal reservoir to the luminal reservoir of the device shown in Figure 4A. The top panel shows the concentration profile of the 40 kDa molecule in a plane just above (10 μm above) the luminal surface of the 1002F film. Illustrated is the concentration profile near eight microholes in the central portion of the array 24 hours after adding the molecule (30 ng / mL) to the basal reservoir. The white scale bar represents 300 μm. The bottom panel shows a cross-section of four microholes in the 1002F film. The inset shows a cross-section of one microhole. [Figure 4E] 4B is a graph showing experimentally measured (filled, dashed line) and simulated (unfilled, solid line) diffusion of fluorescein dextran (40 kDa) over time between the luminal reservoir (square) and basal reservoir (circle) of the device of FIG. 4A (fluorescein dextran concentration in micrograms per milliliter (μg / mL) versus time in hours). At time 0, fluorescein dextran was added to the basal reservoir but not to the luminal reservoir. Data points represent the mean and standard deviation of measured data points (n=2), and the line indicates the simulated concentration. [Figure 5]This is a series of confocal microscopy image pairs (fluorescence image on the left, differential interference contrast image on the right) showing the growth of intestinal epithelial cells across the entire top surface of a cell support substrate comprising a collagen-coated photoresist film with four microholes in an array. The image pair on the left (day 2, EM / EM) was taken on day 2 of cell culture with both the luminal and basal sides of the cell support exposed to growth medium (EM). The top center and top right image pairs were taken on days 3 and 4, respectively, of cell culture with media conditions in which growth medium (EM) persisted on both the basal and luminal sides of the cell support (i.e., luminal / basal: EM / EM). The bottom center and bottom right image pairs were taken on days 3 and 4, respectively, of cell culture with the luminal side of the cell support exposed to differentiation medium (DM) and the basal side exposed to EM (i.e., luminal / basal: DM / EM). These images were taken along a single XY plane. Fluorescent images were analyzed for EdU incorporation, ALP activity, and Hoechst 33342 staining. The white scale bar represents 200 μm. All images are at the same magnification. [Figure 6A] 6B is a pair of representative confocal microscopy images of primary mouse intestinal epithelial cells grown on the presently disclosed cell support substrate. Cells were exposed to growth medium (EM) on both the luminal and basal sides of the support (i.e., luminal / basal: EM / EM). The image on the left is a projection image of four planar crypts, and the image on the right is a high-magnification image of one planar crypt. The white line in the center of the image on the right marks the location of the cross-sectional confocal view of the cell monolayer shown in FIG. 6B. The light spot in the center of the crypt represents EdU incorporation, and the off-center gray spot represents ALP activity. The scale bar at the bottom right of the left image represents 200 micrometers (μm), and the scale bar at the bottom right of the right image represents 100 μm. [Figure 6B] 1 is a representative cross-sectional confocal microscopy image of primary mouse intestinal epithelial cells grown in a single planar crypt on a cell support substrate of the present disclosure. Cells were exposed to growth medium (EM) on both the luminal and basal sides of the support (i.e., luminal / basal: EM / EM). Light spots represent EdU incorporation. The scale bar at the bottom right of the image represents 50 micrometers (μm). [Figure 6C] 6D , which shows a pair of representative confocal microscopy images of primary mouse intestinal epithelial cells grown on the presently disclosed cell support substrate. Cells were exposed to differentiation medium (DM) on the luminal side of the support and to proliferation medium (EM) on the basal side of the support (i.e., luminal / basal: DM / EM). The image on the left is a projection image of four planar crypts, and the image on the right is a high-magnification image of one planar crypt. The white line in the center of the image on the right marks the location of the cross-sectional confocal view of the cell monolayer shown in FIG. 6D . Light spots represent EdU incorporation, and gray spots represent ALP activity. The scale bar at the bottom right of the left image represents 200 micrometers (μm), and the scale bar at the bottom right of the right image represents 100 μm. [Figure 6D] 1 is a representative cross-sectional confocal microscopy image of primary mouse intestinal epithelial cells grown in a single planar crypt on a cell support substrate of the present disclosure. Cells were exposed to differentiation medium (DM) on the luminal side of the support and proliferation medium (EM) on the basal side of the support (i.e., luminal / basal: DM / EM). Light spots represent EdU incorporation. The scale bar in the lower right of the image represents 50 micrometers (μm). [Figure 6E] Figure 1 shows a pair of fluorescent images of four planar crypts exposed to differentiation medium (DM) on the luminal side of the support and proliferation medium (EM) on the basal side of the support substrate (i.e., luminal / basal: DM / EM). The image on the left is stained for mucin 2, while the image on the right is stained for chromogranin A and Hoechst 33342. The scale bar at the bottom left of each image represents 200 micrometers (μm). [Figure 6F] Projected segmented image of E-cadherin (left) and β-catenin (right) immunofluorescent staining of a single planar crypt exposed to growth medium (EM) on both the luminal and basal sides of the support substrate (i.e., luminal / basal: EM / EM). The scale bar at the bottom right of the image represents 100 micrometers (μm). [Figure 6G]Projected segmented image of E-cadherin (left) and β-catenin (right) immunofluorescent staining of a single planar crypt exposed to differentiation medium (DM) on the luminal side of the support substrate and proliferation medium (EM) on the basal side of the support substrate (i.e., luminal / basal: DM / EM). The scale bar at the bottom right of the image represents 100 micrometers (μm). [Figure 6H] Representative scanning electron microscopy images of a single planar crypt exposed to differentiation medium (DM) on the luminal side of the support substrate and proliferation medium (EM) on the basal side of the support substrate (i.e., luminal / basal: DM / EM). The black and white boxes indicate the central and edge regions of the crypt area, respectively, which are shown at higher magnification in Figure 6I. The scale bar below the image represents 200 micrometers (μm). [Figure 6I] 6H are a pair of representative high-magnification scanning electron microscope images showing different regions of the planar crypt shown in FIG. 6H at higher magnification. The image on the left shows cells in the center of the crypt, corresponding to the area within the black box in FIG. 6H. The image on the right shows cells in the edge region of the crypt, corresponding to the area within the white box in FIG. 6H. The scale bars at the top right of the left image and at the bottom right of the right image represent 2 micrometers (μm). [Figure 6J] 1 is a graph showing the radial normalized fluorescence intensity (measured in micrometers (μm) outward from the center of the crypt) of incorporated 5-ethynyl-2′-deoxyuridine (EdU) for cells grown on a subject cell support substrate and treated with growth medium (EM) on both the luminal and basal sides of the cell support substrate (luminal / basal: EM / EM, squares), differentiation medium on the luminal side of the cell support substrate and growth medium on the basal side of the cell support substrate (luminal / basal: DM / EM, circles), or differentiation medium on both the luminal and basal sides of the cell support substrate (luminal / basal: DM / DM, triangles). Data points represent the mean, and error bars represent the standard deviation of five samples, where fluorescence images were measured in 5 μm-wide concentric circles from the center of the crypt. [Figure 6K]1 is a graph showing the radial normalized fluorescence intensity (measured in micrometers (μm) outward from the center of the crypt) of internalized alkaline phosphatase (ALP) activity for cells grown on a subject cell support substrate and treated with growth medium (EM) on both the luminal and basal sides of the cell support substrate (luminal / basal: EM / EM, squares), differentiation medium on the luminal side of the cell support substrate and growth medium on the basal side of the cell support (luminal / basal: DM / EM, circles), or differentiation medium on both the luminal and basal sides of the cell support substrate (luminal / basal: DM / DM, triangles). Data points represent the mean, and error bars represent the standard deviation of five samples, where fluorescence images were measured in 5 μm-wide concentric circles from the center of the crypt. [Figure 7A] 1 shows confocal fluorescence microscopy images of four planar crypts cultured on a subject cell support substrate for four days in differentiation medium (DM) on the luminal side of the support substrate and proliferation medium (EM) on the basal side of the support substrate, followed by incubation with 5-ethynyl-2'-deoxyuridine (EdU) for three hours, after which the cells were fixed and stained. The scale bar in the upper left represents 200 micrometers (μm). [Figure 7B] 7A-7C are confocal fluorescence microscopy images of four planar crypts cultured on a subject cell support substrate and pulse-labeled with 5-ethynyl-2'-deoxyuridine (EdU) as described for the cells shown in FIG. 7A, but cultured in medium lacking EdU for an additional two days before fixation and staining. The scale bar in the upper left represents 200 micrometers (μm). [Figure 7C] 7A is a graph showing the number of 5-ethynyl-2'-deoxyuridine (EdU) positive cells cultured as described in FIG. 7A at various distances (measured in micrometers (μm)) from the center of the crypt immediately after a 3-hour pulse-labeling with EdU. N=5. [Figure 7D] 7B is a graph showing the number of 5-ethynyl-2'-deoxyuridine (EdU) positive cells cultured as described in FIG. 7B at various distances (measured in micrometers (μm)) from the center of the crypt after pulse-labeling with EdU. N=5. [Figure 8A] Graph showing 5-ethynyl-2'-deoxyuridine (EdU)-positive area per crypt for planar crypts treated as follows: crypts grown with differentiation medium (DM) on the luminal side of the cell support substrate and growth medium (EM) on the basal side of the cell support substrate (DM / EM; control), crypts grown with DM + acetate on the luminal side and EM on the basal side (DM + acetate / EM), crypts grown with DM + propionate on the luminal side and EM on the basal side (DM + propionate / EM), and crypts grown with DM + butyrate on the luminal side and EM on the basal side (DM + butyrate / EM). "Control" represents crypts treated with differentiation medium (DM) on the luminal side of the cell support substrate and growth medium (EM) on the basal side of the cell support substrate. "Acetate" represents crypts whose DM contained acetate, "propionate" represents crypts whose DM contained propionate, and "butyrate" represents crypts whose DM contained butyrate. The EdU-positive area per crypt was normalized to the Hoechst 33342-stained area per crypt. Ten crypts from each of two different mice (20 crypts total) were analyzed. *, **, and *** represent P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively. [Figure 8B] Figure 8B is a graph showing alkaline phosphatase (ALP)-positive area per crypt for planar crypts treated as described in Figure 8A. "Control" represents crypts treated with differentiation medium (DM) on the luminal side of the cell support substrate and growth medium (EM) on the basal side of the cell support substrate. "Acetate" represents crypts in which the DM contained acetate, "Propionate" represents crypts in which the DM contained propionate, and "Butyrate" represents crypts in which the DM contained butyrate. The ALP-positive area per crypt was normalized to the Hoechst 33342-stained area per crypt. Ten crypts from each of two different mice (20 crypts total) were analyzed. ** and *** represent P < 0.01 and P < 0.001, respectively. [Figure 8C]Figure 8B shows the Hoechst 33342-positive area per crypt for various fatty acids compared to the Hoechst 33342-positive area of ​​control crypts. Crypts were treated as described in Figure 8A. "Control" represents crypts treated with differentiation medium (DM) on the luminal side of the cell support substrate and growth medium (EM) on the basal side of the cell support substrate. "Acetate" represents crypts whose DM contained acetate, "Propionate" represents crypts whose DM contained propionate, and "Butyrate" represents crypts whose DM contained butyrate. Ten crypts from each of two different mice (20 crypts total) were analyzed. *** represents P < 0.001. [Figure 9] 1 is a set of three representative confocal fluorescence microscopy images of four crypts of human epithelial cells grown using one of the disclosed subject devices under three different media conditions: differentiation medium (DM) on the luminal side and proliferation medium (EM) on the basal side (DM / EM, left); EM on both sides (EM / EM, center); and DM on both sides (DM / DM). The scale bar at the bottom left of each image represents 200 micrometers (μm). DETAILED DESCRIPTION OF THE INVENTION

[0019] The presently disclosed subject matter is described in more detail below, but by way of some, but not all, embodiments of the presently disclosed subject matter. Indeed, the presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0020] I. Definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure.

[0021] Unless otherwise defined below, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. References to technology employed herein shall refer to the technology as commonly understood in the art, and include variations of those technologies or equivalent technical substitutions that would be apparent to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to facilitate the description of the presently disclosed subject matter.

[0022] In describing the presently disclosed subject matter, it will be appreciated that several techniques and steps are disclosed, each of which has distinct advantages and each of which may be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0023] Thus, for the sake of clarity, this specification refrains from unnecessarily repeating every possible combination of the individual steps, but the specification and claims should be read with the understanding that all such combinations are fully within the scope of the invention and claims.

[0024] Following long-standing patent law convention, the words "a," "an," and "the" mean "one or more" when used in this application, including the claims. Thus, for example, a reference to "a unit cell" includes a plurality of such unit cells, and so forth. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0025] As used herein, the term "about," when referring to a value or amount of composition, mass, weight, temperature, time, volume, concentration, percentage, and the like, encompasses variations from the stated amount of, in some embodiments, ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1%, where such variations are appropriate in practicing the methods or employing the compositions of the present disclosure.

[0026] The word "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not preclude the addition of unrecited elements or method steps. "Comprising" is a term of art used in claim language that means that the specified elements are required, but that other elements can be added and still form a structure within the scope of the claim.

[0027] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase "consisting of" appears in a clause of a claim rather than immediately following the preamble, it limits only the elements specified in that clause and does not exclude other elements from the claim as a whole.

[0028] As used herein, the phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and to materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0029] With respect to the terms "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the disclosed and claimed subject matter can encompass the use of either of the other two terms.

[0030] As used herein, the term "and / or," when used in the context of a list of entities, refers to the entities present either singly or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, and also all combinations and subcombinations of A, B, C, and D.

[0031] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted as including X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y," and phrases such as "from about X to Y" mean "from about X to about Y."

[0032] The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, 4.24, and 5). Likewise, the recitation herein of numerical ranges by endpoints includes all subranges subsumed within that range (e.g., 1 to 5 includes 1 to 1.5, 1.5 to 2, 2 to 2.75, 2.75 to 3, 3 to 3.90, 3.90 to 4, 4 to 4.24, 4.24 to 5, 2 to 5, 3 to 5, 1 to 4, and 2 to 4).

[0033] It should be understood that when an element is referred to as being "adjacent," "attached," "connected," "coupled," and / or "in contact with," etc., the element may be directly adjacent, connected, coupled, or in contact with the other element, or intervening elements may be present. In contrast, when an element is referred to as being "directly adjacent," "directly attached," "directly connected," "directly coupled," or "in direct contact with," for example, there are no intervening elements present. Additionally, those skilled in the art will understand that when a structure or feature is referred to as being "adjacent" to another feature, the reference may include portions that overlap or underlie the adjacent feature.

[0034] Spatially relative terms such as "under," "below," "lower," "over," "upper," and the like may be used herein for ease of description when describing the relationship of one element or feature to another, as shown in the figures. It should be understood that these spatially relative terms are intended to encompass other orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were inverted, an element described as being "under" or "beneath" another element or feature would then be oriented "over" that other element or feature. Thus, for example, the term "under" can encompass both an orientation of "over" and "under." The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein interpreted accordingly. Similarly, terms such as "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only, unless otherwise specified.

[0035] As used herein, the words "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting example and / or variant embodiments discussed herein, and are not intended to imply that one or more of the embodiments discussed herein are preferred over one or more other embodiments.

[0036] As used herein, the words "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and grammatical variations thereof) refer to an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to a control.

[0037] As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "decrease" (and grammatical variations thereof) refer to a decrease of, for example, at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In certain embodiments, the decrease may result in no, or essentially no, detectable activity or amount (i.e., an insignificant amount, e.g., less than about 10%, or even less than 5%).

[0038] The term "cell culture medium," as used herein, refers to a liquid or gel containing components that support cell growth (e.g., an appropriate energy source and components to regulate the cell cycle). In some embodiments, a basal medium may comprise a minimal essential type of medium (e.g., Dulbecco's Modified Eagle's Medium, Ham's F-12, Eagle's Medium, RPMI, AR8, etc.) to which other components may optionally be added. The term does not exclude media that are prepared or intended for a particular use but that, after modification, can be used for other cell types, etc.

[0039] "Compound," as used herein, means any type of substance or agent generally considered to be a drug, therapeutic agent, pharmaceutical, small molecule, or candidate for use as any of these, as well as combinations and mixtures thereof.

[0040] The term "drug," as used herein, means a compound or composition that has a known biological effect and is used medicinally (e.g., in human and / or veterinary applications) to treat a disease, disorder, and / or condition.

[0041] The term "test compound" as used herein means a compound of suspected or unknown biological efficacy.

[0042] The use of the word "detect" and its grammatical variations shall mean the measurement of a species without quantification, and the use of the words "determine" or "measure" and their grammatical variations shall mean the measurement of a species with quantification. The terms "detect" and "identify" are used interchangeably herein.

[0043] The term "growth factor" as used herein refers to a bioactive molecule that promotes the proliferation of cells or tissues. Growth factors useful in the present disclosure include transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), platelet-derived growth factor (including AA, AB, and BB isoforms) (PDGF), fibroblast growth factor (FGF) (including FGF acidic isoforms 1 and 2, FGF basic form 2, and FGF 4, 8, 9, and 10), nerve growth factor (NGF) (NGF 2.5s, NGF 3.5s, NGF 4.5s, NGF 5.5s, NGF 6.5s, NGF 7.5s, NGF 8.5s, NGF 9.5s, NGF 10.5s, NGF 11.5s, NGF 12.5s, NGF 13.5s, NGF 14.5s, NGF 15.5s, NGF 16.5s, NGF 17.5s, NGF 18.5s, NGF 19.5s, NGF 20.5s, NGF 21.5s, NGF 22.5s, NGF 23.5s, NGF 24.5s, NGF 25.5s, NGF 26.5s, NGF 27.5s, NGF 28.5s, NGF 29.5s, NGF 30.5s, NGF 31.5s, NGF 32.5s, NGF 33.5s, NGF 34.5s, NGF 35.5s, NGF 36.5s, NGF 37.5s, NGF 38.5s, NGF 39.5s, NGF 40.5s, NGF 41.5s, NGF 42.5s, NGF 43. Examples of growth factors include, but are not limited to, TGF-β, 7.0s, and beta-NGF, and neurotrophic factors, brain-derived neurotrophic factor, cartilage-derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), EGFR-VEGF, VEGF-related proteins, Bv8VEGF-E, granulocyte colony-stimulating factor (G-CSF), insulin-like growth factors (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor, stem cell factor (SCF), keratinocyte growth factor (KGF), skeletal growth factor, bone matrix-derived growth factor, and bone-derived growth factor and mixtures thereof. Some growth factors can also promote the differentiation of cells or tissues. For example, TGF can promote the proliferation and / or differentiation of cells or tissues.

[0044] The term "ingredient" refers to any compound, whether of chemical or biological origin, that can be used in a cell culture medium to maintain or promote cell growth, survival, or differentiation. The terms "nutrient," "supplement," and "ingredient" can be used interchangeably and all refer to such compounds. Typical, but non-limiting, ingredients used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, etc. Other ingredients that promote or maintain cell culture ex vivo may be selected by those skilled in the art depending on specific needs.

[0045] The term "inhibit," as used herein, refers to the ability of a compound, agent, or method to decrease or slow a stated function, level, activity, rate, etc., depending on the context in which the term "inhibit" is used. Preferably, inhibition is by at least 10%, more preferably by at least 25%, even more preferably by at least 50%, and most preferably, the function is inhibited by at least 75%. The term "inhibit" is used interchangeably with "reduce" and "block."

[0046] The term "material" as used herein refers to synthetic and natural materials (e.g., matrix components (e.g., synthetic or natural polymers)). The term "materials and compounds" as used herein refers to, among other things, materials, compounds, cells, peptides, nucleic acids, drugs, matrix components, and imaging agents.

[0047] The term "modulate," as used herein, means to change the level of an activity, function, or process. The term "modulate" encompasses both inhibiting and stimulating an activity, function, or process. The term "modulate" is used interchangeably with the term "regulate" herein.

[0048] "Sample," as used herein, means a biological sample obtained from a subject, including, but not limited to, a normal tissue sample, a diseased tissue sample, a biopsy sample, blood, saliva, feces, semen, tears, urine, etc. A sample may also be any other source of material obtained from a subject that contains cells, tissues, or fluids of interest.

[0049] The terms "scaffold," "substrate," "cell support," "surface," "platform," and "cell support substrate" are used herein to refer to a supporting framework, e.g., a supporting framework for the growth of cells or tissues in vivo or in vitro. These terms are used interchangeably to refer to a structural unit of any size, said structural unit or substrate having a surface suitable for immobilizing or modifying molecular structures, said substrate being made of materials such as, but not limited to, metals, thin metal films, glass, fused silica, synthetic polymers, membranes, and the like.

[0050] The term "stimulate," as used herein, means to induce or increase the level of an activity or function above a control value. Stimulation may occur by direct or indirect mechanisms. In one aspect, the activity or function is stimulated by at least 10% compared to a control value, more preferably by at least 25%, and even more preferably by at least 50%. The term "stimulator," as used herein, means any composition, compound, or agent whose application stimulates a process or function of interest, including, but not limited to, wound healing, angiogenesis, fracture healing, osteoblast production and function, and osteoclast production, differentiation, and activity.

[0051] "Tissue," as used herein, means (1) a group of similar cells united to perform a specific function; (2) a part of an organism made up of an aggregate of cells with similar structure and function; and / or (3) a population of cells similarly characterized by structure and function (e.g., muscle tissue or nerve tissue).

[0052] As used herein, "positioned above the bottom wall" may encompass "positioned on the bottom wall." In some embodiments, the bottom wall may comprise a cell support structure.

[0053] By "cell type" herein is meant morphologically or phenotypically distinct cell forms within a species.

[0054] II. Overview Stem cell lineage commitment is influenced by many factors, including biochemical cues (eg, signaling molecules and metabolites) and biophysical properties of tissues (eg, matrix properties, mechanical forces, etc.). Terryn et al., F1000Research 2018, p. 7; Semrau and van Oudenaarden, Annual Review of Cell and Developmental Biology 2015, No. 31, pp. 317-345; Ito and Ito, Annual Review of Cell and Developmental Biology 2016, No. 32, pp. 399-409; Gattazzo et al., Biochimica et Biophysica Acta (BBA), General Subjects See, for example, J. Med. Chem. Soc., 2014, 1840, pp. 2506-2519, and Li et al., J. Med. Chem. Soc., 2011, 6, pp. 229-240. The intestinal epithelium is an example of a system in which lineage commitment is undertaken to maintain proper function. The intestinal epithelium is the outer layer of the intestine, protecting it from constant biochemical, microbial, and physical attacks. The intestinal epithelium is composed of various cell types derived from intestinal stem cells. In the colon, intestinal stem cells are located at the base of crypts, rapidly proliferate, migrate along the crypts, and differentiate into various lineages of functionally distinct cells, such as enterocytes, goblet cells, and enteroendocrine cells. The balance between maintenance of stem cell identity, proliferation, and differentiation in the intestinal epithelium determines intestinal health. This balance is regulated by the local microenvironment, including signaling molecules, microbial-derived compounds, gases, and stiffness.See Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2018, 5(3), pp. 440-453. For example, high concentrations of Wnt, ligands for Wnt / β-catenin signaling, and low levels of the bacterial metabolite butyrate at the base of the crypts can maintain and confine stem cell populations.

[0055] Recent studies using in vivo animal models have demonstrated that gut homeostasis extends beyond the gut to affect host health and pathology more broadly. See Hall et al., Nature Reviews Genetics, 2017, 18, p. 690; Young, BMJ, 2017, 356, p. 831; Shreiner et al., Current Opinion in Gastroenterology, 2015, 31, p. 69; Lynch and Pedersen, New England Journal of Medicine, 2016, 375, p. 2369-2379; and Tremlett et al., Annals of Neurology, 2017. However, due to the complexity of the intestinal tissue, our understanding of how intestinal homeostasis is maintained and regulated remains limited. Intestinal tissue is composed of a variety of cells, including epithelial cells, myofibroblasts, endothelial cells, immune cells, and others. Furthermore, the mammalian intestine hosts a microbial cell community that outnumbers host cells. In animal models, it can be difficult to delineate how these various intestinal players interact with each other to regulate intestinal homeostasis. Furthermore, in vivo animal studies are significantly more time-consuming and expensive than in vitro studies. Therefore, the development of in vitro model systems that recapitulate key components of the intestine is an attractive alternative to in vivo animal models.

[0056] The current gold standard for in vitro primary intestinal cell culture is the organoid system. Primary intestinal epithelial stem cells are isolated from tissue and embedded in hydrogels (e.g., Matrigel). This system has advanced gastrointestinal research by enabling primary intestinal cell culture without donor genetic variability. Organoid systems can generate relatively enriched populations of most intestinal epithelial cell types (see Yin et al., Nature Methods, 2014, 11, p. 106; Van Es et al., Nature Cell Biology, 2012, 14, p. 1099; and Basak et al., Cell Stem Cell, 2017, 20, pp. 177–190). Additionally, several disease models, such as cystic fibrosis (see Dekkers et al., Nature Medicine, 2013, 19, p. 939) and pathogenic infections (see Finkbeiner et al., MBio, 2012, 3, e00159; Karve et al., PloS one, 2017, 12, e0178966; and Bartfeld, Developmental Biology, 2016, 420, pp. 262-270), have been modeled using organoid systems. However, the closed structure of organoid cultures can make the luminal side (the environment of the gut microbiota) difficult to access, potentially limiting the accessibility of luminal manipulations or assays. Furthermore, the three-dimensional structure of organoids poses challenges for imaging and genetic manipulation.

[0057] Two-dimensional cultures of intestinal epithelial cells allow unlimited access to the cells. Primary intestinal epithelial cells have been grown as monolayers on collagen hydrogels (see Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165-182) or on extracellular matrix protein coatings (collagen I or IV or Matrigel) of commercially available porous membrane-supported cell culture substrates. See In et al., Cellular and Molecular Gastroenterology and Hepatology, 2016, 2, pp. 48-62; Tong et al., Biomaterials, 2018, 154, pp. 60-73; and Kozuka et al., Stem Cell Reports, 2017, 9, pp. 1976-1990. These 2D systems are interchangeable with three-dimensional (3D) organoids, allowing unrestricted access to the luminal side of the cultures and enabling easy-to-perform assays, including transepithelial electrical resistance measurements and microscopy. However, in both systems, obtaining intestinal stem cells or both proliferating and differentiated cells in a spatially controlled manner within a single sample is challenging. This is because it is difficult to spatially separate the biochemical cues for proliferation and differentiation within liquid media, and therefore, whether cells in 3D organoids and two-dimensional (2D) monolayer cultures become undifferentiated or differentiated is largely determined by the medium composition.

[0058] Improved replication of the in vivo microenvironment in a 3D in vitro platform has been achieved by culturing primary human and mouse intestinal cells on 3D collagen scaffolds with the same microstructure as in vivo tissues, under a growth factor gradient across the scaffold. See Wang et al., Biomaterials, 2017, 128, pp. 44-55; ACS Biomaterials Science & Engineering, 2017, 3, pp. 2502-2513; and Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2018, 5, pp. 113-130. Primary cells in these platforms polarize into a proliferative cell-rich zone (bottom) and a differentiation zone (surface) in response to the growth factor gradient. Furthermore, short-chain fatty acids, well-known bacterial metabolites, were shown to significantly affect the proliferation and differentiation of intestinal epithelial cells in the platform. However, assays and imaging can still be challenging, as the 3D topology of the microstructure may limit the platform's usability.

[0059] The presently disclosed subject matter, in some embodiments, provides in vitro cell culture platforms, and methods for making and using the same, for use, for example, in generating planar tissue constructs containing two or more regions containing different cell populations, and for evaluating the effects of various stimuli (e.g., drugs, toxins, nutraceuticals, food-derived compounds, microbial-derived compounds (e.g., microbial metabolites), etc.) on cell proliferation. By way of example, in some embodiments, the cell culture platform is an intestinal cell culture platform that replicates the cellular compartmentalization of crypts simply as a monolayer, i.e., as flattened or two-dimensional crypts with controllable dimensions.

[0060] In one exemplary embodiment, the cell culture platform comprises a microdevice with an array of openings or holes (e.g., microholes) in an impermeable / non-porous layer overlaid with a layer comprising a permeable / porous material. See FIG. 4A. This configuration creates two physically distinct regions because the surface properties covering the openings or holes are different from the surface properties covering the impermeable layer. The platform is configured such that the "basal" and "luminal" surfaces of the platform are in contact with separate fluid reservoirs / vessels, thereby providing each reservoir / vessel with a separate composition, allowing selected regions of a cell culture grown on the platform to be treated with one or more compounds that are different from other selected regions of the cell culture.

[0061] The presently disclosed subject matter, in some embodiments, relates to methods for defining multiple cell populations on a substantially planar surface. Accordingly, in some embodiments, the presently disclosed subject matter provides a method for generating a planar tissue construct comprising two or more distinct regions, each comprising a different cell population or cell lineage, the method comprising: (a) providing a support substrate comprising two or more physically distinct regions, wherein the two or more physically distinct regions of the support substrate are distinct from one another; and (b) depositing / disposing one or more cells on the support substrate, wherein the one or more cells settle or adhere to the support substrate or substrate assembly and proliferate on the support substrate, and the one or more cells transform into different cell populations or cell lineages on the two or more distinct regions of the support substrate. The support substrate may comprise a substantially planar surface upon which the one or more cells are deposited. In some embodiments, the two or more physically distinct regions are in two physically distinct locations on the support substrate, e.g., one or more different types of cells can be deposited / placed on the support substrate such that (e.g., upon exposure to different compounds or other conditions) the one or more different cells convert into different cell populations on the surface at the distinct locations.

[0062] In some embodiments, other conditions may include, for example, separate locations of the support substrate having different physical properties. Thus, in some embodiments, two or more physically distinct regions of the substrate have different physical properties, including, but not limited to, porosity, permeability, and / or stiffness. Cells deposited on the support may be converted into different cell populations or cell lineages on two or more distinct regions of the support substrate depending on the different physical properties of the physically distinct regions of the support substrate. For example, one cell type may be deposited / placed on the surface of the support substrate, proliferate, and convert into one cell population or cell lineage in one type of physically distinct region of the substrate, and into another cell population or cell lineage in another type of physically distinct region of the substrate.

[0063] Thus, in some embodiments, the presently disclosed subject matter provides methods for generating two or more (e.g., three or more (e.g., three, four, five, six, seven, eight, nine, ten, or more)) regions of different cell populations or cell lineages on a two-dimensional cell support comprising two or more (e.g., three, four, five, six, seven, eight, nine, ten, or more) material properties in the distinct regions of the two-dimensional cell support. In some embodiments, the distinct regions of the cell support may be, for example, but not limited to, regions of different permeability, porosity, and / or stiffness.

[0064] Exemplary ranges of physical properties for a region of the support substrate of the present disclosure are shown in Table 1 below. [Table 1]

[0065] In addition to the ranges of physical properties set forth in Table 1, in some embodiments, the porosity of a region in the support substrate may range from about 0% to about 10%, or from about 0% to about 20%, or from about 0% to about 30%, or from about 0% to about 40%, or from about 0% to about 50%, or from about 0% to about 75%, or from about 0% to about 90%, or from about 5% to about 90%, or from about 10% to about 80%, or from about 20% to about 70%, or from about 30% to about 60%, or from about 5% to about 100%, or from about 10% to about 100%, or from about 20% to about 100%, or from about 30% to about 100%, or from about 40% to about 100%, or from about 50% to about 100%, or from about 75% to about 100%.

[0066] In addition to the ranges of physical properties shown in Table 1, in some embodiments, the permeability coefficient of a region in the support substrate ranges from about 0 cm / s to about 100 cm / s (i.e., 10 2 cm / s), about 0 cm / s to about 50 cm / s, about 0 cm / s to about 25 cm / s, about 0 cm / s to about 10 cm / s, about 10 cm / s to about 100 cm / s, about 20 cm / s to about 100 cm / s, about 30 cm / s to about 100 cm / s, about 40 cm / s to about 100 cm / s, about 50 cm / s to about 100 cm / s, or about 75 cm / s to about 100 cm / s.

[0067] In addition to the ranges of physical properties shown in Table 1, in some embodiments, the range of shear modulus of a region in the support substrate is about 1×10 0 Pa to approximately 1 × 10 12 Pa, approx. 1×10 0 Pa to approximately 1 × 10 11 Pa, approx. 1×10 0 Pa to approximately 1 × 10 10 Pa, approx. 1×10 0 Pa to approximately 1 × 10 9 Pa, approx. 1×10 0 Pa to approximately 1 × 10 8 Pa, approx. 1×10 0 Pa to approximately 1 × 10 7 Pa, approx. 1×10 0 Pa to approximately 1 × 10 6 Pa, approx. 1×10 0 Pa to approximately 1 × 10 5Pa, approx. 1×10 0 Pa to approximately 1 × 10 4 Pa, approx. 1×10 0 Pa to approximately 1 × 10 3 Pa, approx. 1×10 0 Pa to approximately 1 × 10 2 Pa, approx. 1×10 1 Pa to approximately 1 × 10 12 Pa, approx. 1×10 2 Pa to approximately 1 × 10 12 Pa, approx. 1×10 3 Pa to approximately 1 × 10 12 Pa, approx. 1×10 4 Pa to approximately 1 × 10 12 Pa, approx. 1×10 5 PPa to approximately 1 × 10 12 Pa, approx. 1×10 6 Pa to approximately 1 × 10 12 Pa, approx. 1×10 7 Pa to approximately 1 × 10 12 Pa, approx. 1×10 8 Pa to approximately 1 × 10 12 Pa, approx. 1×10 9 Pa to approximately 1 × 10 12 Pa, approx. 1×10 10 Pa to approximately 1 × 10 12 Pa, or approximately 1 x 10 11 Pa to approximately 1 × 10 12 It may be Pa.

[0068] For purposes of the presently disclosed subject matter, the difference in any physical property between distinct regions in a support substrate may be about 0.1% or more, or from about 0.1% to about 100%, or from about 1% to about 90%, or from about 1% to about 50%.

[0069] Multiple regions of different cell populations or cell lineages can result from cellular responses to the material properties of the cell support, from cellular responses to different accessibility to stimuli through the material in the cell support, or from the combined effect of these two factors. For example, in some embodiments, multiple regions of different cell populations or cell lineages can result from a culture of a single cell population due to differences in physical properties (e.g., stiffness) between different regions on the cell support and due to differences in access to nutrients or growth factors between different regions on the cell support.

[0070] In some embodiments, it is possible to fabricate a cell support substrate with two or more material properties by layering two or more materials. In some embodiments, the thickness of each layer is about 1 millimeter or less, about 500 micrometers (μm) or less, about 250 μm or less, about 100 μm or less, about 50 μm or less, or less than about 25 μm. In some embodiments, it is possible to create a pattern of material properties in the cell support by implementing one or more micropatterned films.

[0071] In some embodiments, the presently disclosed subject matter can be used to generate tissue mimics with distinct zones of different cell types, allowing for the investigation of different cell responses to (for example) signaling molecules, metabolites, cytokines, drugs or test compounds, microorganisms, and gases in a single sample. The presently disclosed subject matter also allows for the exposure of controlled areas of an initially homogeneous cell population to any type of stimulus to achieve a heterogeneous cell population (e.g., proliferating and differentiated cells, or apoptotic and viable cells on a single flat surface). The 2D configuration of the disclosed substrates is easily adaptable to conventional cell culture vessels or microfluidic devices and can be readily scaled up for high-throughput screening.

[0072] The cell support substrate of the present disclosure can be fabricated, for example, by combining two materials with different porosities, permeabilities, or stiffness moduli. Non-limiting examples of arrangements of two different materials are shown in Figures 2A-2F. The two different materials may be joined in a coplanar configuration (see Figures 2A and 2B) or may be layered so that one material contains one or more openings or holes (e.g., micropores). See Figures 2C-2F. Thus, physically distinct regions of the support include areas located above or below the openings or holes and areas not located above or below the openings or holes. The exemplary arrangement of Figure 2C was demonstrated in the examples below using a dried collagen film as the porous material and 1002F epoxy photoresist as the nonporous material. See also Figure 4A.

[0073] In some embodiments, one or more layers of the cell support substrate may include openings or holes. In some embodiments, the openings or holes are micropores. As used herein, "micropores" refers to holes having a small diameter that extend through one or more layers of the cell support substrate, e.g., the holes extend through one layer of the two-layer cell support substrate shown in Figures 2C-2F and 4A, or through one or more layers of a multi-layer structure. The diameter of the micropores may be between about 1 micrometer and about 500 micrometers, or between about 1 micrometer and about 250 micrometers. In some embodiments, the diameter of the micropores may be between about 10 micrometers and about 100 micrometers (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrometers). In some embodiments, the diameter of the micropores is about 50 micrometers. The cell substrate may contain a single micro-hole, or two or more micro-holes. In some embodiments, the micro-holes may be arranged in a regular pattern (e.g., a linear, circular, triangular, etc. pattern). In some embodiments, the micro-holes are arranged in an array comprising two or more rows of micro-holes, with the center of each micro-hole being equidistant from the center of each adjacent micro-hole.

[0074] In some embodiments, cell supports having two or more material properties may be created within a single material (e.g., a single layer of a single material). This may be achieved by a combination of chemical or photochemical reactions and a masking technique to mask selected areas of the material's surface, as shown in FIG. 3 . For example, as shown in FIG. 3 , a porous material may be overlaid with a masking material to cover one surface of one or more regions of the porous material. The masked material may then be chemically treated or exposed to UV light to induce a chemical reaction in the exposed regions of the material, changing the material's properties. For example, chemical crosslinks in a polymeric material may be degraded to increase porosity, or the level of crosslinking in the material may be increased to decrease porosity. For example, FIG. 3 illustrates a process for preparing a cell support, in which a layer containing a porous material is masked and treated to reduce the porosity of the unmasked regions, resulting in a support having regions with relatively low and high porosity. Similarly, a non-porous material may be masked and the unmasked regions exposed to conditions that cause the non-porous material to become porous.

[0075] To provide a cell support with three or more distinct regions with different properties, a support with two different coplanar materials may be overlaid with a third material (optionally containing an array of micro-holes), or a cell support with two different coplanar materials may be masked, and a portion of one or both of the regions may be treated (e.g., with light or chemicals) to change the material properties (e.g., porosity).

[0076] Exemplary porous materials useful in the presently disclosed subject matter may include, but are not limited to, natural or synthetic hydrogels (e.g., collagen, matrigel, gelatin, agarose, chitosan, alginate, polyethylene glycol, polyacrylamide), dried hydrogels prior to salt leaching (e.g., collagen hydrogel is dried and salt leached to generate pores), plastic track-etched membranes (e.g., polycarbonate, polyester), membrane filters (polytetrafluoroethylene, cellulose, polyethersulfone resin), and micromolded meshes (e.g., polydimethylsiloxane, epoxy photoresist). Methods for varying porosity / permeability may include, but are not limited to, adjusting the density of the solid material, crosslink density, and / or manufacturing design.

[0077] Exemplary non-porous materials useful in the presently disclosed subject matter include 1002F epoxy photoresist, cyclic olefin polymers (e.g., those sold under the trade name "ZEONOR®" (Zeon Corporation, Tokyo, Japan)), polycarbonate, acrylate polymers (e.g., poly(methyl methacrylate)), polystyrene, polyethylene, polypropylene, polyvinyl chloride, cellulose, acrylonitrile butadiene styrene (ABS) plastic, nylon, acetal resins (e.g., acetal resins sold under the trade name "Delrin®" (EI du Pont de Nemours and Company, Wilmington, Delaware, USA)), polytetrafluoroethylene (e.g., TEFLON® (The Chemours Co., Wilmington, Delaware, USA), polyesters (e.g., polyethylene terephthalate, polyethylene naphthalate, polybutylene terephthalate, polytrimethylene terephthalate, polyglycolic acid, polylactic acid, polycaprolactone, copolyesters sold under the trade name "TRITAN™" (Eastman Chemical Company, Kingsport, Tennessee, USA)), epoxies (e.g., SU-8, 1001F, 1009F), elastomers (e.g., polydimethylsiloxanes, elastomers sold under the trade name "ECOFLEX™" (Smooth-On Inc., Macungie, Pennsylvania, USA)), glass, ceramic, and / or metal.

[0078] The total thickness of the cell support substrate can be between about 1 mm and about 1 μm, for example, the total thickness can be about 500 μm or less, about 250 μm or less, about 100 μm or less, or about 50 μm or less. The top surface of the cell support substrate can comprise a flat surface, or (e.g., in the case where the top substrate layer has micropores, as in Figures 2E and 2F) can comprise one or more indentations that are less than about 1 mm but greater than 1 μm (e.g., less than about 500 μm, less than about 250 μm, less than about 100 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 10 μm, or less than about 5 μm). In some embodiments, the top surface of the cell support substrate is flat, and any micropores in the substrate are not present in the top layer of the substrate.

[0079] Cells useful in the presently disclosed subject matter may be derived from a eukaryotic cell line and / or may be primary cells, hi some embodiments, the cells may be mammalian cells, and optionally human cells.

[0080] In some embodiments, the one or more cells may be small intestinal epithelial cells, colonic epithelial cells (e.g., mouse and human colonic epithelial cells), gastric epithelial cells, fibroblasts, myofibroblasts, endothelial cells, hepatocytes, adipocytes, muscle cells, bone cells, neural cells, immune cells, and / or stem cells (e.g., embryonic induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells). In some embodiments, the one or more cells may be healthy, inflamed, and / or diseased human or animal cancer cells. In some embodiments, the one or more cells may be, but are not limited to, cells derived from the digestive tract, reproductive tract, respiratory tract, eye, nose, ear, kidney, brain, liver, pancreas, gallbladder, lymphatic system, nervous system, skin, bone, tendon, ligament, cartilage, bone marrow, connective tissue, and / or blood. In some embodiments, the one or more cells may be prokaryotic cells or a biofilm consisting of a mixture of prokaryotic and eukaryotic cells. In some embodiments, the cells may be, for example, cells isolated from a biological sample of a particular subject of interest (e.g., a human or animal having or suspected of having a particular condition).

[0081] In some embodiments, the one or more cells comprise or consist of epithelial cells. In some embodiments, the one or more cells comprise or consist of primary epithelial cells. In some embodiments, the one or more cells comprise or consist of primary mouse or human epithelial cells. In some embodiments, the one or more cells may comprise a single type of cell (e.g., primary cells, epithelial cells, primary epithelial cells, etc.) that differentiate into at least two cell populations after growth on a cell support.

[0082] After deposition / deposition of one or more cells, the cells and / or support substrate may be exposed to one or more culture media (e.g., to support cell survival and / or proliferation). The culture media may be removed and replaced periodically (e.g., every few hours or daily). Any suitable culture format may be used (e.g., patch or monolayer). Two or more physically separate regions of the cell support substrate may be exposed to the same culture media or different culture media. In some embodiments, two or more physically separate regions are exposed to different culture media. In some embodiments, two or more physically separate regions may be exposed to the same culture media for a first period of time after cell deposition / deposition (e.g., the first period of time is from the time the cells are deposited on the support until several hours or about one or more days have passed since deposition / deposition), and then may be exposed to different culture media for a second period of time (e.g., beginning at the end of the first period of time).

[0083] In some embodiments, the cell culture medium comprises one or more growth factors. Growth factors useful in the presently disclosed subject matter can be any growth factor that can be used to generate a cell population. In some embodiments, the cell culture medium comprises one or more growth factors suitable for supporting stem cell proliferation (e.g., Wnt3A, R-spondin3, Noggin, etc.). In some embodiments, the cell culture medium can comprise one or more components, including, but not limited to, proteins or peptides (e.g., growth factors (e.g., Wnt3A, R-spondin3, and Noggin), cytokines, hormones, antibodies), metabolites (e.g., amino acids, fatty acids, lipids, nucleotides, sugars), neurotransmitters (acetylcholine, anandamide, histamine and other trace amines, purines), DNA molecules, RNA molecules (e.g., microRNA, siRNA, shRNA conjugated to lipids or polymers or encapsulated in viral vectors), drugs, test compounds, toxins, anti-cancer drugs, antibiotics, anti-fungals, anti-virals, and / or environmental hazards (e.g., airborne particulate matter and pollutants).

[0084] In some embodiments, the presently disclosed subject matter may use gas stimuli, including, but not limited to, oxygen, nitrogen, carbon dioxide, carbon monoxide, hydrogen, methane, hydrogen sulfide, skatole (a by-product of meat digestion), indole (a by-product of meat digestion), methanethiol (a sulfur compound), dimethyl sulfide (a sulfur compound), volatile amines, volatile sulfur compounds (VSCs), methyl mercaptan (MM) (also known as methanethiol (MT)), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), volatile fatty acids, and / or nitric oxide. In some embodiments, the presently disclosed subject matter may use volatile organic compounds (VOCs), including, but not limited to, aliphatic hydrocarbons, ethyl acetate, glycol ethers and acetone, chlorofluorocarbons, benzene, toluene, methylene chloride, perchloroethylene, methyl tert-butyl ether (MTBE), and / or formaldehyde. Biological stimuli useful in the presently disclosed subject matter include, but are not limited to, immune-related cells, blood cells, circulating tumor cells, microorganisms, viruses, exosomes, bacteria-derived molecules such as lipopolysaccharide (LPS), and / or microorganism-associated molecular patterns (MAMPs) and / or their analogs. The gas stimulus can be used, for example, by placing the cell support or one side thereof in a chamber containing the gas stimulus, or by bubbling the gas stimulus into the cell culture medium in contact with the cell support.

[0085] In some embodiments, the disclosed methods further include exposing one or more of the two or more distinct regions to one or more stimuli of interest, for example, to determine whether the stimuli can modulate (i.e., moderate or moderate) cell proliferation or differentiation. In some embodiments, the one or more stimuli are each selected from the group consisting of a drug, a test compound, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial compound. For example, the disclosed methods may be used to measure the effect of the stimulus of interest on cell proliferation by comparing cells grown in a region exposed to the stimulus with cells grown in a region not exposed to the stimulus. Thus, in some embodiments, the disclosed methods further include detecting or measuring the effect of the one or more stimuli. In some embodiments, the detecting or measuring step comprises comparing one or both of cell differentiation and cell proliferation after exposure to the one or more additives to one or both of cell differentiation and cell proliferation before exposure to the one or more stimuli and / or to one or both of cell differentiation and cell proliferation in a comparable tissue composition that has not been exposed to the one or more stimuli.

[0086] In some embodiments, the presently disclosed subject matter provides methods for preparing a two-dimensional live cell culture model of intestinal crypts or colonic crypts, the method comprising: providing an apparatus including a support substrate comprising two or more physically distinct regions, wherein the two or more physically distinct regions of the support substrate are different from one another, e.g., each distinct region of the support substrate has different physical properties; and depositing / disposing one or more epithelial cells on the cell support substrate, wherein the one or more cells anchor or adhere to the cell support and grow on the cell support substrate (e.g., in the presence of an appropriate cell culture medium provided to the cell support substrate or to one or more physically distinct regions, or portions of the one or more physically distinct regions), and the one or more cells transform into different cell populations or cell lineages on the two or more distinct regions of the support substrate. In some embodiments, the one or more cells transform into different cell populations in response to the different physical properties of the physically distinct regions of the support substrate.

[0087] In some embodiments, the epithelial cells comprise or consist of primary cells. In some embodiments, the epithelial cells comprise or consist of mouse or human epithelial cells. In some embodiments, the epithelial cells comprise or consist of human primary epithelial cells or mouse primary epithelial cells. In some embodiments, the epithelial cells are derived from a biological sample from a subject of interest.

[0088] In some embodiments, the device includes a container (e.g., a luminal container) including a bottom wall and at least one sidewall extending upward from the bottom wall, and the cell support substrate is on the bottom wall of the container (e.g., the luminal container). A medium placed inside the container may be used to mimic an in vivo microenvironment of interest. In some embodiments, a medium placed inside the container may be used to mimic a luminal environment of interest, and the container may be referred to as a "luminal container." The top of the container may be open or may have a removable cover.

[0089] In some embodiments, the device further includes a second container. For example, the second container may contain a medium that contacts the bottom side of the cell support substrate, and this container may be referred to as a base container. The base container may include a bottom wall and at least one side wall extending upward from the bottom wall. In some embodiments, the base container may be cylindrical, but have a larger diameter than the luminal container, thereby allowing the luminal container to be inserted into the base container. Thus, in some embodiments, the base container may be defined between the bottom wall of the base container and the bottom wall of the luminal container, and / or between at least one side wall of the base container and at least one side wall of the luminal container. The medium disposed within the base container may be used to mimic an environment of interest (e.g., a base environment of interest) to which the bottom of the cell support is exposed. As described further later in this specification, the luminal container may have an arm or flange extending from its sidewall (e.g., from or near the top of the luminal container), which allows the luminal container to be inserted into a well containing a base container, and the arm or flange can help hold the luminal container in place within another, larger container (e.g., so that the bottom wall of the luminal container does not contact the bottom wall of the base container). For example, the arm or flange of the luminal container may rest on the top edge of the sidewall of the base container when the luminal container is inserted into the base container.

[0090] In some embodiments, the method includes providing a first growth medium to the base container and a second growth medium to the luminal container. The first and second growth media may be the same or different. The first and second growth media may be provided immediately after one or more cells are deposited / placed on the cell support substrate. In some embodiments, the first and second growth media may be removed and replaced periodically (e.g., every few hours or daily).

[0091] In some embodiments, the first and second growth media are the same for at least a first period of time after one or more cells are deposited / placed. In some embodiments, the first period of time is between about 1 hour and about 2 days (e.g., about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, or about 48 hours). In some embodiments, the first and second growth media each include a growth factor that supports stem cell proliferation (e.g., Wnt-3A, R-spondin, Noggin, etc.). In some embodiments, the method includes replacing the first or second growth medium with a third growth medium after the first period of time has elapsed, wherein the third growth medium is different from the first and second growth media. In some embodiments, the third growth medium lacks a growth factor that supports stem cell proliferation. In some embodiments, the third growth medium includes one or more stimuli of interest (e.g., with known or unknown effect or level of effect on epithelial cells). For example, the stimuli of interest may be, but are not limited to, a drug, a nutraceutical, a microbial-derived compound (e.g., a microbial metabolite), a toxin, an inflammatory mediator, or a test compound (e.g., a compound with suspected or unknown biological efficacy). Additionally or alternatively, in some embodiments, one or both of the first and second growth media include one or more stimuli of interest, e.g., each additive selected from a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial-derived compound. In some embodiments, both the first and second growth media are replaced with another growth medium (e.g., a third and fourth growth medium, which may be the same or different) after a certain period of time. The replacement of both media may be performed at the same time or at different times after the cells are deposited / placed.

[0092] In some embodiments, the effect of one or more stimuli of interest on epithelial cells is unknown, and the method further includes detecting or measuring the effect of the one or more stimuli on one or more of the different epithelial cell populations or cell lineages. In some embodiments, the measuring step includes comparing cell differentiation and / or cell proliferation in one or more different cell populations or cell lineages after exposure to the one or more stimuli with cell differentiation and / or cell proliferation in the one or more cell populations or cell lineages before exposure to the one or more stimuli. In some embodiments, the measuring step includes comparing cell differentiation and / or cell proliferation in the one or more different cell populations or cell lineages with cell differentiation and / or cell proliferation in one or more cell populations or cell lineages in a comparable cell culture model that has not been exposed to the one or more stimuli. Reagents and methods for assaying cell differentiation and cell proliferation are known in the art. For example, cell proliferation may be assayed for incorporation of the thymidine analog EdU. Thus, in some embodiments, the planar crypt model of the present disclosure may be used to detect or measure the modulatory effect of one or more stimuli on enterocytes. For example, the stimulus can increase or stimulate cell proliferation and / or differentiation, or decrease cell viability and / or differentiation.

[0093] III. Equipment In some embodiments, the present disclosure provides devices useful in methods for generating tissue constructs comprising two or more distinct regions, each comprising a different cell population or cell lineage, and / or generating two-dimensional live cell culture models of intestinal or colonic crypts. In some embodiments, the devices include a luminal container comprising a bottom wall, at least one side wall extending upwardly from the bottom wall, and an upper opening defined by the at least one side wall, and a cell support substrate on or including the bottom wall, wherein the cell support substrate comprises two or more physically distinct regions, wherein the two or more physically distinct regions of the support substrate are different from one another, and wherein the two or more physically distinct regions of the support substrate or substrate assembly comprise different physical properties.

[0094] In some embodiments, the cell support substrate may comprise a monolayer comprising two or more physically distinct regions comprising different physical properties; for example, the monolayer is the bottom wall of a luminal vessel. In some embodiments, the cell support substrate comprises two or more layers. In some embodiments, the cell support substrate comprises a first layer and a second layer, the first layer overlaying the second layer, the first layer and the second layer having different physical properties, and one of the first and second layers comprising one or more openings or holes extending from one surface of the layer to the opposite surface of the layer. In some embodiments, one of the first and second layers comprises one or more openings or micropores (e.g., 1, 2, 3, 4, 5, 6, or more micropores (micropores and openings are used interchangeably throughout this specification)) extending from one surface of the layer to the opposite surface of the layer. In some embodiments, the second layer is the bottom wall of a luminal vessel. In some embodiments, one of the first and second layers comprises an array of four or more micropores extending from one surface of the layer to an opposite surface of the layer. In some embodiments, each of the one or more micropores has a diameter between about 10 micrometers and about 100 micrometers (e.g., about 50 micrometers). In some embodiments, the first layer comprises a porous material and the second layer comprises a non-porous material, the second layer comprising one or more micropores.

[0095] As described above, the total thickness of the cell support substrate can be between about 1 mm and about 1 μm, e.g., the total thickness can be about 500 μm or less, about 250 μm or less, about 100 μm or less, or about 50 μm or less. The individual thickness of each layer of the cell support substrate can be between about 1 mm and about 1 μm, e.g., the total thickness can be about 500 μm or less, about 250 μm or less, about 100 μm or less, or about 50 μm or less. Each layer can have the same or different thickness. In some embodiments, each layer can be individually between about 2 μm and about 40 μm (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or about 40 μm).

[0096] As discussed above, exemplary porous materials useful in the presently disclosed subject matter may include, but are not limited to, natural or synthetic hydrogels (e.g., collagen, matrigel, gelatin, agarose, chitosan, alginate, polyethylene glycol, polyacrylamide), dried hydrogels prior to salt leaching (e.g., collagen hydrogel is dried and salt leached to generate pores), plastic track-etched membranes (e.g., polycarbonate, polyester), membrane filters (polytetrafluoroethylene, cellulose, polyethersulfone resin), and micromolded meshes (e.g., polydimethylsiloxane, epoxy photoresist). Methods for varying porosity / permeability may include, but are not limited to, adjusting the density of the solid material, crosslink density, and / or manufacturing design.

[0097] Exemplary non-porous materials useful in the presently disclosed subject matter include 1002F epoxy photoresist, cyclic olefin polymers (e.g., those sold under the trade name "ZEONOR®" (Zeon Corporation, Tokyo, Japan)), polycarbonate, acrylate polymers (e.g., poly(methyl methacrylate)), polystyrene, polyethylene, polypropylene, polyvinyl chloride, cellulose, acrylonitrile butadiene styrene (ABS) plastic, nylon, acetal resins (e.g., acetal resins sold under the trade name "Delrin®" (EI du Pont de Nemours and Company, Wilmington, Delaware, USA)), polytetrafluoroethylene (e.g., TEFLON® (The Chemours Co., Wilmington, Delaware, USA), polyesters (e.g., polyethylene terephthalate, polyethylene naphthalate, polybutylene terephthalate, polytrimethylene terephthalate, polyglycolic acid, polylactic acid, polycaprolactone, copolyesters sold under the trade name "TRITAN™" (Eastman Chemical Company, Kingsport, Tennessee, USA)), epoxies (e.g., SU-8, 1001F, 1009F), elastomers (e.g., polydimethylsiloxanes, elastomers sold under the trade name "ECOFLEX™" (Smooth-On Inc., Macungie, Pennsylvania, USA)), glass, ceramic, and / or metal.

[0098] In some embodiments, one or more layers (e.g., a first layer) comprise a hydrogel. In some embodiments, the hydrogel comprises collagen. In some embodiments, the cell support substrate comprises a first layer overlying a second layer, and the first layer (i.e., the layer having an upper surface onto which one or more cells are deposited) comprises a hydrogel (e.g., collagen).

[0099] In some embodiments, the device further includes a base container including a bottom wall and at least one side wall extending upward from the bottom wall, the bottom wall and the at least one side wall defining a well, the luminal container being held within the well of the base container, the bottom wall of the base container being spaced apart from the bottom wall of the luminal container, and the base container being defined between the bottom wall of the base container and the bottom wall of the luminal container, and / or between at least one side wall of the base container and at least one side wall of the luminal container.

[0100] In some embodiments, one or both of the luminal and base reservoirs of the devices of the present disclosure can be, for example, a petri dish, cell culture dish, vessel, or substrate, hi some embodiments, they can be modified versions of commercially available cell culture dishes, vessels, or substrates, where the commercially available products are modified to contain a cell-support substrate comprising at least two physically distinct regions.

[0101] A cross-sectional view of an exemplary device 100A of the presently disclosed subject matter is shown in FIG. 1A. Device 100A includes a luminal vessel (generally 140). Luminal vessel 140 may be generally cylindrical in shape defined by a sidewall 141, one end of which is connected to a bottom wall 142. Luminal vessel 140 also includes a luminal flange 142, which connects to or is adjacent to the open end of luminal vessel 140 and extends radially from the top of wall 141. Bottom wall 142 also forms the bottom layer of a cell support substrate 145, which further includes a top layer 146 at the bottom of luminal vessel 140 that covers bottom wall 142. Bottom wall / layer 142 includes a non-porous material traversed by micropores 147. Top layer 146 includes a porous material such as a hydrogel (e.g., collagen). Top surface 146' of top layer 146 thus includes the surfaces of two physically distinct substrate regions. Region 146'a represents the surface of one of the physically distinct regions where cell support substrate 145 includes both top layer 146 and bottom wall / bottom layer 142. Region 146'b represents the surface of a second type of physically distinct region where top layer 146 resides above one of the microholes 147.

[0102] FIG. 1B illustrates an exemplary device 100B in which the luminal container 140 of FIG. 1A is inserted into a base container 160. The reference numerals for the luminal container 140 are the same as those described with respect to FIG. 1A. The base container 160 may be generally shaped like a cylinder with one end closed. The base container 160 is generally defined by a sidewall 161, which is connected to a bottom wall 162. When the luminal container 140 is inserted into the base container 160, the bottom wall 142 of the luminal container 140 is positioned above, but not in contact with, the bottom wall 162 of the base container 160. As shown in FIG. 1B, the base container 160 also includes a flange 163, which is connected to or adjacent the open end of the luminal container 140 and extends radially from the top of the wall 141. When the luminal receptacle 140 is inserted into the base receptacle 160, the flange 143 of the luminal receptacle 140 can rest on the flange 163 of the base receptacle 160. Alternatively, the flange 163 can be the top wall of an array including multiple base receptacles.

[0103] FIG. 1C illustrates an exemplary device 100C in which the device 100B of FIG. 1B has been seeded with cells and allowed to grow. The reference numbers for the luminal reservoir 140 and the base reservoir 160 are the same as those described with reference to FIGS. 1A and 1B. In FIG. 1C, the luminal reservoir 140 is filled with a cell culture medium 149, and the base reservoir 160 is filled with a cell culture medium 169 that is different from the cell culture medium 149. For example, the cell culture medium 149 may be a differentiation medium, while the medium 169 may be a proliferation medium (e.g., containing growth factors that support stem cells). The cell culture medium 169 in the base reservoir 160 fills the micropores 147 in the bottom wall / layer 142, allowing the cell culture medium 169 to contact the porous top layer 146 of the cell support 145. As shown in FIG. 1C , cells seeded on the upper surface 146′ of the upper layer 146 of the cell support 145 form two distinct cell populations, illustrated as cells 182 and cells 184. Cells 182 proliferate in area 146′a and are cells that will convert to a differentiated cell population (e.g., when stem cell-supporting growth factors are absent from the luminal medium 149) or an untreated cell population (e.g., when the specific stimulus of interest being screened (e.g., a specific drug or test compound) is absent from the luminal medium 149). Cells 184 proliferate in area 146′b and are cells that may be stem cells (e.g., when stem cell-supporting growth factors are present in the basal medium 169) or treated cells (e.g., when the specific stimulus of interest (e.g., a specific drug or test compound) is present in the basal medium 169).

[0104] The apparatus and methods of the present disclosure are versatile and easy to implement for a number of different applications, including but not limited to: 1) In vitro models for physiological studies (transport of macromolecules, ions, and water between cells, enzyme function, and interactions with bacteria). 2) Screening of drugs, biologicals, food compounds, toxins, mutagens, carcinogens, pathogens, viruses, microbiomes, etc. 3) Translational animal models or disease models by using stem and / or primary cells derived from humans. 4) Pharmacological, pharmacokinetic, and pharmacodynamic models for screening drugs, food compounds, etc., including comprehensive dose-response characterization. 5) In vitro models for studying metabolism. 6) An in vitro model of epithelial wound healing that maintains and restores barrier function. 7) In vitro models for studying microbe-host interactions. 8) Tissue engineering for transplantation to repair damaged epithelium. 9) Personalized medicine through research performed on cells from individual patients with specific genetic backgrounds. 10) Functional assays (absorption and transport of water and electrolytes (sodium, chloride, bicarbonate, protons, potassium, calcium), microbial metabolites (short-chain fatty acids, etc.), recovery of unabsorbed nutrients, etc.). 11) Mucus production, flow, movement, and disease-related effects on mucus (e.g., in cystic fibrosis). 12) Assays of anti-diarrheal drugs and treatments for constipation (e.g., laxatives). 13) Assays for synbiotics, prebiotics, and probiotics. 14) Radiopaque and scintigraphic marker testing and assays of their effects on the epithelium. 15) The effects of immune cells and their products (antibodies and cytokines) on the epithelium. 16) Effects of enteric neurons and their products on the epithelium. 17) The influence of muscle cells, their contraction and relaxation, and their metabolic products on the epithelium. 18) Assay of soluble and insoluble fibers and their effects on epithelium. 19) Understanding epithelial repair as a response to any type of injury. 20) Investigation of bacteria that lead to pseudomembrane formation (e.g., Clostridium difficile). 21) Screening of biological warfare compounds. 22) Research to understand the side effects of drugs and therapies such as NSAID therapy, chemotherapy, and radiation therapy. 23) Study of the role of the innate and adaptive immune systems in epithelial integrity, function, and disease (e.g., inflammatory bowel disease, intestinal disease, cancer, etc.). 24) Radiotherapy and chemotherapy and drug assays to improve off-target effects. 25) Tumor models that mimic hypoxic conditions or oxygen gradients in time and / or space, including liquid cancers (e.g., leukemia and myeloma) and / or solid cancers (e.g., melanoma, carcinoma, sarcoma, lymphoma, germ cell tumors, and / or mixed cancers). 26) Antibacterial, antiviral, and / or antifungal drug assays. 27) Research to understand the effects of bacteriophages on commensal and pathogenic bacteria and their interactions with host cells. 28) In vitro model systems for gram-positive bacterial infections, including gram-negative and gram-positive bacteria, and / or gram-negative bacterial infections. Such bacteria include Acinetobacter baumannii, Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recalentis, Brucella abortus, Brucella canis, Brucella fever blight, Brucella abortus, Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium amycolatum, Corynebacterium diphtheriae, Coxiella burnetii, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus enterocolitica, and Enterococcus faecalis. The pathogenic bacteria may include, but are not limited to, Escherichia faecium, Escherichia coli, enterotoxigenic E. coli, pathogenic E. coli, invasive E. coli, enterohemorrhagic E. coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Parachlamydia, Pseudomonas aeruginosa, Nocardia asteroides, Rocky Mountain spotted fever rickettsia, Salmonella enterica, Shigella spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Group B hemolytic streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Viridans streptococcus, Treponema pallidum, Vibrio cholerae, Vibrio vulnificus, and / or Yersinia spp. 29) In vitro culture systems for understanding infections by viruses, including double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, positive-stranded RNA viruses, negative-stranded RNA viruses, circular single-stranded RNA viruses, RNA reverse-transcribed viruses, and DNA reverse-transcribed viruses, including simplex viruses, varicellaviruses, cytomegaloviruses, roseoloviruses, lymphocryptoviruses, rhadinoviruses, mastadenoviruses, alphapapillomaviruses, betapapillomaviruses, gammapapillomaviruses, mupapillomaviruses, pneupapillomaviruses, polyomaviruses, molluscipoxviruses, orthopoxviruses, parapoxviruses, alphatorqueviruses, betatorqueviruses, gammatorqueviruses, gemicircular viruses, erythroviruses, dependoviruses, bocaviruses, coltiviruses, rotaviruses, sedornaviruses, hepeviruses, alphacoronaviruses, betacoronaviruses, toroviruses, mamastroviruses, noroviruses, sapoviruses, flaviviruses, hepaciviruses, and pegiviruses. Examples of such viruses include, but are not limited to, rabies, cardioviruses, cosaviruses, enteroviruses, hepatoviruses, kobuviruses, parechoviruses, rosaviruses, saliviruses, alphaviruses, rubiviruses, deltaviruses, lyssaviruses, vesiculoviruses, filoviruses, Ebolaviruses, Marburgviruses, paramyxoviruses, henipaviruses, morbilliviruses, respiroviruses, rubulaviruses, metapneumoviruses, pneumoviruses, arenaviruses, peribunyaviruses, orthobunyaviruses, hantaviruses, nairoviruses, fenuiviruses, phleboviruses, influenza A virus, influenza B virus, influenza C virus, thogotoviruses, gammaretroviruses, deltaretroviruses, lentiviruses, spumaviruses, and / or orthohepadnaviruses. 30) In vitro culture system for producing vaccines that require hypoxic conditions. 31) In vitro culture systems for protozoal infections (e.g., Entamoeba histolytica, Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora caetanensis, and / or Giardia lamblia). 32) In vitro culture systems for unicellular and / or multicellular fungal infections, such as fungi of the genera Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma, Rhizopus, Mucor, Lichtheimia, Pneumocystis jiroveci, and / or Sporothrix. 33) Models of biofilm formation, growth, spread, and / or disruption by anti-biofilm agents; and / or 34) Model systems requiring oxygen gradients or hypoxia (in space or time), or combinations of such systems, such as hair follicle niches, post-stroke cerebral hypoxia, cyanide poisoning, scar tissue in dish systems, fibrosis and wound healing, retinopathy, corneal hypoxia and angiogenesis, periodontitis, upper and lower airway models, bronchiolitis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia (bacterial, viral, mycoplasma infection), interstitial pneumonia, pulmonary edema, hypoxic pulmonary vasoconstriction models, liver tissue models, liver regeneration, liver These include models of fibrosis, viral hepatitis, fatty liver disease, nephropathy, nephritis, bone growth and regeneration, cartilage regeneration, bone marrow, fracture healing, thrombosis, hematopoietic stem cell niche, anemia including sickle cell disease, models of muscle during exercise, reproductive organ models, endometriosis, placental development including intrauterine hypoxia, embryonic development models, ischemia (cardiac ischemia, intestinal ischemia, cerebral ischemia, limb ischemia, skin ischemia) and ischemia-reperfusion injury models, anesthesia, and / or obesity.

[0105] Example The following examples are provided to provide guidance to those skilled in the art for practicing representative embodiments of the presently disclosed subject matter. In light of this disclosure and common skill in the art, those skilled in the art will understand that the following examples are merely illustrative, and that various changes, modifications, and alterations may be made without departing from the scope of the presently disclosed subject matter.

[0106] Materials and Methods Microdevice fabrication: A 10 × 10 array of microholes (each 50 μm in diameter) was created in a thin photoresist film by patterning through-holes in the film. The patterned photoresist film was mounted on a modified TRANSWELL® cassette (Corning, Inc., Corning, NY, USA) and overlaid with a thin, continuous layer of collagen for cell culture.

[0107] More specifically, to create a patterned photoresist film, a layer of 1002F50 photoresist (20 μm thick) (see Pai et al., Analytical Chemistry, 2007, 79, pp. 8774-8780) was spin-coated onto a glass slide at 1500 revolutions per minute (rpm) for 30 minutes and then baked at 95°C for 30 minutes to harden it. The photoresist was exposed to UV light (500 millijoules (mJ)) through a photomask containing an array of open rings (50 μm in diameter). The exposed photoresist was developed in propylene glycol methyl ether acetate (PGMEA) and baked at 95°C for 12 hours to create an array of very shallow (20 μm) microwells. The film on the glass slide was then soaked in water for over 15 hours to weaken the film's adhesion to the glass and allow for easier transfer of the film to a TRANSWELL® frame (Corning, Inc., Corning, NY, USA). After completely removing the membrane from the base of a standard 12-well TRANSWELL® insert array (Corning, Inc., Corning, NY, USA) with tweezers, the TRANSWELL® frame (Corning, Inc., Corning, NY, USA) was attached to the photoresist side of the patterned microwell array with double-sided medical tape (3M, Maplewood, MN, USA). At this point, the TRANSWELL® frame (Corning, Inc., Corning, NY, USA) was firmly attached to the 1002F film that was placed over the glass slide. The slide was removed from the frame and 1002F film by gently lifting the TRANSWELL® frame (Corning, Inc., Corning, NY, USA) with the attached film from the slide, and then trimming any film that protruded from the frame.

[0108] To prepare the final microdevice for mouse colon cell culture, a thin layer of collagen was formed over the entire surface of the microwell array. First, the 1002F film in a TRANSWELL® insert (Corning, Inc., Corning, NY, USA) was plasma-treated for 5 minutes to improve its hydrophilicity and water wettability. Next, the plasma-treated insert, along with the patterned 1002F film, was placed on a Petri dish coated with polydimethylsiloxane (PDMS) (PDMS was prepared using a silicone elastomer kit sold under the trade name "SYLGARD™ 184" (Dow Corning, MI, USA)). Neutralized collagen (200 μL of rat tail collagen I (see Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165–182) in a 0.02 N acetic acid solution (Corning, Inc., Corning, NY, USA) neutralized with sodium hydroxide, sodium bicarbonate, HEPES, and PBS) was dispensed onto the 1002F film surface within the insert. The collagen was incubated at 37°C for 1 hour to gel. The collagen gel was dried in an oven at 40°C for 16 hours, resulting in a thin layer of collagen coated with salt crystals, which was then overlaid on the photopatterned 1002F film. This thin collagen membrane spanned the gap across the entire hole in the 1002F film. The collagen-coated 1002F surface of the insert was gently rinsed with water to remove salts, sterilized with 70% ethanol, and washed with sterile PBS. To further enhance cell adhesion to the collagen film, 50 μg / mL rat tail collagen I (Corning) was added to the insert in PBS immediately before cell culture and incubated at 37°C for at least 1 hour. Simulation and diffusion studies:

[0109] COMSOL Multiphysics (COMSOL Inc., Burlington, MA, USA) was used to model the diffusion of growth factors through collagen-covered micropores. The geometry of the micropore array and the lumen / basal reservoir were incorporated into the model. The diffusion coefficient in the thin collagen layer was assumed to be equivalent to that in aqueous media. This assumption was reasonable because the collagen layer in this platform was only 5 μm thick (and because the average time for even large growth factors to permeate the membrane was expected to be less than 1 second). The diffusion of fluorescein dextran (40 kDa) was used to simulate the diffusion of growth factors (39.7 kDa (Wnt-3A) and 40 kDa (R-spondin)) through the micropore array. Fluorescein dextran (Sigma, St. Louis, MO, USA) was dissolved in PBS and placed in the basal reservoir of the microdevice (1.5 mL, 200 μg / mL), and PBS without fluorescein dextran (0.5 mL) was placed in the luminal reservoir. Samples (50 μL) were taken from the basal and luminal reservoirs every 24 hours. The fluorescence intensity of the samples was measured to estimate the concentration of fluorescent dextran in each reservoir. The measured concentration was calculated based on the previously reported diffusion rate of 40 kDa fluorescein dextran, i.e., 7.4 × 10 -11 m 2 The experimental data for fluorescent dextran diffusing through collagen in microdevices was compared with the simulated values ​​using ρ / s. See Ahmad et al., RSC Advances, 2015, 5, pp. 74881-74891. Because the simulated values ​​were comparable to the experimental data for fluorescent dextran diffusing through collagen in microdevices, the same diffusion coefficient was used to simulate the diffusion of growth factors in microdevices.

[0110] Cell culture and immunofluorescence staining Crypts collected from the intestines of male and female mice were isolated in a buffer solution (2.0 mM EDTA and 0.5 mM DTT) as previously described (see Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165–182). To ensure chromosomal integrity, all cells used were cultured for fewer than five passages. Mouse intestinal cells were cultured as monolayers on the flat surface of neutralized collagen gel (1 mL in a 6-well plate) in growth medium (EM). EM contains Wnt3A, R-spondin 3, and noggin to support stem cells in culture. The composition of EM is detailed in Table 2 below. Chemicals used in EM were purchased from the following suppliers: Advanced DMEM / F12, GlutaMAX, and HEPES were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA), mouse EGF from PeproTech US (Rocky Hill, NJ, USA), A83-01 from Sigma-Aldrich (St. Louis, MO, USA), N-acetylcysteine ​​from MP Bio (MP Biomedical, Santa Ana, CA, USA), ROCK inhibitor (Y-27632) from ApexBio Technology, LLC (Houston, TX, USA), and an antibiotic sold under the trade name PRIMOCIN™ from InvivoGen (San Diego, CA, USA).

[0111] The EM was replaced with fresh EM every two days. Cells were subcultured every three to five days. This was achieved by digesting collagen with collagenase and dissociating cells into clumps with 0.5 mM EDTA. These monolayers did not completely dissociate into single cells, as this resulted in high stem cell mortality. After cells were placed on the surface of the microdevice, the cell suspension was diluted 1-2 times in EM and spread onto the surface of the microdevice. EM was placed in the luminal and basal reservoirs of the transwell cassette and the cells were cultured for two days. On day two, the luminal and basal media were changed as shown in Table 3, and the cells were incubated for one day. On day three, the luminal and basal media were replaced with fresh media (the same as the media changed on day two), and the cells were incubated for another day. In some experiments, the media in the luminal compartment was replaced with differentiation media (DM, see Table 2). DM is similar to EM but lacks key growth factors necessary to support intestinal stem cells. In experiments with short-chain fatty acids, each short-chain fatty acid was added to the luminal medium at a concentration of 24 mM for acetate, 6 mM for propionate, and 1 mM for butyrate. [Table 2] [Table 3]

[0112] Cells in the S phase of the cell cycle were incubated with a 5-ethynyl-2'-deoxyuridine (EdU) pulse according to the manufacturer's protocol (Click-iT EdU Alexa Fluor 647 Imaging Kit, C10340, Thermo Fisher Scientific, Inc., Waltham, MA, USA). EdU is a nucleotide analogue that is incorporated into DNA during S phase replication. Briefly, EdU (10 μM) was added to the luminal and basal media, and cells were incubated at 37°C for 3 h. Next, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.5% Triton-X for 20 min at 25°C. EdU incorporated into cellular DNA was visualized by reaction with Alexa Fluor 647 via click chemistry according to the manufacturer's protocol (Click-iT EdU Alexa Fluor 647 Imaging Kit, C10340, Thermo Fisher Scientific, Inc., Waltham, MA, USA).

[0113] For pulse-chase experiments with EdU, cells were grown in EM for 2 days. On day 2, the luminal and basal media were removed, followed by the addition of DM to the luminal compartment and EM to the basal compartment, and the cells were incubated for 1 day. The media was replaced with fresh media (the same as the media replaced on day 2), and the cells were incubated for an additional day. On day 4, EdU was added to the luminal and basal media, and the cells were incubated for 3 hours as described above. The luminal and basal media were then replaced with fresh DM and EM, respectively, and the cells were incubated for an additional 2 days, with daily media changes.

[0114] On day 4 of culture, cell monolayers were assayed for alkaline phosphatase activity by incubating with ALP substrate mixture (Vector Red AP Substrate Kit SK-5100, Vector Laboratories Inc., Burlingame, CA, USA) in Tris buffer (pH 8.4) at 37°C for 30 minutes. Cells were then washed with PBS and fixed and permeabilized as described above. To label cellular DNA, Hoechst 33342 (2 μg / mL, B2261, Sigma-Aldrich, St. Louis, MO, USA) was incubated with the cells (1 hour at 25°C).

[0115] For immunofluorescence staining, primary antibodies against E-cadherin (1:200, 20874-1-AP, ProteinTech, Rosemont, IL, USA), β-catenin (1:200, sc-7963, Santa Cruz Biotechnology Inc., Dallas, TX, USA), mucin 2 (1:200, sc-15334, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and chromogranin A (1:1500, ab15160, Abcam, Cambridge, UK) were used, as well as Alexa 488-conjugated goat anti-rabbit antibody (1:500, A11008, Life Technologies, Carlsbad, CA, USA; substitute for E-cadherin, chromogranin A, and mucin 2) or Alexa 488-conjugated goat anti-rabbit antibody (1:500, A11008, Life Technologies, Carlsbad, CA, USA). A 647-conjugated donkey anti-mouse antibody (1:500, 715-605-150, Jackson ImmunoResearch, West Grove, PA, USA, surrogate for β-catenin) was used. Cells were grown on the microdevice in the EM for 2 days and further cultured as described below. On day 4, cells were fixed with 4% paraformaldehyde in PBS for 15 minutes and permeabilized with 0.5% Triton-X for 20 minutes at 25°C. To minimize nonspecific antibody binding, cells were blocked with 3% BSA in PBS for 1 hour at 4°C. Next, each primary antibody, diluted in 3% BSA in PBS at the manufacturer's recommended dilution ratio, was added to the cells and incubated for at least 16 hours at 4°C. The cells were then washed three times with PBS containing 3% BSA, incubated for 1 hour at 25°C with secondary antibody and Hoechst 33342, both diluted 1:500 in PBS containing 3% BSA, and finally washed twice with PBS containing 3% BSA and then with PBS alone. Cell imaging:

[0116] TRANSWELL® inserts (Corning, Inc., Corning, NY, USA) with cell layers from the microdevices were placed in 12-well plates under PBS and imaged using a FLUOVIEW® FV3000® confocal microscope (Olympus Corporation, Tokyo, Japan) (10x objective, 0.4 numerical aperture). Alexa 647, Vector-Red-ALP, and Hoechst 33342 were excited with 640, 561, and 405 nm lasers, respectively, and fluorescence emissions were collected in the ranges of 650-750 nm, 570-590 nm, and 430-470 nm, respectively. These images were analyzed using the Fiji software package. See Schindelin et al., Nature Methods, 2012, 9, 676. For all experiments, sample size was estimated by statistical power analysis based on data from Wang et al. (Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165–182), who measured EdU+ and ALP activity using G*Power under various conditions (α = 0.05, β = 0.85). See Faul et al., Behavior Research Methods, 2007, 39, pp. 175–191. One-way analysis of variance was used for all statistical analyses of data obtained from fluorescence microscopy images. Scanning electron microscopy (SEM) was performed using an FEI QUANTA™ 200 ESEM microscope (Thermo Fisher Scientific, Inc., Waltham, MA, USA).For SEM imaging, samples were first fixed in 4% paraformaldehyde, dried in a critical point dryer (PVT-3, Tousimis Semidri, Rockville, MD, USA), and coated with 10 nm of metal using a sputter coater (Cressington 108, Cressington Scientific Instruments, Watford, UK). Stiffness measurements:

[0117] The stiffness of the collagen described above, as well as the stiffness of the 1002F film adjacent to the microholes, was measured in fluid (1x PBS) using an atomic force microscope (MFP3D, Asylum Research, Morrisville, NC, USA). Force vs. displacement curves were collected (two samples, 10 locations above or adjacent to the microholes for each sample). Silicon cantilevers (nominal spring constant 0.03 N / m) fitted with polystyrene beads (4.5 μm diameter) were purchased from Novascan Technologies, Inc. (Ames, IA, USA). All calibrations, data collection, and data analysis were performed using Asylum software. The cantilever spring constant (0.03175 N / m) was determined more precisely by recording its thermal motion. A force of 10 nN was applied to the sample, and the force versus indentation curve was recorded. The stiffness (kPa) of the sample was obtained by fitting 90% of each curve (average indentation of 140 nm) to the Hertz model.

[0118] Example 1 Fabrication and characterization of planar intestinal crypt-supported microdevices An exemplary fabrication process for a microdevice supporting planar intestinal crypts is shown in Figure 4A. To fabricate this device, a layer of 1002F (40 μm thick by SEM) was coated onto a glass slide and photopatterned using a mask with a 10 × 10 array of opaque circles (50 μm diameter, 300 μm edge-to-edge spacing). See Figure 4B. The 1002F was removed from the slide by incubating in water, and the film was transferred to the bottom of a TRANSWELL® insert (Corning, Inc., Corning, NY, USA) (without the attached membrane). The insert with the 1002F film attached was placed on the PDMS surface, and collagen was loaded into the reservoirs to form a 1 mm-thick collagen gel on top of the micropatterned 1002F. The collagen was then dehydrated, leaving a condensed collagen layer that spread over the surface and bridged the micropores, which were then covered with salt crystals. See the upper panel of Figure 4C. Rehydration of this collagen resulted in a dense yet porous collagen film (4.7 μm thick as measured by confocal fluorescence microscopy) across the entire top surface of the device (see the lower panel of Figure 4C). The stiffness of the collagen layer in the micropores and in the region above the 10002F surface was significantly different at 55.83 ± 19.21 kPa and 146.38 ± 42.27 kPa, respectively, as measured by an AFM with a 4.5 μm diameter probe tip (n = 3, 10 locations per sample, p < 0.0001). During the dehydration and rehydration process, collagen density increased across the entire 1002F surface. This was due to a reduction in collagen thickness from 1 mm to 4.7 μm during this step. This increase in density also resulted in an increase in collagen stiffness, which has been reported to range from 10 to 1000 Pa when uncondensed, depending on the concentration. See Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165-182.The increased stiffness of the collagen-coated 1002F surface suggests that the collagen coating does not completely mask the stiffness of the 1002F film. EPON resins have typical stiffnesses of several GPa. See Engelberg and Tesoro, Polymer Engineering & Science, 1990, 30, pp. 303–307, and Vallo et al., Polymer Gels and Networks, 1993, 1, pp. 257–266. AFM measurements revealed an indentation depth of approximately 150 nm, which is shallower than the indentation depth typically perceived by cells. However, without being bound by any theory, it is possible that cells can “feel” the stiffness of the underlying 1002F film beyond that detected by the AFM tip. See Qu et al., Scientific Reports, 2018, 8, p. 3295; and Franck et al., PloS one, 2011, 6, p. e17833.

[0119] The ability of growth factors to move through 1002F micropores and collagen films was simulated in COMSOL (see Figures 4D and 4E). The diffusion coefficient of 40 kDa fluorescein dextran (which has a molecular weight comparable to Wnt-3A and R-spondin) in the hydrogel was previously measured to be 7.4 × 10 -11 m 2 / s. See Ahmad et al., RSC Advances, 2015, 5, pp. 74881–74891. As a control, the time course of fluorescein dextran (40 kDa) transfer from the basal compartment to the luminal compartment was measured by sampling these reservoirs and compared with simulation predictions. The results were consistent (see Figure 4E). Simulation predicted that 24 h after loading only the basal compartment with fluorescein dextran (30 ng / mL), the luminal compartment's fluorescein dextran concentration reached a maximum level (7.3–8.3 ng / mL) above the collagen-coated microholes (10 μm above the surface) and rapidly decreased in all directions to 4.5–5.5 ng / mL at equidistant points between the microholes. The steepest gradient of the growth factor concentration decline was 18 pg / mL / μm. Without being bound by any theory, this is believed to be sufficient to induce colonic epithelial cells to form two cell compartments: a stem cell / proliferative cell compartment and a differentiated cell compartment. Previous measurements have reported that a gradient of 75 pg / mL / μm was sufficient to polarize 200 μm-long mouse colon organoids (see Ahmad et al., RSC Advances, 2015, 5, pp. 74881-74891). However, this previous study did not incorporate stiffness or porosity gradients. This may indicate that steeper gradients than those measured in accordance with the presently disclosed subject matter are required. Because the above simulations did not incorporate turbulent or convective mixing, the gradients predicted by the simulations also represent minimal concentration differences along the 1002F surface. However, simulations suggest that arrays of collagen-coated microholes can act as localized growth factor sources that establish growth factor gradients outward from the center of the microholes.

[0120] Example 2 Growth of primary mouse intestinal epithelial cells on collagen-coated and patterned films Cells were cultured for 4 days on collagen-coated 1002F films with patterned micropores, with EM or stem cell support medium placed in the luminal (upper) and basal (lower) reservoirs (EM / EM). The 4-day culture period mimicked the typical lifespan of mouse colonic epithelial cells (3-5 days). See Tsubouchi, Developmental Dynamics, 1981, 161, pp. 239-246. Cell proliferation and differentiation, as indicated by EdU incorporation and alkaline phosphatase (ALP) activity, respectively, were tracked over time. See Figure 5. By day 2, cells showed robust proliferation, as indicated by EdU incorporation across the entire film surface. However, ALP activity was barely detectable within the epithelial cells, suggesting that differentiated cells were scarce under these conditions and at this time point. On day 3 of culture, EdU+ cells remained present throughout the array, accompanied by low levels of ALP activity. However, on day 4 of culture, a distinctive cellular patterning was observed, with circular regions of EdU+ cells extending 50–60 μm from the center of the microholes (see Figure 5 and Figures 6A and 6J). No EdU+ cells were present at distances greater than 70 μm from the center of the microholes (see Figure 5 and Figures 6A and 6J). Meanwhile, low levels of ALP activity remained present in cells greater than 90 μm from the center of the microholes (see Figure 5 and Figures 6A and 6K). Cells within 70 μm of the microhole center did not express measurable ALP activity. Cells in the transition zone between 60 and 70 μm from the center of the microholes showed little EdU incorporation or ALP activity. This suggests that proliferation and differentiation signals were equally balanced in these regions.

[0121] To understand why such distinctive patterning occurred despite the application of growth factor-containing EM to all cells, we cultured cells under EM / EM on a collagen-coated, non-porous 1002F film. This ensured that the stiff, impermeable 1002F surface underlay all cells. After 4 days of culture, the cells showed no EdU incorporation, suggesting that under these conditions the influence of the 1002F surface was dominant, thereby halting cell division. Cells were also cultured under EM / EM for 4 days on a collagen layer without an underlying 1002F film. Nearly all of these cells remained EdU+ throughout this period. In the presence of a low-stiffness, highly porous substrate, growth factors had the ability to drive cell proliferation. Thus, the change in the material properties of the underlying matrix alone was sufficient to create two cellular zones on the array: a highly proliferative region and a nonproliferative compartment with evidence of cell differentiation. Porosity significantly influences stem cell behavior, but in hydrogel systems, this effect is often coupled with stiffness. This is because crosslinking frequency is used to increase stiffness (and simultaneously decrease pore size). For example, it has been shown that mesenchymal stem cell migration through PEG hydrogels depends on the porosity of the scaffold, as well as its stiffness and adhesive strength. See Peyton et al., Biotechnology and Bioengineering, 2011, 108, pp. 1181-1193. Furthermore, the porosity of rigid polystyrene can alter the outgrowth of human stem cell-derived neurites. See Hayman et al., Journal of Biochemical and Biophysical Methods, 2005, 62, pp. 231-240. Porosity may also affect cell morphology and cytoskeleton organization in epithelial cells: MDCK II cells, a model epithelial cell line, adopt a more flattened and spread-out morphology and form thicker actin stress fibers on non-porous substrates than on porous substrates.See Rother et al., Journal of the Royal Society Interface, 2015, 12, 20141057; and Janshoff et al., Journal of Adhesion Science and Technology, 2010, 24, 2287-2300. The small change in stiffness between collagen-covered micropores and collagen-coated 1002F may also potentially affect cell behavior. Stiffer surfaces have been shown to reduce the protein levels of Oct3 / 4 or Nanog pluripotent stem cell markers in embryonic stem cells compared to softer surfaces, suggesting that stiffer surfaces may increase differentiation. See Chowdhury et al., PloS One, 2010, 5, p. e15655; and Lu et al., Biomaterials, 2014, 35, pp. 3945-3955. Finally, it has been shown that mouse and human intestinal stem cells cease to proliferate and appear to adopt a more differentiated phenotype on very hard surfaces, such as polystyrene or poly(dimethylsiloxane), compared to soft surfaces. See Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165-182. Taken together, the work disclosed here is consistent with previous studies showing that changes in porosity and stiffness can combine to alter cell fate. Of note, EM contains the ROCK inhibitor Y-27632, which is commonly used to inhibit anoikis in primary intestinal cell cultures.Because ROCK signaling is known to regulate mechanosensing and mechanotransduction (see Yim and Sheetz, Stem Cell Research & Therapy, 2012, 3, 41; and Provenzano and Keely, J. Cell Sci, 2011, 124, 1195-1205), the presence of Y-27632 may alter responses to substrate stiffness.

[0122] To gain insight into the effect of the surface on cell behavior, we further investigated the characteristics of cells in different regions of the disclosed arrays. Z-axis cell cross-sections confirmed that all regions on the surface grew as a monolayer (see Figure 6B). Cell contours visualized by E-cadherin staining in Figure 6F indicate a cell density (2.2 ± 0.1 cells / 100 μm) above the microholes. 2 ) is the cell density in the area between the micropores (0.8±0.1 cells / 100 μm 2) (n = 3 crypts, p < 0.0005), suggesting that the shape of cells above the micropores was more columnar (similar to intestinal epithelium in vivo), while cells in the intermediate region adopted a more spread-out, flattened shape. These results are consistent with the known responses of epithelial cells to porous and nonporous substrates. However, cells in all regions of the surface expressed the adherens junction marker E-cadherin, suggesting that cell-cell adhesion was maintained across the entire surface (see Figure 6F). β-catenin, a downstream effector of Wnt signaling that binds to cadherins within adherens junction complexes (see Valenta et al., The EMBO Journal, 2012, 31, 2714-2736), was also prominent in cells in all regions (see Figure 6F). This indicates that all cells remain subject to Wnt-3A binding and signaling, even if cells far from the micropores are not driven to proliferate by Wnt-3. These data indicate that stiffness and / or porosity were sufficient to initiate cell separation into two regions: i) a proliferative region with few enterocytes and high cell density, and ii) a non-proliferative region where enterocyte marker ALP was higher and the enterocytes adopted a flatter morphology.

[0123] Example 3 Creation of stem cell / proliferative cell compartments and differentiated cell zones Without being bound by any theory, the absence of high ALP activity in differentiated cells was likely due to the continued presence of growth factors Wnt-3, Noggin, and R-spondin. Cells were cultured on the arrays in EM / EM for two days to allow cells to attach and spread across the collagen-coated surface, then switched to DM / EM (luminal / basal) for an additional two days. DM (differentiation medium) does not contain Wnt3A, R-spondin, or Noggin, and forces intestinal epithelial cells to differentiate toward enterocyte or absorptive cell lineages. See Wang et al., ACS Biomaterials Science & Engineering, 2017, 3, pp. 2502–2513. To investigate the fate of cells exposed to DM / EM, cell proliferation and differentiation were tracked over time. See Figure 5. On the third day of culture, proliferating cells were largely localized to the surface area above the microholes, while cells with ALP activity were confined to other areas. On the fourth day, two distinct cellular compartments were readily visualized. There was no statistical difference in size between the proliferating cell compartment located only above the microholes and the EM / EM-derived compartment created after four days. See Figures 6C, 6D, and 6J. This suggests that the cells above the microholes were able to diffuse through the microholes and readily utilize the growth factors in the EM. In contrast, cellular ALP activity began to increase 60 μm from the center of the microholes and reached maximum activity at 130 μm. In these outer regions, ALP activity significantly increased after EM / EM (4.5-fold, n = 5 crypts, p < 0.0005). See Figure 6K. The DM in the upper reservoir, along with the underlying surface, directed intestinal epithelial cells toward a non-proliferative phenotype, significantly increased ALP activity, and created a differentiated cell zone (>130 μm) surrounding the proliferative cell zone (0–50 μm) above the micropores. This compartmentalized culture was continued until at least day 6.

[0124] To confirm that the central proliferation zone was created by growth factors diffusing through the micropores from the underlying EM, several controls were performed. In each control experiment, cells were cultured on the surface described below for two days in EM / EM, followed by an additional two days in the medium described below. In the first experiment, intestinal cells were cultured on a flexible collagen membrane without the 1002F film underneath, then placed in DM / EM on days 3 and 4. By day 4, proliferating cells were present across the entire array surface, again suggesting that growth factors diffused effortlessly through the collagen layer. In the second control experiment, cells were cultured on a collagen-coated 1002F film without micropores, and then placed in DM / EM on days 3 and 4. As expected, no EdU+ cells were present, but ALP activity was observed across the entire surface. In the third control experiment, cells were cultured on a collagen-coated micropore array, then placed in DM / DM on days 3 and 4. On day 4, these devices lacked EdU+ cells, indicating that the collagen film over the microholes was not sufficient to support cell proliferation in the absence of growth factors. Maximal ALP activity was observed on these arrays, with ALP activity 100–110 μm from the center of the microholes being significantly greater than that observed in DM / EM cultures (2.2-fold, n = 5 crypts, p < 0.005). See Figure 6K. The significant difference in ALP activity between DM / EM and DM / DM suggests that less growth factor likely diffused into the differentiated cell zone in DM / EM cultures.

[0125] Planar crypts with proliferation and differentiation zones were further characterized by immunofluorescence and SEM. As shown in Figure 6G, cells within planar crypts under DM / EM maintained cell-cell adhesion throughout the area, as indicated by the presence of E-cadherin, while Wnt signaling, visualized by β-catenin, was mostly localized near the micropores. Under DM / EM, cells within the differentiation zone lost access to growth factors that would enable them to progress further down the enterocyte differentiation pathway. SEM images revealed that microvilli, characteristic of absorptive colonocytes, were prominent in cells away from the micropores within the differentiation zone and were less dense and shorter in cells above the center of the micropores (suggesting a less differentiated cell type). See Figures 6H and 6I. The presence of goblet cells within planar crypts was also confirmed by immunofluorescent staining for mucin 2. See the left image in Figure 6E. Unlike enterocytes, embryonic cells were primarily located near micropores, consistent with the large number of goblet cells located deep within crypts in vivo (see Birchenough et al., Science, 2016, 352, pp. 1535–1542). Finally, planar crypts contained low levels of chromogranin A (approximately 2 per planar crypt), representing enteroendocrine cells (see the right image in Figure 6E). This is consistent with the sparse presence of this cell type in vivo (see Sternini et al., Current Opinion in Endocrinology, Diabetes, and Obesity, 2008, 15, 73). Thus, the planar crypt arrays of the present disclosure recapitulate the cell separation seen in vivo, but in two dimensions and in a format that is easy to fabricate and maintain.

[0126] Example 4 Migration and death of intestinal epithelial cells on planar crypt arrays In the in vivo intestine, proliferation of stem cells at the base of crypts triggers the migration of proliferative cells, which continue to divide and ascend the long axis of the crypt toward the luminal surface of the intestine. As cells approach the lumen, they differentiate into non-dividing lineages (enterocytes, germ cells, and enteroendocrine cells). See Barber, Nature Reviews Molecular Cell Biology, 2014, 15, p. 19. To examine whether cells on planar crypt arrays recapitulate this ordered cell migration from the proliferative cell compartment to the differentiated cell region, mouse endothelial cells on collagen-coated microhole arrays were cultured in EM / EM for 2 days (days 1 and 2) and then polarized in DM / EM for an additional 2 days (days 3 and 4). The cells were then incubated with EdU for 3 hours, after which the EdU was washed out and cultured in DM / EM for an additional 2 days (days 5 and 6). The location of EdU+ cells was measured immediately after EdU incubation on day 4 and again on day 6 (after a 2-day chase period). On day 4, as expected, all EdU+ cells were located above the microholes in the proliferative cell zone (mean cell location was 36 ± 15 μm from the microhole center, n = 165 cells). See Figures 7A and 7C. However, on day 6, the majority of EdU+ cells were located within the differentiated cell zone (mean cell location was 105 ± 44 μm from the microhole center, n = 386 cells, see Figures 7B and 7D). This indicates that these cells migrated from the proliferative zone (pulse location) to the differentiated cell zone during the intervening 2 days. The location of EdU+ cells on day 6 was statistically different from that on day 4 (p < 0.0001). Many of the EdU+ cells also colocalized with ALP activity measured on day 6, indicating that these day 4 proliferating cells differentiated during their outward migration. The average velocity of these cells was 1.6 ± 1.1 μm / h (the difference between the average EdU+ cell positions at 0 and 44 h after EdU washout divided by 44 h), which is comparable to the cell migration velocity within the lower crypts in vivo (1.3 μm / h).See Tsubouchi, Developmental Dynamics, 1981, 161, pp. 239-246. The upward flow of cells from the stem / proliferative niche at the crypt base to the luminal surface of the intestine was restored in planar crypt arrays.

[0127] The average survival time of in vivo epithelial cells on the luminal surface of the mouse colon is 5 days. See Tsubouchi, Developmental Dynamics, 1981, 161, pp. 239–246. After death, these luminal cells are discarded into the intestinal lumen, thereby making space for newly arriving cells. See Barkla and Gibson, Pathology, 1999, 31, pp. 230–238. To understand the fate of differentiated cells on planar crypt arrays, cells were cultured in EM / EM for 2 days and then in DM / EM for an additional 2 days. Necrotic and apoptotic cells were visualized by staining the arrays with propidium iodide (to mark all dead cells) and fluorescein-annexin V (to mark apoptotic cells). Few cells in the proliferation zone showed propidium iodide or annexin V fluorescence. In contrast, dead and dying cells were readily observed in the differentiated cell region. In areas away from the through-holes, dying cells may be straying from clusters where exposed areas of collagen-coated photoresist remained. Taken together, these data suggest that the progeny of cells that divide in the proliferation zone migrate outward into the differentiated cell region and mature into enterocytes as they move outward. At the end of their lifespan, these non-dividing cells die by apoptosis and are replaced by newly arrived cells from the proliferation zone. These functions allow the cell life cycle to be repeated as cells migrate from the crypt base to the luminal epithelium in vivo.

[0128] Example 5 Effects of short-chain fatty acids on proliferation and differentiation of mouse intestinal epithelial cells Colonic epithelial proliferation and differentiation are influenced by microbial products, such as short-chain fatty acids, produced during bacterial fermentation of fibrous materials. See Koh et al., Cell, 2016, 165, pp. 1332-1345. For example, propionate and butyrate inhibit proliferation in intestinal tumor cell lines. See Gamet et al., International Journal of Cancer, 1992, 52, pp. 286-289, and Whitehead et al., Gut, 1986, 27, pp. 1457-1463. Butyrate also reduces proliferation in primary mouse intestinal organoids. See Kaiko et al., Cell, 2016, 165, pp. 1708-1720. To evaluate the effects of short-chain fatty acids on the proliferation and differentiation of intestinal epithelial cells on planar crypt arrays, short-chain fatty acids (24 mM acetate, 6 mM propionate, or 1 mM butyrate) were added to the DM luminal medium during 2 days of polarization, i.e., on days 3 and 4 of cell culture on the arrays. The areas of EdU+ and ALP+ were measured and normalized to the area positive for Hoechst 33342 (total area of ​​cell nuclei as a proxy for cell number). Acetate significantly increased the normalized area of ​​EdU+ cells. Propionate and butyrate significantly decreased the normalized area of ​​EdU+ cells compared to the control without short-chain fatty acids. See Figure 8B. This is consistent with the behavior of mouse intestinal cells cultured as monolayers on collagen gels (see Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2017, 4, pp. 165-182), mouse intestinal organoids in Matrigel putty (see Kaiko et al., Cell, 2016, 165, pp. 1708-1720), and HT29 human colon cancer cells in tissue culture.See Gamet et al., International Journal of Cancer, 1992, 52, pp. 286-289. In contrast, acetate significantly decreased ALP activity, while propionate and butyrate significantly increased ALP activity relative to the control. See Figure 8C. Butyrate and propionate significantly increased ALP activity, similar to the effect of these two short-chain fatty acids when applied as a gradient to 3D crypts formed from primary human colonocytes. See Wang et al., Cellular and Molecular Gastroenterology and Hepatology, 2018, 5, pp. 113-130. Butyrate significantly increased ALP activity compared with the sole short-chain fatty acid and reduced the number of Hoechst 33342+ cells per crypt by approximately 17% (p<0.001) (Figure 8D). This is consistent with previous observations that butyrate induces apoptosis in human colon cancer cell lines. See Ruemmele et al., Gut, 2003:52, pp. 94-100; Fung et al., Journal of Proteome Research, 2011:10, pp. 1860-1869; and Xu et al., Signal Transduction and Targeted Therapy, 2017:2, pp. 16035. These data indicate that planar crypt arrays recapitulate important intestinal epithelial cell responses to microbial metabolites.

[0129] Example 6 Formation of planar crypts for a human intestinal epithelial model system Human planar crypts were generated using the same microdevice described in Example 1 and used in Example 2, using primary human intestinal epithelial cells. Primary human epithelial cells were cultured on this platform for four days in the presence of growth factors in the luminal and basal media. On day four, the cells were incubated for an additional four days. This was performed under three conditions: 1) EM / EM conditions, in which growth factors were present in both the luminal and basal media; 2) DM / EM conditions, in which growth factors were present only in the basal media; and 3) DM / DM conditions, in which growth factors were absent in any compartment. The medium was changed every two days during culture. On day eight, cells were pulse-labeled with EdU for three days, then subjected to a 30-minute ALP array, followed by fixation to label proliferating cells and colonocytes, respectively. Goblet cells were detected by immunofluorescence using a MUC2 antibody. EdU was detected by conjugating a Cy5 fluorophore via click chemistry. Figure 9 shows cells on this platform under these three different media conditions. Only cells under DM / EM conditions exhibited both EdU+ proliferating cells and MUC2+ goblet cells, with the EdU+ proliferating cells confluently forming a proliferative zone above and near the holes.

[0130] It will be understood that various details of the disclosed subject matter may be changed without departing from the scope of the disclosed subject matter. Moreover, the foregoing description has been given by way of example only and not by way of limitation. [Appendix 1] 1. A method for generating a tissue construct comprising two or more distinct regions, each containing a different cell population or cell lineage, comprising: (a) providing a support substrate including two or more physically distinct regions, wherein the two or more physically distinct regions of the support substrate are different from one another; (b) depositing / disposing one or more cells on the support substrate, wherein the one or more cells settle or adhere to the support substrate and grow on the support substrate; Including, the one or more cells transform into different cell populations or cell lineages on the two or more physically distinct regions of the support substrate; method. [Appendix 2] 2. The method of claim 1, wherein the two or more physically distinct regions of the support substrate comprise different physical properties, and the one or more cells are transformed on the two or more physically distinct regions of the support substrate into different cell populations or cell lineages in response to the different physical properties of the two or more physically distinct regions of the support substrate. [Appendix 3] 3. The method of claim 2, wherein the different physical properties are porosity, permeability, shear modulus, or a combination thereof. [Appendix 4] 4. The method of any one of claims 1 to 3, wherein the one or more cells comprise primary cells, and optionally, the one or more cells comprise primary epithelial cells. [Appendix 5] 5. The method of any one of claims 1-4, further comprising exposing one or more of the two or more physically distinct regions to one or more stimuli, each of the one or more stimuli selected from the group consisting of drugs, nutraceuticals, signaling molecules, toxins, inflammatory mediators, and microbially derived compounds. [Appendix 6] 6. The method of claim 5, further comprising detecting or measuring an effect of the one or more stimuli, optionally wherein the detecting or measuring comprises comparing one or both of cell differentiation and cell proliferation after exposure to the one or more stimuli with one or both of cell differentiation and cell proliferation before exposure to the one or more stimuli and / or with one or both of cell differentiation and cell proliferation in a comparable tissue construct not exposed to the one or more stimuli. [Appendix 7] 1. An apparatus for generating a tissue construct comprising two or more distinct regions, comprising: a luminal container including a bottom wall, at least one side wall extending upwardly from the bottom wall, and an upper opening defined by the at least one side wall; a cell support substrate on or including the bottom wall, wherein the cell support substrate comprises two or more physically distinct regions, the two or more physically distinct regions of the cell support substrate being different from one another, and the two or more physically distinct regions of the cell support substrate comprising different physical properties; An apparatus comprising: [Appendix 8] 8. The device of claim 7, wherein the cell support substrate comprises a single layer of material comprising two or more physically distinct regions comprising different physical properties. [Appendix 9] 8. The device of claim 7, wherein the cell support substrate comprises a first layer of material and a second layer of material, the first layer overlaying the second layer, the first layer and the second layer having different physical properties, and one of the first layer and the second layer comprising one or more openings extending from one surface of the first layer or the second layer to an opposite surface of the first layer or the second layer, and optionally the one or more openings are micropores. [Appendix 10] 10. The device of claim 9, wherein the first layer comprises a porous material, the second layer comprises a non-porous material, the second layer comprises the one or more microholes, and optionally, the second layer is the bottom wall of the luminal container. [Appendix 11] 11. The device of claim 10, wherein the first layer comprises a hydrogel, and optionally, the first layer comprises collagen. [Appendix 12] the base container further includes a bottom wall and at least one side wall extending upward from the bottom wall, the bottom wall and the at least one side wall defining a well; the luminal container is held within the well of the base container; the bottom wall of the base container is spaced from the bottom wall of the luminal container; a base container is defined between the bottom wall of the base container and the bottom wall of the luminal container, and / or between the at least one side wall of the base container and the at least one side wall of the luminal container; 12. The device according to any one of appendices 7 to 11. [Appendix 13] 1. A method for preparing a two-dimensional live cell culture model of intestinal crypts or colonic crypts, comprising: providing a device according to any one of appendices 7 to 12; depositing / disposing one or more epithelial cells on the cell support substrate, wherein the one or more cells anchor or adhere to the cell support and proliferate on the cell support substrate or substrate assembly, and the one or more cells transform into different cell populations or cell lineages on the two or more distinct regions of the support substrate or substrate assembly depending on the different physical properties of the physically distinct regions of the support substrate or substrate assembly; A method comprising: [Appendix 14] 14. The method of claim 13, wherein the device includes a base container defined between the bottom wall of the base container and the bottom wall of the luminal container, and / or between the at least one side wall of the base container and the at least one side wall of the luminal container, and the method includes the steps of supplying a first growth medium to the base container and supplying a second growth medium to the luminal container, wherein the first growth medium and the second growth medium may be the same or different. [Appendix 15] 15. The method of claim 14, wherein the first growth medium and the second growth medium are the same for at least a first period of time after the one or more cells are deposited / placed. [Appendix 16] 16. The method of claim 15, wherein the first and second growth media comprise growth factors that support stem cell growth. [Appendix 17] 17. The method of claim 15 or 16, comprising replacing the first or second growth medium with a third growth medium after the first period of time, wherein the third growth medium is different from the first and second growth media, and optionally the third growth medium lacks growth factors that support stem cell proliferation. [Appendix 18] 18. The method of claim 17, wherein the third growth medium comprises one or more stimuli, each selected from a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial-derived compound. [Appendix 19] 15. The method of claim 14, wherein one or both of the first and second growth media comprise one or more stimuli, each stimulus selected from a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbial-derived compound. [Appendix 20] 20. The method of claim 18 or 19, further comprising detecting or measuring an effect of the one or more stimuli on one or more of the different cell populations or cell lineages, optionally wherein the detecting or measuring comprises comparing one or both of cell differentiation and cell proliferation in the one or more different cell populations or cell lineages after exposure to the one or more stimuli with one or both of cell differentiation and cell proliferation in the one or more different cell populations or cell lineages before exposure to the one or more stimuli and / or with one or both of cell differentiation and cell proliferation in one or more cell populations or cell lineages in a comparable cell culture model that has not been exposed to the one or more stimuli.

Claims

1. (a) a base container including a first bottom wall and at least one side wall extending upwardly from the first bottom wall and defining a first well, the first well containing a first cell culture medium; (b) a luminal container held within the first well of the base container; (i) a second bottom wall traversed by one or more micro-perforations; (ii) a porous layer for epithelial cell culture overlying the second bottom wall, the second bottom wall having a reduced porosity compared to the porous layer; (iii) at least one sidewall extending upwardly from the second bottom wall and defining a second well, the second well containing a second cell culture medium; and the luminal container, (c) a first epithelial cell population formed in a first region of the porous layer located above the one or more microholes; (d) a second population of epithelial cells formed in a second region of the porous layer that is not located above the one or more microholes; and Including, the second epithelial cell population is different from the first epithelial cell population; Living cell constructs.

2. The living cell composition of claim 1 , wherein the epithelial cells are primary epithelial cells.

3. The living cell composition of claim 1 , wherein the epithelial cells are intestinal epithelial cells.

4. 4. The living cell construct of claim 1, wherein the distance from the center of one microhole to the center of each adjacent microhole is 350 micrometers.

5. The living cell construct of claim 1 , wherein the micropores have a diameter of 10 micrometers to 100 micrometers.

6. The living cell construct of claim 1 , wherein the second bottom wall comprises a non-porous material.

7. The living cell construct of claim 1 , wherein the porous layer comprises a hydrogel or collagen.

8. A living cell construct according to any one of claims 1 to 7, wherein the porosity of the porous layer is at most 10%.

9. 9. The living cell construct of claim 1, wherein the porous layer has a permeability coefficient of at most 100 cm / s.

10. 10. The living cell construct of claim 1, wherein the porous layer has a modulus of rigidity in the range of 1x10<0> Pa to 1x10<12> Pa.

11. The living cell construct of claim 1 , wherein the porous layer has a thickness of 50 micrometers or less.

12. 12. The living cell composition of claim 1, wherein the first cell culture medium and the second cell culture medium may be the same or different.

13. 13. The living cell composition of claim 1, wherein the first cell culture medium is a differentiation medium and the second cell culture medium is a proliferation medium.

14. 14. The living cell composition of claim 1, wherein the first cell culture medium and the second cell culture medium comprise one or more growth factors.

15. The living cell composition of claim 14 , wherein the one or more growth factors support stem cells, proliferating cells, and / or proliferating cells.

16. The one or more growth factors may be WNT-3A, R-spondin, noggin, transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), AA isoform platelet-derived growth factor (AA PDGF), AB isoform platelet-derived growth factor (AB PDGF), BB isoform platelet-derived growth factor (BB PDGF), acidic isoform 1 fibroblast growth factor (FGF), acidic isoform 2 fibroblast growth factor (FGF), FGF basic form 2, FGF 4, FGF 8, FGF 9, FGF 10, nerve growth factor (NGF) 2.5s, NGF 7.0s, beta-NGF, neurotrophic factors, brain-derived neurotrophic factors, cartilage-derived factors, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), EG-VEGF, VEGF-related proteins, Bv8, VEGF-E, granulocyte colony-stimulating factor (G-CSF), insulin-like growth factor (IGF) I, insulin-like growth factor (IGF) II, hepatocyte growth factor, glial neurotrophic growth factor, stem cell factor (SCF), keratinocyte growth factor (KGF), skeletal growth factor, bone matrix-derived growth factor, and bone-derived growth factor.

17. 17. The living cell composition of claim 1, wherein the first epithelial cell population and the second epithelial cell population form a continuous cell layer.

18. 18. The living cell construct of any one of claims 1 to 17, wherein the pre-living cell construct replicates the human crypt system.

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