Cell co-culture systems and methods of use

Abstract

Described herein are methods for co-culturing cells, co-culture cell constructs and methods for producing co-culture cell constructs.

Description

Attorney Docket No. 00009.008.1801CELL CO-CULTURE SYSTEMS AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U. S. Provisional Application No.63 / 774,005, filed on March 18, 2025; the entire content of which is hereby incorporated by reference herein in its entireties for all purposes.SUMMARY

[0002] The intestinal microenvironment represents a complex and dynamic ecosystem, comprising a diverse range of epithelial (e.g., intestinal epithelial cells) and nonepithelial cells (e.g., immune cells), as well as a protective mucus layer, and a diverse community of gut microbiota. Understanding the intricate interplay between these components is essential for uncovering the mechanisms underlying intestinal health and disease.

[0003] While mono-culture provides valuable information for understanding basic biology, coculture of more than one type of cell provides an opportunity to investigate the effects of intercellular communication. To do so, the intestinal organoids, three-dimensional (3-D) miniintestines that closely mimic the architecture, cellular diversity, and functionality of the intestine, have been used. Such systems have been used to co-culture intestinal organoids with other intestinal elements including immune cells, stromal cells, neural cells, and microbes (bacteria and viruses).

[0004] However, co-culturing organoids and immune cells remains nonetheless challenging as the system requirements for each cell type must be satisfied in the culture medium, which in some cases may not be compatible because of cross-reaction to components in the other system’s medium. Thus, current intestinal cell co-culture models with immune cells, while valuable, face limitations including difficulty in replicating the complex gut environment, reproducibility issues, technical challenges, and challenges in maintaining long-term organoid-immune cell interactions. These limitations manifest in various ways, and can result in the following challenges: (1) the variability in media composition between different co-culture models and experiments can lead to inconsistent results; (2) the efficiency of generating organoids can be low, and tissue availability and TIL (tumor-infiltrating lymphocytes) yield can limit co-culture setup; (3) satisfaction of the specific culture requirements of both organoids and immune cells in a single culture medium can be challenging; (4) some models, like primary intestinal cells, exhibit a short lifespan in culture, making it difficult to establish long-term co-cultures; (5) some models, like Caco-2 cells, may not be able to establish proper cell-cell contacts, which are crucial for gut function; (6) access to the1#623862Attorney Docket No. 00009.008.1801 lumen of organoids can be challenging, limiting the ability to study interactions with luminal contents like bacteria or nutrients.

[0005] Accordingly, improved methods and systems for co-culturing intestinal cells and immune cells are needed.

[0006] Described herein in certain embodiments, is method for co-culturing intestinal epithelial cells and immune cells, comprising: (a) culturing the intestinal epithelial cells in an expansion medium followed by a differentiation medium; (b) culturing the immune cells in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA); and (c) combining the intestinal epithelial cells from (a) and the immune cells from (b) in a co-culture cell construct comprising a first co-culture medium.

[0007] In some embodiments, the intestinal epithelial cells are seeded on a hydrogel layer. In some embodiments, the hydrogel layer is Matrigel, optionally about 1% Matrigel.

[0008] In some embodiments, the intestinal epithelial cells are grown to confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are grown to confluence in the expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the expansion medium is exchanged for the differentiation medium once the intestinal epithelial cells reach confluence.

[0009] In some embodiments, the intestinal epithelial cells are primary intestinal epithelial cells.

[0010] In some embodiments, the immune cells are cultured in suspension prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are seeded at a density from up to a range of about 7xl03cells / 12.6 cm2to about 1.12xl05cells / 12.6 cm2in the first cell culture medium.

[0011] In some embodiments, the first cell culture medium does not comprise mercaptoethanol. In some embodiments, the first cell culture medium comprises Roswell Park Memorial Institute 1640 (RPMI-1640) medium. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 10 pM of the PMA.

[0012] In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0013] In some embodiments, the method further comprises removing the first cell culture medium and providing a second cell culture medium that does not contain PMA. In some embodiments, the second cell culture medium does not contain mercaptoethanol.2#623862Attorney Docket No. 00009.008.1801

[0014] In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0015] In some embodiments, the immune cells are exposed to one or more immune celldifferentiating and / or stimulating agents prior to being combined with the intestinal epithelial cells in the co-culture cell construct, wherein the one or more immune cell-differentiating and / or stimulating agents is selected from the group consisting of: lipopolysaccharide (LPS), interferon gamma (IFN-y), interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colonystimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT).

[0016] In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and / or interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS) and / or from up to a range of about 0.5 ng / mL to about 25 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0017] In some embodiments, the immune cells are exposed to interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to IL-4, CRP, GM-CSF, Zymosan, TNFa, Nigericin, and / or CT for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 100 ng / mL IL-4, from up to a range of about 5 pg / mL to about 100 pg / mL CRP, from up to a range of about 1 ng / mL to about 1000 ng / mL GM-CSF, from up to a range of about 1 pg / mL to about 1000 pg / mL Zymosan, from up to a range of about 0.25 ng / mL to about 100 ng / mL of TNFa, from up to a range of about 1 pM to about 100 pM Nigericin, and / or from up to a range of about 1 ng / mL to about 1000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0018] In some embodiments, the immune cells are treated with one or more inhibitors. In some embodiments, the one or more inhibitors is one or more cytokine inhibitors. In some embodiments,3#623862Attorney Docket No. 00009.008.1801 the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin-12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X-C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNY inhibitor. In some embodiments, the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some embodiments, the IFNy inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib. In some embodiments, the IL-8 inhibitor is selected from the group consisting of: MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756). In some embodiments, the IL-ip inhibitor is selected from the group consisting of: Rilonacept (Arcalyst) and Canakinumab (Haris). In some embodiments, the IL-6 inhibitor is selected from the group consisting of: Tocilizumab (Actemra) and Sarilumab (Kevzara). In some embodiments, the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi). In some embodiments, the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418). In some embodiments, the TNFa inhibitor is adalimumab. In some embodiments, the IFNy inhibitor is tofacitinib.

[0019] In some embodiments, the immune cells are Tohoku Hospital Pediatrics-1 (THP-1) cells.

[0020] Described herein, in certain embodiments, is a method for co-culturing intestinal epithelial cells and immune cells comprising: (a) culturing the intestinal epithelial cells in an expansion medium followed by a differentiation medium; (b) culturing the immune cells in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA); and (c) combining the intestinal epithelial cells from (a) and the immune cells from (b) in a co-culture cell construct comprising a first co-culture medium that does not contain growth factors.

[0021] In some embodiments, the first co-culture medium does not contain epidermal growth factor (EGF). In some embodiments, the first co-culture medium does not contain N-acetylcysteine (NAC). In some embodiments, the first co-culture medium comprises lipopolysaccharide (LPS) and / or interferon gamma (IFN-y). In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS). In some embodiments, the first co-culture medium comprises about 10 ng / mL lipopolysaccharide (LPS). In some embodiments, the first co-culture medium comprises from up to a range of about 0.14#623862Attorney Docket No. 00009.008.1801 ng / mL to about 1 ng / mL interferon gamma (IFN-y). In some embodiments, the first co-culture medium comprises about 0.5 ng / mL interferon gamma (IFN-y).

[0022] In some embodiments, the first co-culture medium comprises one or more TGF-beta / Smad inhibitors. In some embodiments, the one or more TGF-beta / Smad inhibitors is selected from the group consisting of 3,3-dimethyl-l-butanol, A8301, AUDA (12-(3-adamantan-l-yl-ureido) dodecanoic acid), BIBF-0775, DMH1 (dorsomorphin homolog 1), dorsomorphin, dorsomorphin 2HC1, Galunisertib (LY2157299), ginsenoside Rh4, GW788388, halofuginone, ITD-1, K02288, LDN-193189, LDN-193189 2HC1, LDN-212854, LDN-214117, LY2109761, LY 3200882, LY364947, lycopus extract, ML347, PD 169316, PF-06952229, Pirfenidone, R-268712, RepSox (E-616452), SB431542, SB505124, SB525334, SD-208, SIS3, SIS3 HC1, sulfasalazine, TA-02, TGFpRI-IN-3, TP0427736 HC1, and Vactosertib (TEW-7197). In some embodiments, the one or more TGF-beta / Smad inhibitors is A8301.

[0023] In some embodiments, the first co-culture medium comprises one or more inhibitors. In some embodiments, the one or more inhibitors is one or more cytokine inhibitors. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X-C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of a TNFa inhibitor and a IFNy inhibitor. In some embodiments, the TNFa inhibitor is selected from the group consisting of adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some embodiments, the IFNy inhibitor is selected from the group consisting of AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib. In some embodiments, the IL-8 inhibitor is selected from the group consisting of MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756). In some embodiments, the IL-ip inhibitor is selected from the group consisting of Rilonacept (Arcalyst) and Canakinumab (Haris). In some embodiments, the IL-6 inhibitor is selected from the group consisting of Tocilizumab (Actemra) and Sarilumab (Kevzara). In some embodiments, the IL-12 / IL-23 inhibitor is selected from the group consisting of Ustekinumab (Stelara) and Risankizumab (Skyrizi). In some embodiments, the CXCL11 inhibitor is selected from the group consisting of BMS-936559 (MDX-1106) and Vercirnon (TRA-418). In some embodiments, the TNFa inhibitor is adalimumab. In some embodiments, the IFNy inhibitor is tofacitinib.5#623862Attorney Docket No. 00009.008.1801

[0024] Described herein, in certain embodiments, is a co-culture cell construct produced by the method of any of the previous embodiments, the co-culture cell construct comprising: (a) a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well, the first well containing the first co-culture medium; (b) a luminal container held within the first well of the basal container and comprising: (i) a second porous wall; and (ii) at least one side wall extending upwardly from the second wall to define a second well, the second well also containing the first co-culture medium; and wherein the immune cells are cultured in the first well of the basal container and the intestinal epithelial cells are cultured in the second well of the luminal container.

[0025] Described herein, in certain embodiments, is method for producing a co-culture cell construct comprising a co-culture of intestinal epithelial cells and immune cells, the method comprising: (a) providing an apparatus, the apparatus comprising a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well; and a luminal container capable of being held within the first well of the basal container; (b) culturing the intestinal epithelial cells and the immune cells separately, wherein: (i) the intestinal epithelial cells are cultured in the luminal container and in an expansion medium followed by a differentiation medium; and (ii) the immune cells are cultured in the basal container and in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA) followed by a second cell culture medium that does not contain PMA; (c) replacing the differentiation medium in the luminal container and the second cell culture medium in the basal container with a first co-culture medium; and (d) providing the luminal container into the basal container so that the luminal container is held within the first well of the basal container and the intestinal epithelial cells and immune cells are combined in the co-culture; thereby generating the co-culture cell construct comprising the coculture of intestinal epithelial cells and immune cells.

[0026] In some embodiments, the intestinal epithelial cells are seeded on a porous hydrogel layer in the luminal container, optionally wherein the hydrogel layer is Matrigel, further optionally about 1% Matrigel.

[0027] In some embodiments, the intestinal epithelial cells are grown to confluence in the expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the expansion medium is exchanged for the differentiation medium once the intestinal epithelial cells reach confluence.

[0028] In some embodiments, the intestinal epithelial cells are primary intestinal epithelial cells.

[0029] In some embodiments, the immune cells are cultured in suspension in the first cell culture medium and / or in the second cell culture medium prior to being combined with the intestinal6#623862Attorney Docket No. 00009.008.1801 epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are seeded at a density in a range of about 7xl03cells / 12.6 cm2to about 1.12xl05cells / 12.6 cm2in the first cell culture medium.

[0030] In some embodiments, the first cell culture medium does not comprise mercaptoethanol. In some embodiments, the first cell culture medium comprises Roswell Park Memorial Institute 1640 (RPMI-1640) medium. In some embodiments, the first cell culture medium comprises about 1 nM to 10 pM of the PMA.

[0031] In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0032] In some embodiments, the second cell culture medium does not contain mercaptoethanol.

[0033] In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0034] In some embodiments, the immune cells are exposed to one or more immune celldifferentiating and / or stimulating agents prior to being combined with the intestinal epithelial cells in the co-culture cell construct, wherein the one or more immune cell-differentiating and / or stimulating agents is selected from the group consisting of: lipopolysaccharide (LPS), interferon gamma (IFN-y), interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colonystimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT). In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and / or interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / ml to about 1000 ng / mL lipopolysaccharide (LPS) and / or from up to a range of about 0.5 to about 25 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to IL-4, CRP, GM-CSF, Zymosan, TNFa, Nigericin, and / or CT for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal7#623862Attorney Docket No. 00009.008.1801 epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 100 ng / mL IL-4, from up to a range of about 5 pg / mL to about 100 pg / mL CRP, from up to a range of about 1 ng / mL to about 1000 ng / mL GM-CSF, from up to a range of about 1 pg / mL to about 1000 pg / mL Zymosan, from up to a range of about 0.25 ng / mL to about 100 ng / mL of TNFa, from up to a range of about 1 pM to about 100 pM Nigericin, and / or from up to a range of about 1 ng / mL to about 1000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0035] In some embodiments, the immune cells are treated with one or more inhibitors. In some embodiments, the one or more inhibitors is one or more cytokine inhibitors. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin-12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X-C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNY inhibitor. In some embodiments, the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some embodiments, the IFNy inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib. In some embodiments, the IL-8 inhibitor is selected from the group consisting of: MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756). In some embodiments, the IL-ip inhibitor is selected from the group consisting of: Rilonacept (Arcalyst) and Canakinumab (Haris). In some embodiments, the IL-6 inhibitor is selected from the group consisting of: Tocilizumab (Actemra) and Sarilumab (Kevzara). In some embodiments, the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi). In some embodiments, the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418). In some embodiments, the TNFa inhibitor is adalimumab. In some embodiments, the IFNy inhibitor is tofacitinib.

[0036] In some embodiments, the immune cells are Tohoku Hospital Pediatrics-1 (THP-1) cells.

[0037] In some embodiments, the first co-culture medium does not contain any growth factors. In some embodiments, the first co-culture medium does not contain epidermal growth factor (EGF). In some embodiments, the first co-culture medium does not contain N-acetylcysteine (NAC). In some embodiments, the first co-culture medium comprises lipopolysaccharide (LPS) and / or interferon gamma (IFN-y). In some embodiments, the first co-culture medium comprises from up8#623862Attorney Docket No. 00009.008.1801 to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS). In some embodiments, the first co-culture medium comprises about 10 ng / mL lipopolysaccharide (LPS). In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 1 ng / mL interferon gamma (IFN-y). In some embodiments, the first co-culture medium comprises about 0.5 ng / mL interferon gamma (IFN-y).

[0038] In some embodiments, the first co-culture medium comprises one or more TGF-beta / Smad inhibitors. In some embodiments, the one or more TGF-beta / Smad inhibitors is selected from the group consisting of 3, 3 -dimethyl- 1 -butanol, A8301, AUDA, BIBF-0775, DMH1, dorsomorphin, dorsomorphin 2HC1, Galunisertib (LY2157299), ginsenoside Rh4, GW788388, halofuginone, ITD-1, K02288, LDN-193189, LDN-193189 2HC1, LDN-212854, LDN-214117, LY2109761, LY 3200882, LY364947, lycopus extract, ML347, PD 169316, PF-06952229, Pirfenidone, R-268712, RepSox (E-616452), SB431542, SB505124, SB525334, SD-208, SIS3, SIS3 HC1, sulfasalazine, TA-02, TGFPRI-IN-3, TP0427736 HC1, and Vactosertib (TEW-7197). In some embodiments, the one or more TGF-beta / Smad inhibitors is A8301.

[0039] In some embodiments, the first co-culture medium comprises one or more inhibitors. In some embodiments, the one or more inhibitors is one or more cytokine inhibitors. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X-C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor. In some embodiments, the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some embodiments, the IFNy inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib. In some embodiments, the IL-8 inhibitor is selected from the group consisting of: MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756). In some embodiments, the IL-ip inhibitor is selected from the group consisting of: Rilonacept (Arcalyst) and Canakinumab (Haris). In some embodiments, the IL-6 inhibitor is selected from the group consisting of: Tocilizumab (Actemra) and Sarilumab (Kevzara). In some embodiments, the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi). In some embodiments, the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and9#623862Attorney Docket No. 00009.008.1801 Vercirnon (TRA-418). In some embodiments, the TNFa inhibitor is adalimumab. In some embodiments, the IFNy inhibitor is tofacitinib.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 shows bright field images of THP-1 cells after 24 hours of PMA-differentiation in a T25 flask (“With PMA T25 flask (direct addition of PMA)”) versus in a 96-well plate (“With PMA 96WP (detaching the cells)”). THP-1 cells not treated with PMA (“no PMA”) are also shown.

[0041] FIG. 2 shows bright field images of THP-1 cells after 48 hours of PMA-differentiation in THP-1 media or in RMM without EGF, NAC, or A-8301 (RMM-EGF-NAC-A8301), comparing direct addition of PMA versus detaching of cells.

[0042] FIG. 3 shows bright field images of THP-1 cells after 48 hours of PMA-differentiation in RMM with EGF, NAC and A8301 (RMM+EGF+NAC+A8301) and RMM with EGF (RMM+EGF) comparing direct addition of PMA against detaching of cells.

[0043] FIG. 4 shows a bar graph depicting the results of a luminescence assay demonstrating the viability of PMA differentiated THP-1 cells in media variants.

[0044] FIG. 5 shows TEER curves of Donor 1 (DI) Transverse Colon (TC) cells cultured in THP-1 media vs RMM media.

[0045] FIG. 6 shows TEER curves of Donor 1 (DI) Transverse Colon (TC) cells in RMM without EGF media variants.

[0046] FIG. 7 shows TEER curves of Donor 1 (DI) Transverse Colon (TC) cells in RMM vs RMM without A8301 and without NAC.

[0047] FIG. 8 shows bar graphs depicting the percentage change of TEER in Donor 1 (DI) Transverse Colon (TC) cells treated with either 10 ng / ml (top graph) or 30 ng / ml (bottom graph) of TNFa for 48 hours.

[0048] FIG. 9 shows an exemplary co-culture timeline corresponding to the co-culture method described in Example 3. TC and THP-1 monocultures are shown on the left side of the figure. Coculture of TC (in the apical compartment) and PMA differentiated THP-1 cells (in the basal compartment) is shown on the right side of the figure.

[0049] FIG. 10 shows bright field images of THP-1 cells at different seeding densities ( / .<., at IX, 2X, 4X, and 8X) after 24 hour PMA differentiation.

[0050] FIG. 11 shows the TEER curves of different THP-1 seeding densities in TC / THP-1 coculture throughout the 48 hour co-culture period. An n of 4 technical replicates was used for measuring the TEER curve.10#623862Attorney Docket No. 00009.008.1801

[0051] FIG. 12 shows a bar graph depicting the percent change in TEER from FIG. 11, calculated as the co-culture TEER divided by the TC monoculture TEER at 48 hours of co-culture. An n of 3 technical replicates was used for determining the percentage change in TEER.

[0052] FIG. 13 shows a bar graph depicting the results of a luminescence viability assay performed on THP-1 macrophages after 48 hours of TC / THP-1 co-culture for different THP-1 seeding densities in co-culture. An n of 4 technical replicates was used for determining co-culture viability via the luminescence viability assay. An n of 2 technical replicates was used for determining monoculture viability.

[0053] FIG. 14 shows a bar graph depicting the percentage of cytotoxicity as determined by a colorimetric LDH cytotoxicity assay for different THP-1 seeding densities in TC / THP-1 coculture. An n of 4 technical replicates was used for determining the percentage of cytotoxicity.

[0054] FIG. 15 shows TEER curves of three LPS+fFNy combo doses (100 ng / mL and 0.5 ng / mL, 100 ng / mL and 1 ng / mL, 1000 ng / mL and 1 ng / mL) tested in monoculture, 2X co-culture THP-1 seeding density, and 4X co-culture THP-1 seeding density.

[0055] FIG. 16 shows TEER curves of two different THP1 seeding densities (2X and 4X).

[0056] FIG. 17 shows TEER curves of single doses of LPS (100 ng / mL and 1000 ng / mL) and IFNy (0.5 ng / mL and 1 ng / mL).

[0057] FIG. 18 shows bar graphs depicting the percentage change in TEER for TC / THP-1 coculture and TC monoculture at 48 hours for both seeding densities (2X and 4X).

[0058] FIG. 19 shows bar graphs depicting the percentage of cytotoxicity as determined by a colorimetric LDH cytotoxicity assay for different THP-1 seeding densities in TC / THP-1 co-culture and in monoculture.

[0059] FIGs. 20A-20D show bar graphs depicting the results of a luminescence viability assay performed for both apical (TC) and basal (THP-1) compartments at two different seeding densities (2X and 4X). FIG.20A shows the results for the apical compartment (TC) with 2X THP-1 seeding density. FIG. 20B shows the results for the apical compartment (TC) with 4X THP-1 seeding density. FIG. 20C shows the results for the basal compartment (THP-1) with 2X THP-1 seeding density. FIG. 20D shows the results for the basal compartment (THP-1) with 4X THP-1 seeding density.

[0060] FIG. 21 shows an exemplary co-culture timeline corresponding to the co-culture method described in Example 5. TC and THP-1 monocultures are shown on the left side of the figure. Co-culture of TC (in the apical compartment) and PMA differentiated THP-1 cells (in the basal compartment) is shown on the right side of the figure.11#623862Attorney Docket No. 00009.008.1801

[0061] FIG. 22 shows the TEER curves of 4 hour pretreatment in coculture, overnight pretreatment in coculture, and TC monocultures. 4 hour pretreatment of THP-1 cells was conducted using 100 ng / mL LPS and 25 ng / mL IFNy. Overnight pretreatment of THP-1 cells was conducted using 100 ng / mL LPS and 0.5 ng / mL IFNy.

[0062] FIG. 23 shows bar graphs depicting the percent change in TEER from FIG. 22, calculated as the co-culture TEER divided by the TC monoculture TEER at 48 hours, for 4 hour pretreatment and overnight pretreatment of THP-1 cells.

[0063] FIG. 24 shows bar graphs depicting the results of a luminescence assay demonstrating the viability of cells from the apical (TC cells) and basal (THP-1 cells) compartments after 4 hour pretreatment with LPS and IFNy.

[0064] FIG. 25 shows a bar graph depicting the percentage of cytotoxicity as determined by a colorimetric LDH cytotoxicity assay for cells that received a 4 hour pretreatment with LPS and IFNy.

[0065] FIG. 26 shows a bar graph depicting the percent change in TEER as compared to vehicle at 48 hours post-treatment for conditions tested in Example 6. (CCM = Co-Culture Medium; RMM = maturation medium)

[0066] FIG. 27 shows a bar graph depicting the results of a luminescence assay, where ATP activity for transverse colon (TC) epithelial cells (from the apical compartment) was measured in different groups ( / .<., from the conditions described in Example 6). (CCM = Co-Culture Medium; RMM = maturation medium)

[0067] FIG. 28 shows a bar graph depicting the results of a luminescence assay, where ATP activity from THP-1 cells (from the basal compartment) was measured in different groups ( / .<., from the conditions described in Example 6). (CCM = Co-Culture Medium; RMM = maturation medium)

[0068] FIG. 29 shows the TEER curves for THP-l / TC cell lot A co-culture and TC cell lot A monoculture. The cells of TC cell lot A were in Day 2 of RMM at the beginning of co-culture, while THP-1 cells were pre-treated 4 hours prior to co-culture with three different combinations of LPS and IFNy (10+25, 25+25, and 100+25 ng / mL). During co-culture, a lower IFNy concentration of 0.5 ng / mL was used for all groups, while the LPS concentration (100 ng / ml) remained consistent with the pre-treatment concentration for each respective group.

[0069] FIG. 30 shows the TEER curves for THP-l / TC cell lot B co-culture and TC cell lot B monoculture. The cells of TC cell lot B were in Day 2 of RMM at the beginning of co-culture, while THP-1 cells were pre-treated 4 hours prior to co-culture with three different combinations of LPS and IFNy (10+25, 25+25, and 100+25 ng / mL). During co-culture, a lower IFNy concentration12#623862Attorney Docket No. 00009.008.1801 of 0.5 ng / mL was used for all groups, while the LPS concentration (100 ng / ml) remained consistent with the pre-treatment concentration for each respective group, specifically 10, 25, and 100 ng / mL.

[0070] FIG. 31 shows the TEER curves for THP-l / TC cell lot B co-culture and TC cell lot B monoculture. The cells of TC cell lot B were in Day 3 of RMM at the beginning of co-culture, while THP-1 cells were pre-treated 4 hours prior to co-culture with three different combinations of LPS and IFNy (10+25, 25+25, and 100+25 ng / mL). During co-culture, a lower IFNy concentration of 0.5 ng / mL was used for all groups, while the LPS concentration was maintained at the same level as the pre-treatment concentration for each group, specifically 10, 25, and 100 ng / mL.

[0071] FIG. 32 shows bar graphs depicting the percentage change in TEER for TC cell lot A (Day 2 in RMM) and TC cell lot B (Day 2 and Day 3 in RMM). Percent change in TEER is calculated relative to the vehicle at 48 hours of co-culture. The X axis values represent the pretreatment concentrations in ng / ml. LPS concentrations are 100, 10 and 25 ng / ml. IFNy concentration is constant for all the groups which is 25 ng / ml.

[0072] FIG. 33 shows bar graphs depicting the results of a luminescence assay from the apical and basal compartments of TC cell lot B, where ATP activity was measured for cell lot B transverse colon (TC) epithelial cells in mono and co-cultures pretreated with one of three concentrations of LPS and IFNy (10+25, 25+25, 100+25 ng / mL) or with LPS and IFNy (100+25 ng / mL) together with Tofa. Day 2 and Day 3 represent the day in RMM at the time of initiation of co-culture.

[0073] FIG. 34 shows bar graphs depicting the results of a luminescence assay from the apical and basal compartments of TC cell lot A, where ATP activity was measured for cell lot A transverse colon (TC) epithelial cells in mono and co-cultures pretreated with one of three concentrations of LPS and IFNy (10+25, 25+25, 100+25 ng / mL).DETAILED DESCRIPTION

[0074] This disclosure is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented, or all the features that may be added to the instant disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant disclosure. Hence, the following specification is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations, and variations thereof.13#623862Attorney Docket No. 00009.008.1801

[0075] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.Definitions

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0077] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0078] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0079] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0080] It will be further understood that the terms “comprises” and / or “comprising,” which are synonymous with the terms “including,” “containing,” or “characterized by,” when used in this specification, specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, elements, components, and / or groups thereof.

[0081] As used herein, the term “and / or” when used in the context of a listing of entities, includes any and all combinations of one or more of the associated listed items. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0082] The term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this disclosure, a composition, mass, weight, dose, time,14#623862Attorney Docket No. 00009.008.1801 temperature, volume, concentration, percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0083] Numerical ranges recited herein by endpoints include 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). Similarly, numerical ranges recited herein by endpoints include subranges subsumed within that range (e.g.1 to 5 includes 1-1.5, 1.5-2, 2-2.75, 2.75-3, 3-3.90, 3.90-4, 4-4.24, 4.24-5, 2-5, 3-5, 1-4, and 2-4).

[0084] It will be understood that when an element is referred to as being “on,” “attached” to, “connected” to, “coupled” with, “contacting,” etc., another element, it can be directly on, attached to, connected to, coupled with and / or contacting the other element or intervening elements can also be present. In contrast, when an element is referred to as being, for example, “directly on,” “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature can have portions that overlap or underlie the adjacent feature.

[0085] As used herein, phrases such as “between X and Y,” “between about X and Y,” “from up to a range of X to Y,” and “from up to a range of about X to about Y,” should be interpreted to include 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."

[0086] Spatially relative terms, such as “under,” “below,” “lower,” “over,” “upper” and the like, can be used herein for ease of description to describe an element's or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different 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 is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device can otherwise be oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly,” “downwardly,” “vertical,” “horizontal” and the like are used herein for the purpose of explanation only, unless specifically indicated otherwise.

[0087] The term “cell medium” or “cell media,” as used herein, refers to a liquid or gel comprising components to support the growth of cells (e.g., a suitable energy source and compounds to regulate cell cycle). In some embodiments, the cell medium can comprise a minimum essential type of medium, such as Dulbecco’s Modified Eagle’s Medium, Ham’s F12, Eagle’s Medium, RPMI, AR8, etc., to which other ingredients can optionally be added. The term15#623862Attorney Docket No. 00009.008.1801 does not exclude media which have been prepared or are intended for specific uses, but which upon modification can be used for other cell types, etc.

[0088] The term “ingredient” refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the proliferation, survival, or differentiation of cells. The terms “nutrient”, “supplement”, and ingredient” can be used interchangeably and are all meant to refer to such compounds. Typical non-limiting ingredients that are used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins and the like. Other ingredients that promote or maintain cultivation of cells ex vivo can be selected by those of skill in the art, in accordance with the particular need.

[0089] The term “growth factor” as used herein means a bioactive molecule that promotes the proliferation of a cell or tissue. Growth factors useful in the present disclosure include, but are not limited to, WNT-3 A, R-spondin, noggin, transforming growth factor-alpha (TGF-a), transforming growth factor-beta (TGF-P), platelet-derived growth factors including the AA, AB and BB isoforms (PDGF), fibroblast growth factors (FGF), including FGF acidic isoforms 1 and 2, FGF basic form 2, and FGF 4, 8, 9 and 10, nerve growth factors (NGF) including NGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brain derived neurotrophic factor, cartilage derived factor, bone growth factors (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), EG-VEGF, VEGF-related protein, Bv8, VEGF-E, granulocyte colony stimulating factor (G-CSF), insulin like growth factor (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 factors, and bone derived growth factors and mixtures thereof. Some growth factors can also promote differentiation of a cell or tissue. TGF, for example, can promote growth and / or differentiation of a cell or tissue.

[0090] As used herein, the terms “example,” “exemplary,” and grammatical variations thereof are intended to refer to non-limiting examples and / or variant embodiments discussed herein, and are not intended to indicate preference for one or more embodiments discussed herein compared to one or more other embodiments.

[0091] As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more as compared to a control.

[0092] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least16#623862Attorney Docket No. 00009.008.1801 about 5%, about 10%, about 15%, about 20%, about 25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0093] The term “inhibit,” as used herein, refers to the ability of a compound, agent, or method to reduce or impede a described function, level, activity, rate, etc., based on the context in which the term “inhibit” is used. In some embodiments, inhibition is by at least 10%, by at least 25%, by at least 50%, or by at least 75%. The term “inhibit” is used interchangeably with “reduce” and “block.”

[0094] The term “material”, as used herein, refers to synthetic and natural materials, such as matrix components (e.g., synthetic or natural polymers). The term “materials and compounds” as used herein, refers to, inter alia, materials, compounds, cells, peptides, nucleic acids, drugs, matrix components, and imaging agents.

[0095] As used herein, “scaffold”, “substrate”, “cell support”, “surface”, “platform”, and “cell support substrate” refer to a supporting framework, such as one for cell or tissue growth, either in vivo or in vitro. The terms can be used interchangeably herein and refer to a structural unit of any size, where said structural unit or substrate has a surface suitable for immobilization of molecular structure or modification of said structure and said substrate is made of a material such as, but not limited to, metal, metal films, glass, fused silica, synthetic polymers, and membranes.

[0096] Tissue,” as used herein, means (1) a group of similar cells united to perform a specific function; (2) a part of an organism consisting of an aggregate of cells having a similar structure and function; and / or (3) a grouping of cells that are similarly characterized by their structure and function, such as muscle or nerve tissue.

[0097] A “cell type” as used herein refers to morphologically or phenotypically distinct cell forms within a species.

[0098] As used herein, “co-culture” and “co-culturing” refers culturing two or more different cell types together in a shared cell-culture vessel, flask and / or container.1. Methods for culture of cells and producing a co-culture cell construct

[0099] Described herein, in certain embodiments, are methods for co-culturing intestinal epithelial cells and immune cells, comprising: a) culturing the intestinal epithelial cells in an expansion medium followed by a differentiation medium; b) culturing the immune cells in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA); and c) combining the17#623862Attorney Docket No. 00009.008.1801 intestinal epithelial cells from (a) and the immune cells from (b) in a co-culture cell construct comprising a first co-culture medium.

[0100] Further described herein, in certain embodiments, are methods for co-culturing intestinal epithelial cells and immune cells comprising: (a) culturing the intestinal epithelial cells in an expansion medium followed by a differentiation medium; (b) culturing the immune cells in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA); and (c) combining the intestinal epithelial cells from (a) and the immune cells from (b) in a co-culture cell construct comprising a first co-culture medium that does not contain growth factors.

[0101] Further described herein, in certain embodiments, are methods for producing a co-culture cell construct comprising a co-culture of intestinal epithelial cells and immune cells, the method comprising: (a) providing an apparatus, the apparatus comprising a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well; and a luminal container capable of being held within the first well of the basal container; (b) culturing the intestinal epithelial cells and the immune cells separately, wherein: (i) the intestinal epithelial cells are cultured in the luminal container and in an expansion medium followed by a differentiation medium; and (ii) the immune cells are cultured in the basal container and in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA) followed by a second cell culture medium that does not contain PMA; (c) replacing the differentiation medium in the luminal container and the second cell culture medium in the basal container with a first co-culture medium; and (d) providing the luminal container into the basal container so that the luminal container is held within the first well of the basal container and the intestinal epithelial cells and immune cells are combined in the co-culture; thereby generating the co-culture cell construct comprising the coculture of intestinal epithelial cells and immune cells.

[0102] In some embodiments, the methods for co-culturing cells and producing a co-culture cell construct comprise a co-culture of two or more different cell types. In some embodiments, the methods for co-culturing cells and producing a co-culture cell construct comprise a co-culture of two different cell types.

[0103] In some embodiments, the two different cell types are first cultured separately ( / .<., the two different cell types are cultured in separate cell-culture vessels, flasks and / or containers that do not share media) before being combined to define a co-culture. In some embodiments, one or both of the two different cell types are grown to confluence before being combined to define a coculture. In some embodiments, one or both of the two different cell types are differentiated before being combined to define a co-culture.18#623862Attorney Docket No. 00009.008.1801

[0104] In some embodiments, the two different cell types are combined to define a co-culture (i.e., the two different cell types are co-cultured). In some embodiments, combining the two different cell types comprises bringing the two different cell types together in a shared cell-culture vessel, flask and / or container. In some embodiments, the two different cell types are kept (or are located) in separate chambers, regions, and / or compartments of the shared cell-culture vessel, flask, and / or container. In some embodiments, the two different cell types are exposed to a shared source of media ( / .<., a co-culture media).

[0105] In some embodiments, the immune cells are Tohoku Hospital Pediatrics-1 (THP-1) cells.1.1. Culture of intestinal epithelial cells prior to co-culture

[0106] Provided herein, in certain embodiments are conditions for culturing intestinal epithelial cells prior to being combined with the immune cells in the co-culture cell construct.1.1.1. Seeding of intestinal epithelial cells

[0107] In some embodiments, the intestinal epithelial cells are seeded on a hydrogel layer prior to being cultured. In some embodiments, the hydrogel layer comprises a natural or synthetic hydrogel (e.g., collagen, Matrigel, gelatin, agarose, chitosan, alginate, polyethylene glycol, polyacrylamide), dried hydrogel followed by salt leaching (e.g. collagen hydrogel is dried and the salts are leach out to create pores). In some embodiments, the hydrogel layer comprises collagen. In some embodiments, the hydrogel layer comprises Matrigel. In some embodiments, the hydrogel layer comprises gelatin. In some embodiments, the hydrogel layer comprises agarose. In some embodiments, the hydrogel layer comprises chitosan. In some embodiments, the hydrogel layer comprises alginate. In some embodiments, the hydrogel layer comprises polyethylene glycol. In some embodiments, the hydrogel layer comprises polyacrylamide.

[0108] In some embodiments, the intestinal epithelial cells are seeded on a porous hydrogel layer in the luminal container prior to being cultured. In some embodiments, the hydrogel layer comprises a natural or synthetic hydrogel (e.g., collagen, Matrigel, gelatin, agarose, chitosan, alginate, polyethylene glycol, polyacrylamide), dried hydrogel followed by salt leaching (e.g. collagen hydrogel is dried and the salts are leach out to create pores). In some embodiments, the hydrogel layer comprises collagen. In some embodiments, the hydrogel layer comprises Matrigel. In some embodiments, the hydrogel layer comprises gelatin. In some embodiments, the hydrogel layer comprises agarose. In some embodiments, the hydrogel layer comprises chitosan. In some embodiments, the hydrogel layer comprises alginate. In some embodiments, the hydrogel layer comprises polyethylene glycol. In some embodiments, the hydrogel layer comprises polyacrylamide.19#623862Attorney Docket No. 00009.008.1801

[0109] In some embodiments, the hydrogel layer is Matrigel. In some embodiments, the hydrogel layer is between about 0.1% to about 5% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 5% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 4.5% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 4% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 3.5% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 3% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 2.5% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 2% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 1.5% Matrigel. In some embodiments, the hydrogel layer is between about 0.5% to about 1% Matrigel. In some embodiments, the hydrogel layer is about 0.1% Matrigel. In some embodiments, the hydrogel layer is about 0.2% Matrigel. In some embodiments, the hydrogel layer is about 0.3% Matrigel. In some embodiments, the hydrogel layer is about 0.4% Matrigel. In some embodiments, the hydrogel layer is about 0.5% Matrigel. In some embodiments, the hydrogel layer is about 0.6% Matrigel. In some embodiments, the hydrogel layer is about 0.7% Matrigel. In some embodiments, the hydrogel layer is about 0.8% Matrigel. In some embodiments, the hydrogel layer is about 0.9% Matrigel. In some embodiments, the hydrogel layer is about 1% Matrigel. In some embodiments, the hydrogel layer is about 1.1% Matrigel. In some embodiments, the hydrogel layer is about 1.2% Matrigel. In some embodiments, the hydrogel layer is about 1.3% Matrigel. In some embodiments, the hydrogel layer is about 1.4% Matrigel. In some embodiments, the hydrogel layer is about 1.5% Matrigel.1.1.2. Growth of intestinal epithelial cells

[0110] In some embodiments, the intestinal epithelial cells are grown to confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are grown to between about 70% and about 100% confluence, between about 80% and about 100% confluence, between about 90% and about 100% confluence, between about 95% and about 100% confluence, between about 96% and about 100% confluence, between about 97% and about 100% confluence, between about 98% and about 100% confluence, or between about 99% and about 100% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 70% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 80% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 90% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 95%20#623862Attorney Docket No. 00009.008.1801 confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 96% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 97% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 98% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 99% confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 100% confluence prior to being combined with the immune cells in the co-culture cell construct.

[0111] In some embodiments, the intestinal epithelial cells are grown to confluence in expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are grown to between about 70% and about 100% confluence, between about 80% and about 100% confluence, between about 90% and about 100% confluence, between about 95% and about 100% confluence, between about 96% and about 100% confluence, between about 97% and about 100% confluence, between about 98% and about 100% confluence, or between about 99% and about 100% confluence in expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 70% confluence in expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 80% confluence in expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 90% confluence in expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 95% in expansion medium confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 96% in expansion medium confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 97% in expansion medium confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 98% in expansion medium confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the intestinal epithelial cells are to about 99% in expansion medium confluence prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the21#623862Attorney Docket No. 00009.008.1801 intestinal epithelial cells are to about 100% in expansion medium confluence prior to being combined with the immune cells in the co-culture cell construct.

[0112] In some embodiments, the intestinal epithelial cells are grown to confluence in expansion medium prior to being combined with the immune cells in the co-culture cell construct. In some embodiments, the expansion medium is exchanged for the differentiation medium once the intestinal epithelial cells reach confluence.

[0113] In some embodiments, the intestinal epithelial cells are primary intestinal epithelial cells.1.2. Culture of immune cells prior to co-culture

[0114] Described herein, in certain embodiments, are conditions for culturing immune cells prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0115] In some embodiments, the immune cells are Tohoku Hospital Pediatrics-1 (THP-1) cells.1.2.1. Seeding of immune cells

[0116] In some embodiments, the immune cells are cultured in suspension prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0117] In some embodiments, the immune cells are cultured in suspension in a first cell culture medium and / or in a second cell culture medium prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in suspension in a first cell culture medium prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in suspension in a second cell culture medium prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0118] In some embodiments, the immune cells are seeded at a density from up to a range of about 1×103cells / 12.6 cm2to about 1×106cells / 12.6 cm2, of about 1×103cells / 12.6 cm2to about 1×105cells / 12.6 cm2, of about 1×103cells / 12.6 cm2to about 1×104cells / 12.6 cm2, of about 1×104cells / 12.6 cm2to about 1×106cells / 12.6 cm2, of about 1×105cells / 12.6 cm2to about 1×106cells / 12.6 cm2, or of about 1×104cells / 12.6 cm2to about 1×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 1×103cells / 12.6 cm2to about 1×106cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 1×103cells / 12.6 cm2to about 1×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 1×103cells / 12.6 cm2to about 1×104cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 1×104cells / 12.6 cm2to about 1×106cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density22#623862Attorney Docket No. 00009.008.1801 from up to a range of about 1×105cells / 12.6 cm2to about 1×106cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range or of about 1×104cells / 12.6 cm2to about 1×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 5×103cells / 12.6 cm2to about 1.12×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 6×103cells / 12.6 cm2to about 1.12×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 7×103cells / 12.6 cm2to about 1.12×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 8×103cells / 12.6 cm2to about 1.12×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 9×103cells / 12.6 cm2to about 1.12×105cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density from up to a range of about 7×103cells / 12.6 cm2to about 1.12×105cells / 12.6 cm2in the first cell culture medium.

[0119] In some embodiments, the immune cells are seeded at a density of about 1.4×104cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density of about 2.8×104cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density of about 5.6×104cells / 12.6 cm2in the first cell culture medium. In some embodiments, the immune cells are seeded at a density of about 1.12×105cells / 12.6 cm2in the first cell culture medium.1.2.2. Growth and PMA-induced differentiation of immune cells in a first cell culture medium

[0120] In some embodiments, the immune cells are cultured in a first cell culture medium prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the first cell culture medium does not comprise mercaptoethanol. In some embodiments, the first cell culture medium comprises Roswell Park Memorial Institute 1640 (RPMI-1640) medium. In some embodiments, the first cell culture medium comprises Advanced Dulbecco's Modified Eagle Medium / Ham's F-12 (DMEM / F-12) medium.

[0121] In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 10 pM, about 1 nM to 1 pM, about 1 nM to 900 nM, about 1 nM to 800 nM, about 1 nM to 700 nM, about 1 nM to 600 nM, about 1 nM to 500 nM, about 1 nM to 400 nM, about 1 nM to 300 nM, about 1 nM to 200 nM, about 1 nM to 100 nM, about 1 nM to 90 nM, about 1 nM to 80 nM, about 1 nM to 70 nM, about 1 nM to 60 nM, about 1 nM to 50 nM, about 1 nM to 40 nM, about 1 nM to 30 nM, about 1 nM to 20 nM, about 1 nM to 10 nM, about 1 nM to 5 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about23#623862Attorney Docket No. 00009.008.1801 1 nM to 100 pM, about 10 nM to 100 pM, about 50 nM to 100 pM, about 100 nM to 100 pM, about 150 nM to 100 pM, about 200 nM to 100 pM, about 250 nM to 100 pM, about 300 nM to 100 pM, about 350 nM to 100 pM, about 400 nM to 100 pM, about 450 nM to 100 pM, about 500 nM to 100 pM, about 550 nM to 100 pM, about 600 nM to 100 pM, about 650 nM to 100 pM, about 700 nM to 100 pM, about 750 nM to 100 pM, about 800 nM to 100 pM, about 850 nM to 100 pM, about 900 nM to 100 pM, about 950 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 pM to 100 pM, about 10 pM to 100 pM, about 20 pM to 100 pM, about 30 pM to 100 pM, about 40 pM to 100 pM, about 50 pM to 100 pM, about 60 pM to 100 pM, about 70 pM to 100 pM, about 80 pM to 100 pM, or about 90 pM to 100 pM of the PMA.

[0122] In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 10 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 1 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 900 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 800 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 700 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 600 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 500 nM. In some embodiments, the first cell culture medium comprises from up to a range of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 400 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 300 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 200 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 100 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 90 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 80 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 70 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 60 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 50 nM of the PMA. about 1 nM to 40 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 30 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 20 nM of the PMA. In some embodiments, the first cell culture medium comprises24#623862Attorney Docket No. 00009.008.1801 from up to a range of about 1 nM to 10 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 5 nM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 10 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 100 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 200 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 300 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 400 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 500 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 600 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 700 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 800 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 900 nM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 1 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 10 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 20 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 30 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 40 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 50 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 60 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 70 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 80 pM to 100 pM of the PMA. In some embodiments, the first cell culture medium comprises from up to a range of about 90 pM to 100 pM of the PMA.

[0123] In some embodiments, the first cell culture medium comprises about 1 nM, about 10 nM, about 50 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM of the PMA. In some embodiments, the first cell culture medium comprises about 1 pM, about 1025#623862Attorney Docket No. 00009.008.1801 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM of the PMA. In some embodiments, the first cell culture medium comprises about 10 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM of the PMA.

[0124] In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 12 hours to about 48 hours, about 12 hours to about 42 hours, about 12 hours to about 36 hours, about 12 hours to about 30 hours, about 12 hours to about 24 hours, about 18 hours to about 48 hours, about 24 hours to about 48 hours, about 30 hours to about 48 hours, about 36 hours to about 48 hours, about 42 hours to about 48 hours, about 12 hours to about 24 hours, about 13 hours to about 24 hours, about 14 hours to about 24 hours, about 15 hours to about 24 hours, about 16 hours to about 24 hours, about 17 hours to about 24 hours, about 18 hours to about 24 hours, about 19 hours to about 24 hours, about 20 hours to about 24 hours, about 21 hours to about 24 hours, about 22 hours to about 24 hours, or about 23 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0125] In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0126] In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 20 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 21 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 22 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 23 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the first cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.26#623862Attorney Docket No. 00009.008.1801 1.2.3. Growth of immune cells in a second cell culture medium

[0127] In some embodiments, the first cell culture medium is removed and replaced with a second cell culture medium. In some embodiments, the second cell culture medium does not contain PMA. In some embodiments, the second cell culture medium does not contain mercaptoethanol.

[0128] In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 12 hours to about 48 hours, about 12 hours to about 42 hours, about 12 hours to about 36 hours, about 12 hours to about 30 hours, about 12 hours to about 24 hours, about 18 hours to about 48 hours, about 24 hours to about 48 hours, about 30 hours to about 48 hours, about 36 hours to about 48 hours, about 42 hours to about 48 hours, about 12 hours to about 24 hours, about 13 hours to about 24 hours, about 14 hours to about 24 hours, about 15 hours to about 24 hours, about 16 hours to about 24 hours, about 17 hours to about 24 hours, about 18 hours to about 24 hours, about 19 hours to about 24 hours, about 20 hours to about 24 hours, about 21 hours to about 24 hours, about 22 hours to about 24 hours, or about 23 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0129] In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0130] In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 20 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 21 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 22 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 23 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are cultured in the second cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.27#623862Attorney Docket No. 00009.008.1801 1.2.4. Exposure of immune cells to immune cell-differentiating and / or stimulating agents

[0131] In some embodiments, the immune cells are exposed to one or more immune celldifferentiating and / or stimulating agents prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0132] In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is selected from the group consisting of: lipopolysaccharide (LPS), interferon gamma (IFN-y), interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT). In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is lipopolysaccharide (LPS). In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is interferon gamma (IFN-y). In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is interleukin-4 (IL-4). In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is C-reactive protein (CRP). In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is Zymosan. In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is Tumor Necrosis Factor-alpha (TNFa). In some embodiments, the one or more immune celldifferentiating and / or stimulating agents is Nigericin. In some embodiments, the one or more immune cell-differentiating and / or stimulating agents is Cholera toxin (CT).

[0133] In some embodiments, the immune cells are exposed to the one or more immune celldifferentiating and / or stimulating agents for a period of from about 1 hours to about 48 hours, about 1 hours to about 40 hours, about 1 hours to about 36 hours, about 1 hours to about 30 hours, about 1 hours to about 24 hours, about 1 hours to about 18 hours, about 1 hours to about 12 hours, about 1 hours to about 6 hours, about 1 hours to about 24 hours, about 2 hours to about 24 hours, about 3 hours to about 24 hours, about 4 hours to about 24 hours, about 5 hours to about 24 hours, about 6 hours to about 24 hours, about 7 hours to about 24 hours, about 8 hours to about 24 hours, about 9 hours to about 24 hours, about 10 hours to about 24 hours, about 11 hours to about 24 hours, about 12 hours to about 24 hours, about 13 hours to about 24 hours, about 14 hours to about 24 hours, about 15 hours to about 24 hours, about 16 hours to about 24 hours, about 17 hours to about 24 hours, about 18 hours to about 24 hours, about 19 hours to about 24 hours, about 20 hours to about 24 hours, about 21 hours to about 24 hours, about 22 hours to about 24 hours, or about 23 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.28#623862Attorney Docket No. 00009.008.1801

[0134] In some embodiments, the immune cells are exposed to the one or more immune celldifferentiating and / or stimulating agents for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 5 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 6 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 7 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 8 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune celldifferentiating and / or stimulating agents for a period of from about 9 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 10 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 11 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 12 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 13 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune celldifferentiating and / or stimulating agents for a period of from about 14 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 15 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the29#623862Attorney Docket No. 00009.008.1801 immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 16 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 17 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 18 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune celldifferentiating and / or stimulating agents for a period of from about 19 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 20 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 21 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 22 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to the one or more immune cell-differentiating and / or stimulating agents for a period of from about 23 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0135] In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and / or interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0136] In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 1 hours to about 48 hours, about 1 hours to about 40 hours, about 1 hours to about 36 hours, about 1 hours to about 30 hours, about 1 hours to about 24 hours, about 1 hours to about 18 hours, about 1 hours to about 12 hours, about 1 hours to about 6 hours, about 1 hours to about 24 hours, about 2 hours to about 24 hours, about 3 hours to about 24 hours, about 4 hours to about 24 hours, about 5 hours to about 24 hours, about 6 hours to about 24 hours, about 7 hours to about 24 hours, about 8 hours to about 24 hours, about 9 hours to about 24 hours, about 10 hours to about 24 hours, about 11 hours to about 24 hours, about 12 hours to about 24 hours, about 13 hours to about 24 hours, about 14 hours to about 24 hours, about 1530#623862Attorney Docket No. 00009.008.1801 hours to about 24 hours, about 16 hours to about 24 hours, about 17 hours to about 24 hours, about 18 hours to about 24 hours, about 19 hours to about 24 hours, about 20 hours to about 24 hours, about 21 hours to about 24 hours, about 22 hours to about 24 hours, or about 23 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0137] In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 5 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 6 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 7 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 8 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 9 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 10 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 11 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 12 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 13 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 14 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed31#623862Attorney Docket No. 00009.008.1801 to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 15 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 16 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 17 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 18 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 19 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 20 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 21 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 22 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 23 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0138] In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0139] In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 4 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 5 hours prior to32#623862Attorney Docket No. 00009.008.1801 being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 6 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 7 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 8 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 9 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 10 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 11 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 12 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 13 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 14 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 15 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 16 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 17 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 18 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma33#623862Attorney Docket No. 00009.008.1801 (IFN-y) for a period of about 19 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 20 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 21 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 22 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 23 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0140] In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 5000 ng / mL, about 10 ng / mL to about 5000 ng / mL, about 10 ng / mL to about 4500 ng / mL, about 10 ng / mL to about 4000 ng / mL, about 10 ng / mL to about 3500 ng / mL, about 10 ng / mL to about 3000 ng / mL, 10 ng / mL to about 2500 ng / mL, about 10 ng / mL to about 2000 ng / mL, about 10 ng / mL to about 1500 ng / mL, about 10 ng / mL to about 1000 ng / mL, 10 ng / mL to about 900 ng / mL, about 10 ng / mL to about 800 ng / mL, about 10 ng / mL to about 700 ng / mL, about 10 ng / mL to about 600 ng / mL, about 10 ng / mL to about 500 ng / mL, about 10 ng / mL to about 400 ng / mL, about 10 ng / mL to about 300 ng / mL, about 10 ng / mL to about 200 ng / mL, about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 90 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, or about 10 ng / mL to about 20 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0141] In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 900 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 800 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the34#623862Attorney Docket No. 00009.008.1801 co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 700 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 600 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 500 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 400 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 300 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 200 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 10 ng / mL to about 100 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0142] In some embodiments, the immune cells are exposed to about 10 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 50 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 100 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 200 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 300 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 400 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 500 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 600 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the co-35#623862Attorney Docket No. 00009.008.1801 culture cell construct. In some embodiments, the immune cells are exposed to about 700 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 800 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 900 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 1000 ng / mL lipopolysaccharide (LPS) prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0143] In some embodiments, the immune cells are exposed to from up to a range of about 0.1 ng / mL to about 50 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 50 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 45 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 40 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 35 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 30 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 25 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 20 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 15 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 10 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 5 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 4.5 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 4 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 3.5 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 3 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 2.5 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 2 ng / mL interferon gamma (IFN-y), about 0.5 ng / mL to about 1.5 ng / mL interferon gamma (IFN-y), or about 0.5 ng / mL to about 1 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0144] In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 25 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 20 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 15 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 10 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the36#623862Attorney Docket No. 00009.008.1801 immune cells are exposed to from up to a range of about 0.5 ng / mL to about 5 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 4.5 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 4 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 3.5 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 3 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 2.5 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 2 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 1.5 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.5 ng / mL to about 1 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0145] In some embodiments, the immune cells are exposed to about 0.5 ng / mL interferon gamma (IFN-y), about 1 ng / mL interferon gamma (IFN-y), about 1.5 ng / mL interferon gamma (IFN-y), about 2 ng / mL interferon gamma (IFN-y), about 2.5 ng / mL interferon gamma (IFN-y), about 3 ng / mL interferon gamma (IFN-y), about 3.5 ng / mL interferon gamma (IFN-y), about 4 ng / mL interferon gamma (IFN-y), about 4.5 ng / mL interferon gamma (IFN-y), about 5 ng / mL interferon gamma (IFN-y), about 10 ng / mL interferon gamma (IFN-y), about 15 ng / mL interferon gamma (IFN-y), about 20 ng / mL interferon gamma (IFN-y), about 25 ng / mL interferon gamma (IFN-y), about 30 ng / mL interferon gamma (IFN-y), about 35 ng / mL interferon gamma (IFN-y), about 40 ng / mL interferon gamma (IFN-y), about 45 ng / mL interferon gamma (IFN-y), or about 50 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.37#623862Attorney Docket No. 00009.008.1801

[0146] In some embodiments, the immune cells are exposed to about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, or about 25 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0147] In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 150 ng / mL IL-4, about 5 ng / mL to about 150 ng / mL IL-4, about 5 ng / mL to about 140 ng / mL IL-4, about 5 ng / mL to about 130 ng / mL IL-4, about 5 ng / mL to about 120 ng / mL IL-4, about 5 ng / mL to about 110 ng / mL IL-4, about 5 ng / mL to about 100 ng / mL IL-4, about 5 ng / mL to about 90 ng / mL IL-4, about 5 ng / mL to about 80 ng / mL IL-4, about 5 ng / mL to about 70 ng / mL IL-4, about 5 ng / mL to about 60 ng / mL IL-4, about 5 ng / mL to about 50 ng / mL IL-4, about 5 ng / mL to about 45 ng / mL IL-4, about 5 ng / mL to about 40 ng / mL IL-4, about 5 ng / mL to about 35 ng / mL IL-4, about 5 ng / mL to about 30 ng / mL IL-4, about 5 ng / mL to about 25 ng / mL IL-4, about 5 ng / mL to about 20 ng / mL IL-4, about 5 ng / mL to about 15 ng / mL IL-4, or about 5 ng / mL to about 10 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0148] In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 100 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 90 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 80 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 70 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 60 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 50 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 40 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 30 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 20 ng / mL IL-4 prior to being combined with the38#623862Attorney Docket No. 00009.008.1801 intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 ng / mL to about 10 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0149] In some embodiments, the immune cells are exposed to about 5 ng / mL IL-4, about 10 ng / mL IL-4, about 15 ng / mL IL-4, about 20 ng / mL IL-4, about 25 ng / mL IL-4, about 30 ng / mL IL-4, about 35 ng / mL IL-4, about 40 ng / mL IL-4, about 45 ng / mL IL-4, about 50 ng / mL IL-4, about 60 ng / mL IL-4, about 70 ng / mL IL-4, about 80 ng / mL IL-4, about 90 ng / mL IL-4, about 100 ng / mL IL-4, about 110 ng / mL IL-4, about 120 ng / mL IL-4, about 130 ng / mL IL-4, about 140 ng / mL IL-4, or about 150 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0150] In some embodiments, the immune cells are exposed to about 5 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 10 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 20 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 30 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 40 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 50 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 60 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 70 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 80 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 90 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 100 ng / mL IL-4 prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0151] In some embodiments, the immune cells are exposed to from up to a range of about 1 µg / mL to about 150 µg / mL, about 5 µg / mL to about 150 µg / mL, about 5 µg / mL to about 140 µg / mL, about 5 µg / mL to about 130 µg / mL, about 5 µg / mL to about 120 µg / mL, about 5 µg / mL to about 110 µg / mL, about 5 µg / mL to about 100 µg / mL, about 5 µg / mL to about 90 µg / mL, about39#623862Attorney Docket No. 00009.008.1801 5 µg / mL to about 80 µg / mL, about 5 µg / mL to about 70 µg / mL, about 5 µg / mL to about 60 µg / mL, about 5 µg / mL to about 50 µg / mL, about 5 µg / mL to about 45 µg / mL, about 5 µg / mL to about 40 µg / mL, about 5 µg / mL to about 35 µg / mL, about 5 µg / mL to about 30 µg / mL, about 5 µg / mL to about 25 µg / mL, about 5 µg / mL to about 20 µg / mL, about 5 µg / mL to about 15 µg / mL, or about 5 µg / mL to about 10 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0152] In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 100 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 90 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 80 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 70 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 60 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 50 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 40 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 30 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 20 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 5 µg / mL to about 10 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0153] In some embodiments, the immune cells are exposed to about 5 µg / mL, about 10 µg / mL, about 15 µg / mL, about 20 µg / mL, about 25 µg / mL, about 30 µg / mL, about 35 µg / mL, about 40 µg / mL, about 45 µg / mL, about 50 µg / mL, about 60 µg / mL, about 70 µg / mL, about 80 µg / mL, about 90 µg / mL, about 100 µg / mL, about 110 µg / mL, about 120 µg / mL, about 130 µg / mL, about 140 µg / mL, or about 150 µg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct.40#623862Attorney Docket No. 00009.008.1801

[0154] In some embodiments, the immune cells are exposed to about 5 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 10 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 20 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 30 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 40 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 50 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 60 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 70 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 80 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 90 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 100 pg / mL CRP prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0155] In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 2000 ng / mL, about 1 ng / mL to about 1750 ng / mL, about 1 ng / mL to about 1500 ng / mL, about 1 ng / mL to about 1250 ng / mL, about 1 ng / mL to about 1000 ng / mL, about 1 ng / mL to about 900 ng / mL, about 1 ng / mL to about 800 ng / mL, about 1 ng / mL to about 700 ng / mL, about 1 ng / mL to about 600 ng / mL, about 1 ng / mL to about 500 ng / mL, about 1 ng / mL to about 400 ng / mL, about 1 ng / mL to about 300 ng / mL, about 1 ng / mL to about 200 ng / mL, about 1 ng / mL to about 100 ng / mL, about 1 ng / mL to about 50 ng / mL, or about 1 ng / mL to about 25 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0156] In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 1000 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 900 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 800 ng / mL GM-CSF prior to being combined with41#623862Attorney Docket No. 00009.008.1801 the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 700 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 600 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 500 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 400 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 300 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 200 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 100 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 50 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 25 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0157] In some embodiments, the immune cells are exposed about 1 ng / mL, about 25 ng / mL, about 50 ng / mL, about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1000 ng / mL, about 1250 ng / mL, about 1500 ng / mL, about 1750 ng / mL, or about 2000 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0158] In some embodiments, the immune cells are exposed about 1 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 25 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 50 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 100 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed about 200 ng / mL GM-42#623862Attorney Docket No. 00009.008.1801 CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 300 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 400 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 500 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 600 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 700 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 800 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 900 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed about 1000 ng / mL GM-CSF prior to being combined with the intestinal epithelial cells in the coculture cell construct.

[0159] In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 2000 pg / mL, about 1 pg / mL to about 1750 pg / mL, about 1 pg / mL to about 1500 pg / mL, about 1 pg / mL to about 1250 pg / mL, about 1 pg / mL to about 1000 pg / mL, about 1 pg / mL to about 900 pg / mL, about 1 pg / mL to about 800 pg / mL, about 1 pg / mL to about 700 pg / mL, about 1 pg / mL to about 600 pg / mL, about 1 pg / mL to about 500 pg / mL, about 1 pg / mL to about 400 pg / mL, about 1 pg / mL to about 300 pg / mL, about 1 pg / mL to about 200 pg / mL, about 1 pg / mL to about 100 pg / mL, about 1 pg / mL to about 50 pg / mL, or about 1 pg / mL to about 25 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0160] In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 1000 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 900 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 800 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 700 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some43#623862Attorney Docket No. 00009.008.1801 embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 600 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 500 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 400 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 300 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 200 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 100 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 50 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pg / mL to about 25 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0161] In some embodiments, the immune cells are exposed to about 1 pg / mL, about 25 pg / mL, about 50 pg / mL, about 100 pg / mL, about 200 pg / mL, about 300 pg / mL, about 400 pg / mL, about 500 pg / mL, about 600 pg / mL, about 700 pg / mL, about 800 pg / mL, about 900 pg / mL, about 1000 pg / mL, about 1250 pg / mL, about 1500 pg / mL, about 1750 pg / mL, or about 2000 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0162] In some embodiments, the immune cells are exposed to about 1 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 25 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 50 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 100 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 200 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 300 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments,44#623862Attorney Docket No. 00009.008.1801 the immune cells are exposed to about 400 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 500 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 600 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 700 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 800 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 900 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 1000 pg / mL Zymosan prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0163] In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 200 ng / mL, about 0.25 ng / mL to about 175 ng / mL, about 0.25 ng / mL to about 150 ng / mL, about 0.25 ng / mL to about 125 ng / mL, about 0.25 ng / mL to about 100 ng / mL, about 0.25 ng / mL to about 90 ng / mL, about 0.25 ng / mL to about 80 ng / mL, about 0.25 ng / mL to about 70 ng / mL, about 0.25 ng / mL to about 60 ng / mL, about 0.25 ng / mL to about 50 ng / mL, about 0.25 ng / mL to about 40 ng / mL, about 0.25 ng / mL to about 30 ng / mL, about 0.25 ng / mL to about 20 ng / mL, about 0.25 ng / mL to about 10 ng / mL, about 0.25 ng / mL to about 5 ng / mL, about 0.25 ng / mL to about 4 ng / mL, about 0.25 ng / mL to about 3 ng / mL, about 0.25 ng / mL to about 2 ng / mL, or about 0.25 ng / mL to about 1 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0164] In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 100 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 90 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 80 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 70 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 60 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell45#623862Attorney Docket No. 00009.008.1801 construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 50 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 40 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 30 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 20 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 10 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 0.25 ng / mL to about 5 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0165] In some embodiments, the immune cells are exposed to about 0.25 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 125 ng / mL, about 150 ng / mL, about 175 ng / mL, about 200 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0166] In some embodiments, the immune cells are exposed to about 0.25 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 5 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 10 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 20 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 30 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 40 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 50 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 60 ng / mL of TNFa prior to being combined with the intestinal epithelial cells46#623862Attorney Docket No. 00009.008.1801 in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 70 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 80 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 90 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 100 ng / mL of TNFa prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0167] In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 200 pM, about 1 pM to about 175 pM, about 1 pM to about 150 pM, about 1 pM to about 125 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, or about 1 pM to about 10 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0168] In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 100 pM Nigericin prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 90 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 80 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 70 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 60 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 50 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 40 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 30 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 20 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-47#623862Attorney Docket No. 00009.008.1801 culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 pM to about 10 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0169] In some embodiments, the immune cells are exposed to about 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, or about 200 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0170] In some embodiments, the immune cells are exposed to about 1 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 10 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 20 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 30 pM Nigericin prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 40 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 50 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 60 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 70 pM Nigericin prior to being combined with the intestinal epithelial cells in the coculture cell construct. In some embodiments, the immune cells are exposed to about 80 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 90 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 100 pM Nigericin prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0171] In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 2000 ng / mL, about 1 ng / mL to about 1750 ng / mL, about 1 ng / mL to about 1500 ng / mL, about 1 ng / mL to about 1250 ng / mL, about 1 ng / mL to about 1000 ng / mL, about 1 ng / mL to about 900 ng / mL, about 1 ng / mL to about 800 ng / mL, about 1 ng / mL to about 700 ng / mL, about 1 ng / mL to about 600 ng / mL, about 1 ng / mL to about 500 ng / mL, about 1 ng / mL to about 400 ng / mL, about 1 ng / mL to about 300 ng / mL, about 1 ng / mL to about 200 ng / mL, about 1 ng / mL to about 100 ng / mL, about 1 ng / mL to about 50 ng / mL, about 1 ng / mL to about 25 ng / mL, or about48#623862Attorney Docket No. 00009.008.1801 1 ng / mL to about 10 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0172] In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 1000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 900 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 800 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 700 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 600 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 500 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 400 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 300 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 200 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 100 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to from up to a range of about 1 ng / mL to about 10 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

[0173] In some embodiments, the immune cells are exposed to about 1 ng / mL, about 10 ng / mL, about 50 ng / mL, about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1000 ng / mL, about 1250 ng / mL, about 1500 ng / mL, about 1750 ng / mL, or about 2000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct, construct.

[0174] In some embodiments, the immune cells are exposed to about 1 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 10 ng / mL CT prior to being combined with the intestinal49#623862Attorney Docket No. 00009.008.1801 epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 100 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 200 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 300 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 400 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 500 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 600 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 700 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 800 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 900 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct. In some embodiments, the immune cells are exposed to about 1000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.1.2.5. Exposure of immune cells to inhibitors

[0175] In some embodiments, the immune cells are treated with one or more inhibitors. In some embodiments, the immune cells are exposed to one or more inhibitors.

[0176] In some embodiments, the one or more inhibitors is one or more cytokine inhibitors. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X-C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor.

[0177] In some embodiments, the one or more cytokine inhibitors is a Tumor Necrosis Factoralpha (TNFa) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interferongamma (IFNY) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin-8 (IL-8) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin-1 beta (IL-ip) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin-6 (IL-6) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor. In some embodiments, the one or more50#623862Attorney Docket No. 00009.008.1801 cytokine inhibitors is a C-X-C motif chemokine 11 (CXCL11) inhibitor. In some embodiments, the one or more cytokine inhibitors is Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor.

[0178] In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor.

[0179] In some embodiments, the one or more cytokine inhibitors is a Tumor Necrosis Factoralpha (TNFa) inhibitor. In some embodiments, the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some embodiments, the TNFa inhibitor is adalimumab. In some embodiments, the TNFa inhibitor is certolizumab pegol. In some embodiments, the TNFa inhibitor is etanercept. In some embodiments, the TNFa inhibitor is golimumab. In some embodiments, the TNFa inhibitor is infliximab.

[0180] In some embodiments, the one or more cytokine inhibitors is an interferon-gamma (IFNy) inhibitor. In some embodiments, the IFNy inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib. In some embodiments, the IFNy inhibitor is AMG 811. In some embodiments, the IFNy inhibitor is baricitinib. In some embodiments, the IFNy inhibitor is filgotinib. In some embodiments, the IFNy inhibitor is fontolizumab. In some embodiments, the IFNy inhibitor is oclacitinib. In some embodiments, the IFNy inhibitor is ruxolitinib. In some embodiments, the IFNy inhibitor is tofacitinib. In some embodiments, the IFNy inhibitor is upadacitinib.

[0181] In some embodiments, the one or more cytokine inhibitors is an IL-8 inhibitor. In some embodiments, the IL-8 inhibitor is selected from the group consisting of: MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756). In some embodiments, the IL-8 inhibitor is MS-986253. In some embodiments, the IL-8 inhibitor is Ab-8 (GSK2140935). In some embodiments, the IL-8 inhibitor is Danirixin (GSK1325756).

[0182] In some embodiments, the one or more cytokine inhibitors is an IL-ip inhibitor. In some embodiments, the IL-ip inhibitor is selected from the group consisting of: Rilonacept (Arcalyst) and Canakinumab (Haris). In some embodiments, the IL-ip inhibitor is Rilonacept (Arcalyst). In some embodiments, the IL-ip inhibitor is Canakinumab (Haris).

[0183] In some embodiments, the one or more cytokine inhibitors is an IL-6 inhibitor. In some embodiments, the IL-6 inhibitor is selected from the group consisting of: Tocilizumab (Actemra) and Sarilumab (Kevzara). In some embodiments, the IL-6 inhibitor is Tocilizumab (Actemra). In some embodiments, the IL-6 inhibitor is Sarilumab (Kevzara).

[0184] In some embodiments, the one or more cytokine inhibitors is an IL-12 / IL-23 inhibitor. In some embodiments, the IL-12 / IL-23 inhibitor is selected from the group consisting of:51#623862Attorney Docket No. 00009.008.1801 Ustekinumab (Stelara) and Risankizumab (Skyrizi). In some embodiments, the IL-12 / IL-23 inhibitor is Ustekinumab (Stelara). In some embodiments, the IL-12 / IL-23 inhibitor is Risankizumab (Skyrizi).

[0185] In some embodiments, the one or more cytokine inhibitors is a CXCL11 inhibitor. In some embodiments, the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418). In some embodiments, the CXCL11 inhibitor is BMS-936559 (MDX-1106). In some embodiments, the CXCL11 inhibitor is Vercimon (TRA-418).1.3 Co-culture o f intestinal cells and immune cells

[0186] Provided herein, in certain embodiments, are conditions for co-culturing cells ( / .<., intestinal epithelial cells and immune cells).1.3.1 Co-culture in a first co-culture medium

[0187] In some embodiments, the cells being co-cultured are co-cultured in a first co-culture medium.

[0188] In some embodiments, the first co-culture medium contains growth factors. In some embodiments, the first co-culture medium does not contain growth factors. In some embodiments, the first co-culture medium does not contain epidermal growth factors (EGF).

[0189] In some embodiments, the first co-culture medium does not contain N-acetylcysteine (NAC).

[0190] In some embodiments, the first co-culture medium comprises Roswell Park Memorial Institute 1640 (RPMI-1640) medium. In some embodiments, the first co-culture medium comprises Advanced Dulbecco's Modified Eagle Medium / Ham's F-12 (DMEM / F-12) medium.

[0191] In some embodiments, the first co-culture medium comprises lipopolysaccharide (LPS) and / or interferon gamma (IFN-y).

[0192] In some embodiments, the first co-culture medium comprises LPS.

[0193] In some embodiments, the first co-culture medium comprises from up to a range of about 1 ng / mL to about 2000 ng / mL, about 10 ng / mL to about 2000 ng / mL, about 10 ng / mL to about 1750 ng / mL, about 10 ng / mL to about 1500 ng / mL, about 10 ng / mL to about 1250 ng / mL, about 10 ng / mL to about 1000 ng / mL, about 10 ng / mL to about 900 ng / mL, about 10 ng / mL to about 800 ng / mL, about 10 ng / mL to about 700 ng / mL, about 10 ng / mL to about 600 ng / mL, about 10 ng / mL to about 500 ng / mL, about 10 ng / mL to about 400 ng / mL, about 10 ng / mL to about 300 ng / mL, about 10 ng / mL to about 200 ng / mL, about 10 ng / mL to about 100 ng / mL, about 10 ng / mL to about 90 ng / mL, about 10 ng / mL to about 80 ng / mL, about 10 ng / mL to about 70 ng / mL, about 10 ng / mL to about 60 ng / mL, about 10 ng / mL to about 50 ng / mL, about 10 ng / mL to about 40 ng / mL, about 10 ng / mL to about 30 ng / mL, about 10 ng / mL to about 20 ng / mL, about 1 ng / mL to52#623862Attorney Docket No. 00009.008.1801 about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, about 1 ng / mL to about 7 ng / mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL LPS.

[0194] In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 1000 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 900 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 800 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 700 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 600 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 500 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 400 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 300 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 200 ng / mL LPS. In some embodiments, the first co-culture medium comprises from up to a range of about 10 ng / mL to about 100 ng / mL LPS.

[0195] In some embodiments, the first co-culture medium comprises about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1000 ng / mL, about 1250 ng / mL, about 1500 ng / mL, about 1750 ng / mL, or about 2000 ng / mL LPS.

[0196] In some embodiments, the first co-culture medium comprises about 10 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 100 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 200 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 300 ng / mL LPS. In some embodiments, the first coculture medium comprises about 400 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 500 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 600 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 700 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 800 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 900 ng / mL LPS. In some embodiments, the first co-culture medium comprises about 1000 ng / mL LPS.53#623862Attorney Docket No. 00009.008.1801

[0197] In some embodiments, the first co-culture medium comprises interferon gamma (IFN-y).

[0198] In some embodiments, the first co-culture medium comprises interferon gamma (IFN-y). In some embodiments, the first co-culture medium comprises from up to a range of about 0.01 ng / mL to about 10 ng / mL, about 0.1 ng / mL to about 10 ng / mL, about 0.1 ng / mL to about 9 ng / mL, about 0.1 ng / mL to about 8 ng / mL, about 0.1 ng / mL to about 7 ng / mL, about 0.1 ng / mL to about 6 ng / mL, about 0.1 ng / mL to about 5 ng / mL, about 0.1 ng / mL to about 4 ng / mL, about 0.1 ng / mL to about 3 ng / mL, about 0.1 ng / mL to about 2 ng / mL, about 0.1 ng / mL to about 1 ng / mL, about 0.1 ng / mL to about 0.9 ng / mL, about 0.1 ng / mL to about 0.8 ng / mL, about 0.1 ng / mL to about 0.7 ng / mL, about 0.1 ng / mL to about 0.6 ng / mL, about 0.1 ng / mL to about 0.5 ng / mL, about 0.1 ng / mL to about 0.4 ng / mL, about 0.1 ng / mL to about 0.3 ng / mL, or about 0.1 ng / mL to about 0.2 ng / mL IFN-y.

[0199] In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 1 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.9 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.8 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.7 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.6 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.5 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.4 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.3 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 0.2 ng / mL IFN-y.

[0200] In some embodiments, the first co-culture medium comprises about 0.01 ng / mL, about 0.1 ng / mL, about 0.2 ng / mL, about 0.3 ng / mL, about 0.4 ng / mL, about 0.5 ng / mL, about 0.6 ng / mL, about 0.7 ng / mL, about 0.8 ng / mL, about 0.9 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL IFN-y.

[0201] In some embodiments, the first co-culture medium comprises about 0.1 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.2 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.3 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.4 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.5 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.6 ng / mL IFN-y. In some54#623862Attorney Docket No. 00009.008.1801 embodiments, the first co-culture medium comprises about 0.7 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.8 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 0.9 ng / mL IFN-y. In some embodiments, the first co-culture medium comprises about 1 ng / mL IFN-y.1.3.2 Addition of inhibitors to the first co-culture medium

[0202] In some embodiments, the first co-culture medium comprises one or more inhibitors.

[0203] In some embodiments, the first co-culture medium comprises one or more TGF-beta / Smad inhibitors. In some embodiments, the one or more TGF-beta / Smad inhibitors is selected from the group consisting of 3, 3 -dimethyl- 1 -butanol, A8301, AUDA, BIBF-0775, DMH1, dorsomorphin, dorsomorphin 2HC1, Galunisertib (LY2157299), ginsenoside Rh4, GW788388, halofuginone, ITD-1, K02288, LDN-193189, LDN-193189 2HC1, LDN-212854, LDN-214117, LY2109761, LY 3200882, LY364947, lycopus extract, ML347, PD 169316, PF-06952229, Pirfenidone, R-268712, RepSox (E-616452), SB431542, SB505124, SB525334, SD-208, SIS3, SIS3 HC1, sulfasalazine, TA-02, TGFpRI-IN-3, TP0427736 HC1, and Vactosertib (TEW-7197). In some embodiments, the one or more TGF-beta / Smad inhibitors is 3,3-dimethyl-l-butanol. In some embodiments, the one or more TGF-beta / Smad inhibitors is A8301. In some embodiments, the one or more TGF-beta / Smad inhibitors is AUDA. In some embodiments, the one or more TGF-beta / Smad inhibitors is BIBF-0775. In some embodiments, the one or more TGF-beta / Smad inhibitors is DMH1. In some embodiments, the one or more TGF-beta / Smad inhibitors is dorsomorphin. In some embodiments, the one or more TGF-beta / Smad inhibitors is dorsomorphin 2HC1. In some embodiments, the one or more TGF-beta / Smad inhibitors is Galunisertib (LY2157299). In some embodiments, the one or more TGF-beta / Smad inhibitors is ginsenoside Rh4. In some embodiments, the one or more TGF-beta / Smad inhibitors is GW788388. In some embodiments, the one or more TGF-beta / Smad inhibitors is halofuginone. In some embodiments, the one or more TGF-beta / Smad inhibitors is ITD-1. In some embodiments, the one or more TGF-beta / Smad inhibitors is K02288. In some embodiments, the one or more TGF-beta / Smad inhibitors is LDN-193189. In some embodiments, the one or more TGF-beta / Smad inhibitors is LDN-193189 2HC1. In some embodiments, the one or more TGF-beta / Smad inhibitors is LDN-212854. In some embodiments, the one or more TGF-beta / Smad inhibitors is LDN-214117. In some embodiments, the one or more TGF-beta / Smad inhibitors is LY2109761. In some embodiments, the one or more TGF-beta / Smad inhibitors is LY 3200882. In some embodiments, the one or more TGF-beta / Smad inhibitors is LY364947. In some embodiments, the one or more TGF-beta / Smad inhibitors is lycopus extract. In some embodiments, the one or more TGF-beta / Smad inhibitors is ML347. In some embodiments, the one or more TGF-beta / Smad inhibitors is PD 169316. In some55#623862Attorney Docket No. 00009.008.1801 embodiments, the one or more TGF-beta / Smad inhibitors is PF-06952229. In some embodiments, the one or more TGF-beta / Smad inhibitors is Pirfenidone. In some embodiments, the one or more TGF-beta / Smad inhibitors is R-268712. In some embodiments, the one or more TGF-beta / Smad inhibitors is RepSox (E-616452). In some embodiments, the one or more TGF-beta / Smad inhibitors is SB431542. In some embodiments, the one or more TGF-beta / Smad inhibitors is SB505124. In some embodiments, the one or more TGF-beta / Smad inhibitors is SB525334. In some embodiments, the one or more TGF-beta / Smad inhibitors is SD-208. In some embodiments, the one or more TGF-beta / Smad inhibitors is SIS3. In some embodiments, the one or more TGF-beta / Smad inhibitors is SIS3 HC1. In some embodiments, the one or more TGF-beta / Smad inhibitors is sulfasalazine. In some embodiments, the one or more TGF-beta / Smad inhibitors is TA-02. In some embodiments, the one or more TGF-beta / Smad inhibitors is TGFpRI-IN-3.

[0204] In some embodiments, the one or more TGF-beta / Smad inhibitors is TP0427736 HC1.

[0205] In some embodiments, the one or more TGF-beta / Smad inhibitors is Vactosertib (TEW-7197).

[0206] In some embodiments, the co-culture media comprises one or more cytokine inhibitors. In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X-C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor. In some embodiments, the one or more cytokine inhibitors is a Tumor Necrosis Factor-alpha (TNFa) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interferon-gamma (IFNy) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin-8 (IL-8) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin- 1 beta (IL-1P) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin-6 (IL-6) inhibitor. In some embodiments, the one or more cytokine inhibitors is a C-X-C motif chemokine 11 (CXCL11) inhibitor. In some embodiments, the one or more cytokine inhibitors is an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor. In some embodiments, the one or more cytokine inhibitors is a Granulocyte Colony-Stimulating Factor (G-CSF) inhibitor.

[0207] In some embodiments, the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor.

[0208] In some embodiments, the one or more cytokine inhibitors is a TNFa inhibitor. In some embodiments, the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab. In some embodiments, the TNFa56#623862Attorney Docket No. 00009.008.1801 inhibitor is adalimumab. In some embodiments, the TNFa inhibitor is certolizumab pegol. In some embodiments, the TNFa inhibitor is etanercept. In some embodiments, the TNFa inhibitor is golimumab. In some embodiments, the TNFa inhibitor is infliximab.

[0209] In some embodiments, the one or more cytokine inhibitors is a IFNy inhibitor. In some embodiments, the IFNy inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib. In some embodiments, the IFNy inhibitor is AMG 811. In some embodiments, the IFNy inhibitor is baricitinib. In some embodiments, the IFNy inhibitor is filgotinib. In some embodiments, the IFNy inhibitor is fontolizumab. In some embodiments, the IFNy inhibitor is oclacitinib. In some embodiments, the IFNy inhibitor is ruxolitinib. In some embodiments, the IFNy inhibitor is tofacitinib. In some embodiments, the IFNy inhibitor is upadacitinib.

[0210] In some embodiments, the one or more cytokine inhibitors is an IL-8 inhibitor. In some embodiments, the IL-8 inhibitor is selected from the group consisting of: MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756). In some embodiments, the IL-8 inhibitor is MS-986253. In some embodiments, the IL-8 inhibitor is Ab-8 (GSK2140935). In some embodiments, the IL-8 inhibitor is Danirixin (GSK1325756).

[0211] In some embodiments, the one or more cytokine inhibitors is an IL-ip inhibitor.

[0212] In some embodiments, the IL-ip inhibitor is selected from the group consisting of: Rilonacept (Arcalyst) and Canakinumab (Haris). In some embodiments, the IL-ip inhibitor is Rilonacept (Arcalyst). In some embodiments, the IL-ip inhibitor is Canakinumab (Haris).

[0213] In some embodiments, the one or more cytokine inhibitors is an IL-6 inhibitor. In some embodiments, the IL-6 inhibitor is selected from the group consisting of: Tocilizumab (Actemra) and Sarilumab (Kevzara). In some embodiments, the IL-6 inhibitor is Tocilizumab (Actemra). In some embodiments, the IL-6 inhibitor is Sarilumab (Kevzara).

[0214] In some embodiments, the one or more cytokine inhibitors is an IL-12 / IL-23 inhibitor. In some embodiments, the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi). In some embodiments, the IL-12 / IL-23 inhibitor is Ustekinumab (Stelara). In some embodiments, the IL-12 / IL-23 inhibitor is Risankizumab (Skyrizi).

[0215] In some embodiments, the one or more cytokine inhibitors is a CXCL11 inhibitor. In some embodiments, the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418). In some embodiments, the CXCL11 inhibitor is BMS-936559 (MDX-1106). In some embodiments, the CXCL11 inhibitor is Vercimon (TRA-418).57#623862Attorney Docket No. 00009.008.1801 2. Co-culture cell culture constructs

[0216] Described herein, in certain embodiments, are co-culture cell constructs produced by the method of any of the embodiments described herein, the co-culture cell construct comprising: (a) a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well, the first well containing a first co-culture medium; (b) a luminal container held within the first well of the basal container and comprising: (i) a second porous wall; and (ii) at least one side wall extending upwardly from the second wall to define a second well, the second well also containing the first co-culture medium; and wherein the immune cells are cultured in the first well of the basal container and the intestinal epithelial cells are cultured in the second well of the luminal container.

[0217] Further described herein, in certain embodiments, are apparatuses for co-culturing cells. In some embodiments, the apparatus comprises a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well; and a luminal container capable of being held within the first well of the basal container.

[0218] In some embodiments, the co-culture cell constructs and / or apparatuses for co-culturing cells described herein provide for long-term co-culture, improved confluence of cells in co-culture, improved differentiation of cells prior to and / or during co-culture, or combinations thereof. In some embodiments, cells grown using the co-culture cell constructs and / or apparatuses for co-culturing cells described herein are maintained in culture for at least 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or more. In some embodiments, cells grown using the co-culture cell constructs and / or apparatuses for co-culturing cells described herein are maintained in culture for at least 24 hours.2.1. Basal / luminal containers

[0219] In some embodiments, the co-culture cell constructs and / or apparatuses for co-culturing cells comprises one or more containers. In some embodiments, the co-culture cell constructs and / or apparatuses for co-culturing cells comprises a basal container and a luminal container.

[0220] In some embodiments, the basal container comprises a first wall (i.e., a bottom wall of the basal container) and at least one side wall extending upwardly from the first wall to define a first well. In some embodiments, the first wall is a bottom wall of the basal container. In some embodiments, the basal container is cylindrical in shape. In some embodiments, the basal container is cylindrical in shape and has a larger diameter than the luminal container, such that the luminal container can be inserted into the basal container.58#623862Attorney Docket No. 00009.008.1801

[0221] In some embodiments, the luminal contain comprises a second wall (e.g. a bottom wall of the luminal container) and at least one side wall extending upwardly from the second wall to define a second well. In some embodiments the second wall is a bottom wall of the luminal container. In some embodiments, the luminal container can be cylindrical in shape. In some embodiments, the luminal container is cylindrical in shape and has a smaller diameter than the basal container, such that the luminal container can be inserted into the basal container.

[0222] In some embodiments, the luminal container is held within the well of the basal container. In some embodiments, the bottom wall of the basal container is spaced apart from the bottom wall of the luminal container, and a basal compartment is defined between the bottom wall of the basal container and the bottom wall of the luminal container and / or between the at least one side wall of the basal container and the at least one side wall of the luminal container.2.2. Second wall o f the luminal container

[0223] In some embodiments, the second wall of the luminal container is a second porous wall. In some embodiments, the second porous wall comprises a porous material. In some embodiments, the second porous wall comprises a material with reduced porosity that comprises one or more openings or holes (e.g., microholes). In some embodiments, the second porous wall comprises a non-porous material that comprises one or more openings or holes (e.g., microholes). In some embodiments, the one or more openings or holes (e.g., microholes) extend through the second porous wall of the luminal container (z.e., extend across from one surface of the second porous wall of the luminal container through to a second surface of the second porous wall of the luminal container). In some embodiments, the one or more openings or holes (e.g., microholes) are distributed randomly throughout the second porous wall of the luminal container. In some embodiments, the one or more openings or holes (e.g., microholes) are distributed evenly throughout the second porous wall of the luminal container.2.3. Composition o f the second wall o f the luminal container

[0224] In some embodiments, the second wall of the luminal container comprises a material with reduced porosity or a non-porous material. In some embodiments, the material with reduced porosity or the non-porous material includes, but is not limited to, 1002F epoxy photoresist, cyclic olefin polymer (COC), polycarbonate (PC), acrylate polymers (e.g., poly(methyl methacrylate)), poly-methyl-meta-acrylate (PMMA), polystyrene, polyethylene, polyimide, polypropylene, polyvinyl chloride, cellulose, acrylonitrile butadiene styrene (ABS) plastic, nylon, acetal resin, polytetrafluoroethylene, polyesters (e.g., polyethylene terephthalate (PET), polyethylene naphthalate, polybutylene terephthalate, polytrimethylene terephthalate, polyglycolide, polylactic acid, poly caprolactone, copolyesters), epoxy (e.g., SU-8, 100 IF, 1009F), elastomers (e.g.,59#623862Attorney Docket No. 00009.008.1801 polydimethylsiloxane (PDMS), glass, ceramics, and / or metals. In some embodiments, the material with reduced porosity or the non-porous material comprises 1002F epoxy photoresist. In some embodiments, the material with reduced porosity or the non-porous material comprises cyclic olefin polymer (COC). In some embodiments, the material with reduced porosity or the non-porous material comprises polycarbonate (PC In some embodiments, the material with reduced porosity or the non-porous material comprises acrylate polymers. In some embodiments, the material with reduced porosity or the non-porous material comprises poly(methyl methacrylate). In some embodiments, the material with reduced porosity or the non-porous material comprises poly-methyl-meta-acrylate (PMMA). In some embodiments, the material with reduced porosity or the non-porous material comprises polystyrene. In some embodiments, the material with reduced porosity or the non-porous material comprises polyethylene. In some embodiments, the material with reduced porosity or the non-porous material comprises polyimide. In some embodiments, the material with reduced porosity or the non-porous material comprises polypropylene. In some embodiments, the material with reduced porosity or the non-porous material comprises polyvinyl chloride. In some embodiments, the material with reduced porosity or the non-porous material comprises cellulose. In some embodiments, the material with reduced porosity or the non-porous material comprises acrylonitrile butadiene styrene (ABS) plastic. In some embodiments, the material with reduced porosity or the non-porous material comprises nylon. In some embodiments, the material with reduced porosity or the non-porous material comprises acetal resin. In some embodiments, the material with reduced porosity or the non-porous material comprises polytetrafluoroethylene. In some embodiments, the material with reduced porosity or the non-porous material comprises polyesters. In some embodiments, the material with reduced porosity or the non-porous material comprises polyethylene terephthalate (PET). In some embodiments, the material with reduced porosity or the non-porous material comprises polyethylene naphthalate. In some embodiments, the material with reduced porosity or the non-porous material comprises polybutylene terephthalate. In some embodiments, the material with reduced porosity or the non-porous material comprises polytrimethylene terephthalate. In some embodiments, the material with reduced porosity or the non-porous material comprises polyglycolide. In some embodiments, the material with reduced porosity or the non-porous material comprises polylactic acid. In some embodiments, the material with reduced porosity or the non-porous material comprises polycaprolactone. In some embodiments, the material with reduced porosity or the non-porous material comprises copolyesters. In some embodiments, the material with reduced porosity or the non-porous material comprises epoxy. In some embodiments, the material with reduced porosity or the non-porous material comprises SU-8. In some embodiments, the material with reduced60#623862Attorney Docket No. 00009.008.1801 porosity or the non-porous material comprises 100 IF. In some embodiments, the material with reduced porosity or the non-porous material comprises 1009F. In some embodiments, the material with reduced porosity or the non-porous material comprises elastomers. In some embodiments, the material with reduced porosity or the non-porous material comprises polydimethylsiloxane (PDMS). In some embodiments, the material with reduced porosity or the non-porous material comprises glass. In some embodiments, the material with reduced porosity or the non-porous material comprises ceramics. In some embodiments, the material with reduced porosity or the non-porous material comprises metals.2.4. Porous layer

[0225] In some embodiments, a porous layer is overlaid onto the second wall of the luminal container. In some embodiments, the porous layer overlaying the second wall comprises a porous material. In some embodiments, the porous material comprises a hydrogel. In some embodiments, the hydrogel is a natural or synthetic hydrogel (e.g., collagen, Matrigel, gelatin, agarose, chitosan, alginate, polyethylene glycol, polyacrylamide), dried hydrogel followed by salt leaching (e.g. collagen hydrogel is dried and the salts are leach out to create pores). In some embodiments, the porous material comprises collagen. In some embodiments, the porous material comprises Matrigel. In some embodiments, the porous material comprises gelatin. In some embodiments, the porous material comprises agarose. In some embodiments, the porous material comprises chitosan. In some embodiments, the porous material comprises alginate. In some embodiments, the porous material comprises polyethylene glycol. In some embodiments, the porous material comprises polyacrylamide.

[0226] In some embodiments, the porous layer has a porosity that can range from about 0% to about 10%, or about 0% to about 20%, or about 0% to about 30%, or about 0% to about 40%, or about 0% to about 50%, or about 0% to about 75%, or about 0% to about 90%, or about 5% to about 90%, or about 10% to about 80%, or about 20% to about 70%, or about 30% to about 60%, or about 5% to about 100%, or about 10% to about 100%, or about 20% to about 100%, or about 30% to about 100%, or about 40% to about 100%, or about 50% to about 100%, or about 75% to about 100%. In some embodiments, the porosity of the porous layer is up to about 1%. In some embodiments, the porosity of the porous layer is up to about 2%. In some embodiments, the porosity of the porous layer is up to about 3%. In some embodiments, the porosity of the porous layer is up to about 4%. In some embodiments, the porosity of the porous layer is up to about 5%. In some embodiments, the porosity of the porous layer is up to about 6%. In some embodiments, the porosity of the porous layer is up to about 7%. In some embodiments, the porosity of the porous layer is up to about 8%. In some embodiments, the porosity of the porous layer is up to about 9%.61#623862Attorney Docket No. 00009.008.1801 In some embodiments, the porosity of the porous layer is up to about 10%. In some embodiments, the porosity of the porous layer is up to about 11%. In some embodiments, the porosity of the porous layer is up to about 12%. In some embodiments, the porosity of the porous layer is up to about 13%. In some embodiments, the porosity of the porous layer is up to about 14%. In some embodiments, the porosity of the porous layer is up to about 15%. In some embodiments, the porosity of the porous layer is up to about 20%. In some embodiments, the porosity of the porous layer is up to about 25%. In some embodiments, the porosity of the porous layer is up to about 30%. In some embodiments, the porosity of the porous layer is up to about 35%. In some embodiments, the porosity of the porous layer is up to about 40%. In some embodiments, the porosity of the porous layer is up to about 45%. In some embodiments, the porosity of the porous layer is up to about 50%.

[0227] In some embodiments, the porous layer has a thickness of about 1 millimeter or less, about 500 micrometers (pm) or less, about 250 pm or less, about 100 pm or less, about 50 pm or less, about 25 pm or less, about 20 pm or less, about 15 pm or less, about 10 pm or less, or about 5 pm or less. In some embodiments, the porous layer has a thickness of about 1 millimeter or less. In some embodiments, the porous layer has a thickness of about 500 micrometers (pm) or less. In some embodiments, the porous layer has a thickness of about 250 pm or less. In some embodiments, the porous layer has a thickness of about 100 pm or less. In some embodiments, the porous layer has a thickness of about 50 pm or less. In some embodiments, the porous layer has a thickness of about 25 pm or less. In some embodiments, the porous layer has a thickness of about 20 pm or less. In some embodiments, the porous layer has a thickness of about 15 pm or less. In some embodiments, the porous layer has a thickness of about 10 pm or less. In some embodiments, the porous layer has a thickness of about 5 pm or less.3. Cell types

[0228] In some embodiments, cells useful with the presently disclosed subject matter are from a eukaryotic cell line and / or are primary cells. In some embodiments, the cells useful with the presently disclosed subject matter are mammalian cells. In some embodiments, the cells useful with the presently disclosed subject matter are selected from the group consisting of: intestinal epithelial cells, colonic epithelial cells (e.g., mouse and human colonic epithelial cells), gastric epithelial cells, fibroblasts, myofibroblasts, endothelial cells, liver cells, adipocytes, muscle cells, bone cells, nervous cells, immune cells, and / or stem cells (embryonic, induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells).62#623862Attorney Docket No. 00009.008.1801

[0229] In some embodiments, the cells useful with the presently disclosed subject matter are cancerous cells from healthy, inflamed, and / or diseased human or animal. In some embodiments, the cells useful with the presently disclosed subject matter include, but are not limited to, cells from a digestive tract, a reproductive tract, a respiratory tract, an eye, a nose, an ear, a kidney, a brain, a liver, a pancreas, a gall bladder, a lymphatic system, a nerve system, skin, a bone, a tendon, a ligament, cartilage, bone marrow, connective tissue, and / or blood. In some embodiments, the cells useful with the presently disclosed subject matter are isolated from a biological sample, e.g., from a particular subject of interest, e.g., human or other animal with a particular disease condition or suspected of having a particular disease condition.

[0230] In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of epithelial cells (e.g., primary epithelial cells, human primary epithelial cells, mouse primary epithelial cells, intestinal epithelial cells, colonic epithelial cells, and / or gastric epithelial cells), fibroblasts, myofibroblasts, endothelial cells, liver cells, adipocytes, muscle cells, bone cells, nervous cells, immune cells, and / or stem cells (embryonic, induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells). In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of epithelial cells. In some embodiments, the cells useful with the presently disclosed subject matter are epithelial cells. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of primary epithelial cells. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of murine or human primary epithelial cells.

[0231] In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of intestinal epithelial cells (e.g., human primary intestinal epithelial cells, mouse primary intestinal epithelial cells). In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of intestinal epithelial cells. In some embodiments, the cells useful with the presently disclosed subject matter are intestinal epithelial cells. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of primary intestinal epithelial cells. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of murine or human primary intestinal epithelial cells.

[0232] In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of immune cells (e.g., macrophages, mast cells, neutrophils, eosinophils, basophils cells, natural killer (NK) cells, T lymphocytes (T cells), helper T cells, cytotoxic T cells, B lymphocytes (B cells), plasma cells, memory B cells dendritic cells and / or platelets). In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of immune cells. In some embodiments, the cells useful with the presently disclosed subject matter63#623862Attorney Docket No. 00009.008.1801 are immune cells. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of monocytes. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of murine or human monocytes. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of differentiated monocytes (e.g. macrophages). In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of differentiated macrophages. In some embodiments, the macrophages of the presently disclosed subject matter comprise or consist of M1 macrophages, M2 macrophages, regulatory macrophages, embryonically derived macrophages, adult-derived macrophages, exudate macrophages, resident macrophages, and / or Kupffer cells.

[0233] In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of cells from a cancer-derived cell line. In some embodiments, the cancer derived cell line is a human or murine cell line. In some embodiments, the cancer derived cell line is a leukemia-derived cell line. In some embodiments, the cancer derived cell line is a human leukemia monocytic cell line. In some embodiments, the cells from the human leukemia monocytic cell line comprise or consist of THP-1 cells, MONO-MAC-6 cells, HL-60 cells, TZ-1 cells, U937, and / or Kasumi-3 cells. In some embodiments, the cells useful with the presently disclosed subject matter comprise or consist of THP-1 cells.EXAMPLES

[0234] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.Example 1: Differentiation of THP-1 cells with PMA (phorbol 12-myristate 13-acetate) and monoculture of PMA-differentiated THP-1 cells in different media variants1.1 Objective

[0235] The objectives of the experiments described in this example were: (1) to establish a protocol for PMA (phorbol 12-myristate 13-acetate) differentiation of THP-1 cells, and (2) to test media variants in PMA-differentiated THP-1 monoculture.1.2 Cell cultures

[0236] THP-1 cells were thawed from passage 0 (P0) cells and passaged (i.e., subcultured). At the time of the experiment, THP-1 cells were at passage 5.1.3 Reagents

[0237] The reagents used in the experiments described in this example are shown in Table 1.64#623862Attorney Docket No. 00009.008.1801 Table 1.Component Amount UsedTHP-1 media 100 mLRGM (expansion media) 20 mLPMA (lOOnM) 0.1 plLuminescence assay reagent 1.2 mL1.4 Conditions Tested

[0238] The following two PMA differentiation conditions were tested: (1) direct addition of PMA, and (2) detaching of THP-1 cells.

[0239] The following four media variants were tested: (1) THP-1 media, (2) RMM media (differentiation media), (3) RMM without EGF (epidermal growth factor), NAC (N-acetylcysteine), or A-8301, and (4) RMM media with EGF (epidermal growth factor).1.5 Experimental Procedures

[0240] Both PMA differentiation conditions described above (i.e., (1) direct addition of PMA, and (2) detaching of THP-1 cells) were tested in triplicate (i.e., n = 3) in each of the four media variants described above (i.e., in (1) THP-1 media, (2) RMM media, (3) RMM without EGF, NAC, or A-8301, and (4) RMM media with EGF).1.5,1 Cell culture

[0241] The cell culture timeline for testing the two different PMA differentiation conditions in media variants is described below and summarized in Table 2.Table 2.PMA Differentiation Condition TestedDayDirect Addition of PMA Detaching of THP-1 cellsTHP-1 cells were counted and added to a PMA was added to a flask containing THP-1 1 96 well plate with media containing PMA cells (n = 3)(n = 3)Cells were monitored for adherence Cells were monitored for adherence Cells were detached, counted, and added to 2 96 well plateMedia variants were added to cells in After a 1.5 hour resting phase, media variants were added to cells in 96 well plate3 Cells were monitored Cells were monitoredCells were monitored and a luminescence Cells were monitored and a luminescence 4 assay was performed to determine the assay was performed to determine theviability of THP-1 cells viability of THP-1 cells1,5,2 PMA differentiation via direct addition of PMA

[0242] THP-1 cells (passage 5), which were in suspension, were collected from a T225 flask and transferred to a 15 mL tube using 10 mL pipette. After centrifugation at 600 rpm for 5 minutes, the65#623862Attorney Docket No. 00009.008.1801 cell pellet was rinsed with 5 mL of PBS (phosphate-buffered saline). Cells were counted using Hemocytometer and adjusted to a density of 1.4x104 cells / well in THP-1 media without mercaptoethanol with 100 nM PMA. Cells were seeded directly into the basal compartment of a 96 well plate. Cells were allowed to adhere for 24 hours. Following adhesion, the media was replaced with media variants (i.e., with (1) THP-1 media, (2) RMM media, (3) RMM without EGF, NAC, or A-8301, or (4) RMM media with EGF).1.5.3 PMA differentiation via detaching of THP-1 cells

[0243] THP-1 cells (passage 5) which were in passage in the cell culture incubator were obtained. The final concentration of 100 nM of PMA was directly added to the 25 cm2 flask containing THP-1 cells in THP-1 media without mercaptoethanol. After 48 hours, once the cells had adhered, media was aspirated carefully. 500 pl of cell detachment solution was added to the cells and kept in the incubator for 5 minutes. Once the cells detached, cells suspension was collected, followed by counting the cells. A total of 1.4x104 cells / well was added to the basal compartment in THP-1 media without mercaptoethanol. After 1.5 hours of resting phase and cell adhesion, the media was replaced with media variants (i.e., with (1) THP-1 media, (2) RMM media, (3) RMM without EGF, NAC, or A-8301, or (4) RMM media with EGF).1.5.4 PMA differentiation via detaching of THP-1 cells

[0244] After 48 hours in different media variants, a luminescence assay was performed to determine the viability of THP-1 cells.1.6 Results

[0245] Results for PMA differentiation of THP-1 cells after 24 hours via direct addition of PMA (in a T25 flask) and via detaching of THP-1 cells (in a 96-well plate) are shown in FIG. 1.

[0246] Results for 48 hours of PMA differentiation in THP-1 media and RMM without EGF, NAC, or A-8301 (RMM-EGF-NAC-A8301) comparing direct addition of PMA against detaching of THP-1 cells are shown in FIG. 2.

[0247] Results for 48 hours of PMA differentiation in RMM with EGF, NAC and A8301 (RMM+EGF+NAC+A8301) and RMM with EGF (RMM+EGF) comparing direct addition of PMA against detaching of THP-1 cells are shown in FIG. 3.

[0248] Results from the luminescence assay demonstrating the viability of PMA differentiated THP-1 cells in media variants are shown in FIG. 41.7 Conclusions

[0249] Both protocols for PMA differentiation — i.e., (1) direct addition of PMA during seeding, and (2) adding PMA to the flask followed by detaching and reseeding — resulted in adherence of THP-1 cells, indicating that both protocols for PMA differentiations worked. However, bright field66#623862Attorney Docket No. 00009.008.1801 images (FIGs. 1-3) and visual observations showed a decreased cell number in the detaching condition. The luminescence viability assay (FIG. 4) showed that direct addition of PMA had increased viability of the cells compared to the detachment method. Additionally, differentiated macrophages cultured in THP-1 media have decreased viable cell number compared to RMM media and its variants in both the PMA differentiation conditions.

[0250] Based on these results, direct addition of PMA was chosen over detaching the cells.Example 2: Evaluation of transverse colon (TC) cell culture in different media variants 2.1 Objective

[0251] THP-1 media’s base media component is RPMI 1640 whereas RMM media’s base component is Advanced DMEM / F12 along with additional components such as HEPES, EGF, NAC and A8301 which are absent in THP-1 media. To co-culture transverse colon (TC) cells and THP-1 cells in the same compartment, it was essential to find the right coculture media that sustained both cell populations. The primary objectives of this experiment were: (1) to test Donor 1 (DI) TC cells in THP-1 media variants and RMM media variants and measure the TEER (transepithelial electrical resistance), and (2) to test the response of DI TC cells to two concentrations of TNFa ( / .<., 10 ng / mL and 30 ng / mL TNFa) in THP-1 media and RMM media variants.2.2 Cell cultures

[0252] At the time of the experiment, DI Transverse Colon (TC) cells were at passage 10.2.3 Conditions Tested

[0253] The TNFa concentrations that were tested were 10 ng / mL and 30 ng / mL TNFa. The media conditions that were tested are summarized in Table 3.Table 3.Media Conditions TestedTHP-1 mediaTHP-1 media without mercaptoethanolRPMI + EGF, +NAC, +A8301, + HEPES, + Glutamax, + serumRMM RMM without A8301 (RMM - A8301)RMM without EGF (RMM - EGF)RMM without NAC (RMM - NAC)RMM without A8301 and EGF (RMM - A8301 / EGF)RMM without A8301 and NAC (RMM - A8301 / NAC)RMM without EGF and NAC (RMM - EGF / NAC)RMM without A8301, EGF, and NAC (RMM - A8301 / EGF / NAC)RMM with 2mM Glutamax (total of 4 mM Glutamax)67#623862Attorney Docket No. 00009.008.1801 2.4 Experimental Procedures2,4,1 Cell culture

[0254] DI TC cells were seeded on a 1% Matrigel coated 0.4 pM 96 well transwell plate using RGM (expansion media). Cell monitoring and TEER measurements were performed every day. RGM (expansion media) change was performed 48 hours post-plating. Once the cells were confluent, RMM media was added. 48 hours later, media variants according to Table 3 were added.

[0255] Each media variant was divided into three groups (and each group consisted of a sample size of n = 3): (1) Vehicle, (2) 10 ng / mL of TNFa, and (3) 30 ng / mL of TNFa. 4 hours after media variants were added, cells are treated with either 10 ng / mL or 30 ng / mL of TNFa. 48 hours later, supernatants were collected from TNFa treated cells and terminated. However, vehicle groups were continued, and TEER was measured everyday until the TEER values crashed.2.5 Results

[0256] TEER curves of DI Transverse Colon (TC) cells cultured in THP-1 media vs RMM media are shown in FIG. 5. The results demonstrate that TC cells survived in THP-1 media. However, the TEER plateau was short for cells cultured in THP-1 media compared to cells cultured in RMM media.

[0257] TEER curves of DI Transverse Colon (TC) cells in RMM without EGF media variants are shown in FIG. 6. The results demonstrate that all media variants without EGF resulted in an extended TEER plateau. On Day 8, loss of contrast and texture was noticed in RMM media without EGF.

[0258] TEER curves of DI Transverse Colon (TC) cells in RMM vs RMM with additional 2 mM Glutamax are shown in FIG. 7. The results demonstrate that addition of 2 mM Glutamax had no effect on the TEER curve and was comparable to RMM media.

[0259] The percentage change of TEER in Transverse Colon (TC) cells treated with either 10 ng / mL or 30 ng / mL of TNFa is shown in FIG.8. The results for cells treated with 10 ng / mL TNFa show that cells in THP-1 media were more sensitive to TNFa and responded to TNFa treatment, and had an almost 80% drop in TEER. DI TC cells in RMM media were resistant to 10 ng / mL TNFa treatment and had only approximately a 10% drop in TEER. Removal of EGF, alone or in combination with removal of A-8301 or NAC, from RMM made cells sensitive to treatment with 10 ng / mL TNFa. Removal of NAC from RMM also made cells sensitive to treatment with 10 ng / mL TNFa, but not as strongly as removal of EGF. Addition of Glutamax resulted in a mild response to treatment with 10 ng / mL TNFa. For cells that were treated with 30 ng / mL TNFa, the results show that apart from RMM and RMM without A8301 and RMM with 2mM Glutamax, all68#623862Attorney Docket No. 00009.008.1801 other groups responded strongly to treatment with 30 ng / mL TNFa (more than 80% drop). RMM, RMM without A8301 and RMM with 2mM Glutamax had a 60-70% drop in TEER.2.6 Conclusions

[0260] In this example, the TEER of DI TC cells in THP-1 media and different RMM media variants was measured. DI TC cells survived in all media variants. TEER curves of TC cells in RMM without A8301, without NAC, with additional Glutamax, and in THP-1 media were similar to the TC cells in RMM media. However, removal of EGF resulted in an extended TEER plateau in TC cells.

[0261] Additionally, removal of EGF and NAC from RMM increased the sensitivity of TC cells to TNFa treatment indicating that EGF and NAC contributed to the responsiveness of TC cells to the inflammatory response. This result was inconsistent with what has been previously reported in literature (e.g., see Lee et al. (2024); Nam et al. 2024); Palacio et al. (2011); Wang et al. (2020); Zhang et al. (2016)) where EGF reduced cell sensitivity to TNFa, and NAC inhibited release of inflammatory cytokines from activated macrophages. In this example, TC cells responded equally and strongly to 30 ng / mL TNFa treatment, except RMM and RMM without A8301.

[0262] In summary, DI TC cells were capable of surviving in both THP-1 media (RPML1640 as its base) and RMM media (Advanced DMEM / F12 as its base). These results therefore demonstrate that either base medium may be used to formulate a co-culture media. Additionally, removal of EGF and NAC increased the sensitivity of cells to TNFa. Furthermore, because TGF-P downregulates TNFa and IL-12 release, inclusion of A8301 (which blocks TGF-P signaling) in co-culture media was important in order to block TGF-P signaling and to allow macrophages to secrete TNFa.Example 3: Evaluation of unstimulated TC / THP-1 co-culture3.1 Objective

[0263] The objective of the experiments described in this example was to test different seeding densities of THP-1 cells to establish a stable TC / THP-1 coculture system.3.2 Reagents

[0264] The reagents used in the experiments described in this example are shown in Table 4. Table 4.Component Amount UsedTHP-1 cells 1 vialTHP-1 media 50 mLDonor 1 (DI) TC 1 vialRMM+A8301-NAC-EGF (Co-culture 50 mLMedia)69#623862Attorney Docket No. 00009.008.1801 RGM (expansion media) 20 mLPMA (lOOnM) 0.1 pLRMM 25 mLLuminescence viability assay 1.2 mLColorimetric LDH Cytotoxicity Assay 1 kit3.3 Methods

[0265] An overview of the timeline and steps for preparing the TC / THP-1 co-culture system described in this example is shown in FIG. 9. The steps are described in further detail below.3.3.1 Step 1: Seeding, expansion, and differentiation of DI TC cells

[0266] A vial of DI TC cells was seeded onto 0.4 pM 96-well plates coated with 1% Matrigel. RGM (expansion media) was used as the seeding medium. Upon reaching confluency on day 3 post-plating, the medium was switched to RMM. Daily monitoring of the cells and TEER measurements were performed.3.3.2 Step 2: Seeding, expansion, and differentiation of THP-1 cells

[0267] THP-1 cells were seeded at various densities in the basal compartments ( / .<., in the receiver plates) of 96-well plates, using THP-1 media without mercaptoethanol and supplemented with 100 nM PMA. The seeding densities included IX, 2X, 4X, and 8X (where X represents 1.4 × 104cells per well). Each density was plated at an n of 4. Cells were grown for 3 days before PMA was added.3.3.2.1 PMA di fferentiation protocol

[0268] The working concentration of PMA that was used was 100 nM. To achieve this, 0.1 pL of a 10 mM stock PMA (in DMSO) was added to 10 mL of THP-1 media without mercaptoethanol, resulting in a final concentration of 100 nM PMA.

[0269] Images of THP-1 cells were captured on Day 0, followed by monitoring and additional imaging 24 hours later. On Day 1, after 24 hours of PMA incubation, the THP-1 cells adhered, with no visible floating cells observed. The media was then replaced with fresh THP-1 media lacking both mercaptoethanol and PMA. On Day 2, some of the adhered cells spread and showed a macrophage-like morphology. Co-culture was initiated on day 23.3.3 Step 3: Co-culture of DI TC and THP-1 cells

[0270] TC cells that were differentiated in the apical compartment of a receiver plate for 48 hours, and THP-1 macrophages that where PMA-differentiated in the basal compartment of another receiver for 48 hours were bought together for co-culture. Media was aspirated from both apical and basal compartments. Co-culture media (RMM with A8301 and without NAC and EGF, z.e., RMM+A8301-NAC-EGF) was added to both apical and basal compartments. Cell monitoring and TEER measurements were performed daily for 48 hours.70#623862Attorney Docket No. 00009.008.1801 3.3.4 End point assays

[0271] 48 hours after co-culturing, apical and basal supernatants were collected separately. A colorimetric LDH cytotoxicity assay was performed using both apical and basal supernatants at a ratio of 1:2. A luminescence viability assay was performed on THP-1 macrophages. Remaining supernatants were stored at -80 °C.3.4 Results

[0272] Bright field images of THP-1 cells at different seeding densities ( / .<., at IX, 2X, 4X, and 8X) after 24 hour PMA differentiation are shown in FIG. 10.

[0273] The TEER curve of different THP-1 seeding densities in TC / THP-1 co-culture throughout the period of a 48 hour co-culture is shown in FIG. 11.

[0274] The percent change in TEER from FIG. 11, calculated as the co-culture TEER divided by the TC monoculture TEER at 48 hours of co-culture, is shown in FIG. 12.

[0275] The results of the luminescence viability assay of THP-1 macrophages described above are shown in FIG. 13.

[0276] The percentage of cytotoxicity as determined by the colorimetric LDH cytotoxicity assay described above for different THP-1 seeding densities in TC / THP-1 co-culture is shown in FIG.143.6 Conclusions

[0277] In this example, PMA differentiation was optimized and successfully established. THP-1 monocytes, which are suspension cells, adhered by 24 hours following PMA treatment (FIG. 10) and spread to acquire macrophage morphology by 48 hours, which confirmed successful differentiation.

[0278] Percent change in TEER measurements showed no significant drop between IX and 2X THP-1 seeding densities, but a significant decrease was observed at 4X and 8X seeding densities compared to IX and 2X seeding density (FIG. 12). This was consistent with the literature, as higher THP-1 seeding densities increased the release of TNFa, which may cause barrier damage and lower TEER values (Kampfer et al. (2017)).

[0279] A luminescence cell viability assay was performed on THP-1 cells under both monoculture and co-culture conditions (FIG. 13). The results showed similar viability in both setups, indicating that TC cells had no toxic effect on THP-1 cells. Additionally, while increased THP-1 seeding density led to higher cytotoxicity percentages, the values between monoculture and co-culture were comparable, suggesting that the higher density did not induce toxicity.

[0280] LDH colorimetric cytotoxicity assay was performed on a mixture of apical and basal supernatants at a ratio of 1:2 (FIG. 14), which impeded the ability to analyze the LDH activity of71#623862Attorney Docket No. 00009.008.1801 only TC cells. Hence, in the next example (i.e. in Example 4), LDH activity was assessed separately on apical and basal supernatants.

[0281] In conclusion, a stable co-culture system was successfully optimized and established.Example 4: Evaluation of inflamed TC / THP-1 co-culture4.1 Objective

[0282] The objective of the experiments described in this example was to test dosing of lipopolysaccharide (LPS) and interferon-gamma (IFNy) in combination. Specifically, three different combinations of concentrations were tested to evaluate stimulation of macrophages and the corresponding release of inflammatory cytokines that induce barrier disruption in TC cells.4.2 Cell cultures

[0283] 2X and 4X seeding densities where used in this example (where X represents 1.4 x 104cells per well).

[0284] The concentrations of LPS and IFNy that were tested ( / .<., as a single concentration or combinations of concentrations) are summarized in Table 5. An n of 2 technical replicates was used to evaluate four different test groups: (1) a TC / THP-1 co-culture test group, (2) a THP-1 monoculture test group, (3) a TC monoculture test group, and (4) a no treatment test group.Table 5.Combination Concentrations Tested Single Concentrations TestedLPS IFNy LPS IFNy100 ng / mL 0.5 ng / mL 100 ng / mL100 ng / mL 1 ng / mL 1,000 ng / mL1,000 ng / mL 1 ng / mL 0.5 ng / mL1 ng / mL4.3 Experimental Procedures4,3,1 Cell Culture

[0285] Donor 1 (DI) TC cells were seeded on a 1% Matrigel coated 0.4 pM 96-well transwell plate using RGM (expansion media). Before seeding the DI TC cells, the basal compartment (z.e., the receiver plate) was saved for seeding THP-1 cells. The transwell inserts were kept on an empty receiver plate. Cell monitoring via TEER measurements were performed every day. RGM (expansion media) change was performed 48 hours post-plating. By day 3 post-plating, cells were confluent and RMM media was added to the cells. On Day 2 in RMM, co-culture with THP-1 cells was initiated.72#623862Attorney Docket No. 00009.008.1801 4.3.2 PMA differentiation of THP-1 cells

[0286] On the day of DI TC becoming confluent and RMM was added (differentiation of DI TC cells), THP-1 cells were also differentiated using PMA. THP-1 cells (passage 6) were collected in a 15 mL tube with a 10 mL pipette and were centrifuged at 600 rpm for 1 minute. THP-1 media without mercaptoethanol and without PMA was added to the cell pellet. Cells were counted and adjusted to 2 different seeding densities which were 2X and 4X. 2X corresponds to 2.8xl04cells / 100 pl and 4X corresponds to 5.6xl04cells / 100 pl. 100 pl of THP-1 cell suspension was added to the basal compartment (without inserts) that was saved during seeding of the TC cells.

[0287] Cells were monitored visually under the microscope and bright field images were taken every day.

[0288] Co-culture was initiated 48 hours after DI TC cells were differentiated (in RMM media for 48 hours) and THP-1 cells were differentiated in PMA for 48 hours.4.3.3 Priming

[0289] On the day of co-culture, THP-1 cells were primed 4 hours in advance. LPS (100 ng / ml) and IFNy (25 ng / ml) were added to THP-1 media (without mercaptoethanol and PMA), which was then applied to the THP-1 cells at 100 pl per well.4.3.4 Co-culture

[0290] Prior to coculture, DI TC cells were monitored, and TEER was measured. THP-1 cells were monitored, and bright field images were taken. Media was aspirated from DI TC and THP-1 cells. THP-1 cells from the basal compartment and DI TC cells from the apical compartment were bought together. Co-culture media (RGM without EEC or NAC and with A8301, / .<., RGM-EEC-NAC+A8301) with different concentrations of LPS and / or IFNy (either single concentrations or combinations of concentrations), with DMSO, or with no treatment were prepared according to the conditions shown in Table 6. This was added to both apical (100 pl) and basal (200 pl) compartments.Table 6.Seeding Density Culture type Condition tested2X TC / THP-1 co-culture 100 ng / ml LPS + 0.5 ng / mL IFNy2X TC / THP-1 co-culture 100 ng / ml LPS + 1 ng / mL IFNy2X TC / THP-1 co-culture 1000 ng / ml LPS + 1 ng / mL IFNy2X TC / THP-1 co-culture 100 ng / ml LPS2X TC / THP-1 co-culture 1000 ng / ml LPS2X TC / THP-1 co-culture 0.5 ng / mL IFNy73#623862Attorney Docket No. 00009.008.1801 2X TC / THP-1 co-culture 1 ng / mL IFNy2X TC / THP-1 co-culture DMSO2X TC mono-culture 100 ng / ml LPS + 0.5 ng / mL IFNy 2X TC mono-culture 100 ng / ml LPS + 1 ng / mL IFNy 2X TC mono-culture 1000 ng / ml LPS + 1 ng / mL IFNy 2X TC mono-culture 100 ng / ml LPS2X TC mono-culture 1000 ng / ml LPS2X TC mono-culture 0.5 ng / mL IFNy2X TC mono-culture 1 ng / mL IFNy2X TC mono-culture No treatment2X THP-1 mono-culture 100 ng / ml LPS + 0.5 ng / mL IFNy 2X THP-1 mono-culture 100 ng / ml LPS + 1 ng / mL IFNy 2X THP-1 mono-culture 1000 ng / ml LPS + 1 ng / mL IFNy 2X THP-1 mono-culture 100 ng / ml LPS2X THP-1 mono-culture 1000 ng / ml LPS2X THP-1 mono-culture 0.5 ng / mL IFNy2X THP-1 mono-culture 1 ng / mL IFNy2X THP-1 mono-culture No treatment4X TC / THP-1 co-culture 100 ng / ml LPS + 0.5 ng / mL IFNy 4X TC / THP-1 co-culture 100 ng / ml LPS + 1 ng / mL IFNy 4X TC / THP-1 co-culture 1000 ng / ml LPS + 1 ng / mL IFNy 4X TC / THP-1 co-culture 100 ng / ml LPS4X TC / THP-1 co-culture 1000 ng / ml LPS4X TC / THP-1 co-culture 0.5 ng / mL IFNy4X TC / THP-1 co-culture 1 ng / mL IFNy4X TC / THP-1 co-culture DMSO4X TC mono-culture 100 ng / ml LPS + 0.5 ng / mL IFNy 4X TC mono-culture 100 ng / ml LPS + 1 ng / mL IFNy 4X TC mono-culture 1000 ng / ml LPS + 1 ng / mL IFNy 4X TC mono-culture 100 ng / ml LPS4X TC mono-culture 1000 ng / ml LPS4X TC mono-culture 0.5 ng / mL IFNy4X TC mono-culture 1 ng / mL IFNy74#623862Attorney Docket No. 00009.008.1801 4X TC mono-culture DMSO4X THP-1 mono-culture 100 ng / ml LPS + 0.5 ng / mL IFNy4X THP-1 mono-culture 100 ng / ml LPS + 1 ng / mL IFNy4X THP-1 mono-culture 1000 ng / ml LPS + 1 ng / mL IFNy4X THP-1 mono-culture 100 ng / ml LPS4X THP-1 mono-culture 1000 ng / ml LPS4X THP-1 mono-culture 0.5 ng / mL IFNy4X THP-1 mono-culture 1 ng / mL IFNy4X THP-1 mono-culture DMSO

[0291] Both THP-1 and TC cells were monitored every day. TEER measurements were taken for the co-culture (basal and apical compartments were kept together). After 48 hours of co-culture, TEER was measured, and cells were monitored for TC cells. For THP-1 cells, cells were monitored, and bright field images were taken. Both apical and basal supernatants were collected separately. Pooled supernatants were used to measure LDH activity (cytotoxicity). Remaining apical and basal supernatants were stored at -80 °C. A viability assay was performed on the TC and THP1 cells using a luminescent cell viability assay.4.4 Results4.4.1 PMA differentiation of THP-1 cells

[0292] 24 hours after adding PMA to THP-1 cells, cells adhered at both (2X and 4X) seeding densities. Cell debris was noticed. 48 hours later, cells were still adhered to the plate and 10% of the cells spread.4.4.2 Co-culture barrier integrity (TEER measurements)

[0293] The TEER curves of three LPS+IFNy combination doses (100 ng / mL and 0.5 ng / mL, 100 ng / mL and 1 ng / mL, 1000 ng / mL and 1 ng / mL) tested in monoculture, co-culture with 2X THP-1 seeding density, and co-culture with 4X THP-1 seeding density are shown in FIG. 15. The results demonstrate / show that in co-cultures of both seeding densities (2X and 4X) for all three combinations doses of LPS and IFNy (100+0.5, 100+1, 1000+1) TEER values completely crashed at 48 hours. At 2X THP-1 seeding density, there was no significant TEER drop at 24 hours compared to monocultures. However, at 48 hours the TEER values completely crashed. For 4X THP-1, TEER values started dropping at 24 hours, and completely crashed at 48 hours. Monocultures with three combo doses of LPS and IFNy (100 ng / mL and 0.5 ng / mL, 100 ng / mL and 1 ng / mL, 1000 ng / mL and 1 ng / mL) maintained TEER values and the values did not drop even at 48 hours.75#623862Attorney Docket No. 00009.008.1801

[0294] The TEER curves of two different THP1 seeding densities (2X and 4X) are shown in FIG. 16. The results demonstrate that unprimed 2X and 4X THP-1 seeding densities had no TEER reduction. Primed co-cultures treated with combo dosing of LPS and IFNy (100+0.5, 100+1 and 1000+1) drastically crashed the TEER values at 48 hours.

[0295] The TEER curves of single doses of LPS (100 ng / mL and 1000 ng / mL) and IFNy (0.5 ng / mL and 1 ng / mL) are shown in FIG. 17. The results demonstrate that priming THP-1 cells with individual doses of LPS (100 ng / mL and 1000 ng / mL) and IFNy (0.5 ng / mL and 1 ng / mL) had no effect on the TEER values and the values were comparable to monocultures. The TEER curves between 2X and 4X THP-1 seeding densities were quite similar.

[0296] The percentage change in TEER for TC / THP-1 co-culture and TC monoculture at 48 hours for both seeding densities (2X and 4X) is shown in FIG. 18. The results demonstrate that for both THP-1 seeding densities (2X and 4X), there was 100% reduction in TEER for combination dosing with LPS and IFNy. Individual doses of LPS (i.e., the 100 ng / mL dose of LPS and 1000 ng / mL dose of LPS) and IFNy (0.5 ng / mL of IFNy and 1 ng / mL of IFNy) had no effect on TEER values.

[0297] The percentage of cytotoxicity as determined by a colorimetric LDH cytotoxicity assay for different THP-1 seeding densities in TC / THP-1 co-culture and in monoculture is shown in FIG.19. The results show that at 2X seeding density, compared to monoculture, co-culture had higher cytotoxicity in combination treatment with LPS and IFNy. Individual treatments of LPS and IFNy were not cytotoxic, and the values were comparable to monocultures. However, at 4X seeding density, cytotoxicity was comparable to monocultures and was not statistically different.

[0298] The results of the luminescence viability assay performed for both apical (TC) and basal (THP-1) compartments at two different seeding densities (2X and 4X) is shown in FIGs. 20A-20D. The results show that on the apical side, all the combo dose treatments with LPS and IFNy showed decreased viability of TC cells in co-cultures compared to monocultures for both 2X and 4X seeding densities (FIGs. 20A-20B). For the individual treatments of LPS and IFNy, both cocultures and monocultures had comparable values. On the basal side where THP-1 cells were present, neither combo doses of LPS and IFNy nor the individual doses of LPS and IFNy had a negative effect on the viability of THP-1 cells and the numbers are comparable to monoculture values (FIGs. 20C-20D)4.5 Conclusions

[0299] At both 2X and 4X seeding densities and three combination doses of LPS and IFNy, TEER values crashed at 48 hours, indicating that a lower seeding density may be beneficial to have a slower TEER drop.76#623862Attorney Docket No. 00009.008.1801Example 5: Evaluation of mild inflammatory co-culture conditions and prevention of inflammatory damage using Adalimumab (Ada) and Tofacitinib (Tofa)5.1 Objective

[0300] The objectives of the experiments described in this example were: (1) to test two priming conditions (i.e., overnight priming with 100 ng / mL LPS and 0.5 ng / mL IFNy or 4 hour priming with 100 ng / mL LPS and 25 ng / mL IFNy) to determine the conditions that could create a mild inflammatory co-culture condition, and (2) to test the inclusion of the drugs Adalimumab (a TNFa inhibitor) and Tofacitinib (an IFNy inhibitor) to determine the conditions that could prevent inflammatory damage.5.2 Cell cultures

[0301] At the time of this experiment, Donor 1 (DI) TC cells were at passage 10 and THP-1 cells were at passage 7.5.3 Conditions Tested

[0302] Two different priming conditions were evaluated in this example: (1) overnight priming with 100 ng / mL LPS and 0.5 ng / mL IFNy, and (2) 4 hour priming with 100 ng / mL LPS and 25 ng / mL IFNy.

[0303] One treatment condition was evaluated in this example: 100 ng / mL LPS and 0.5 ng / mL IFNy.

[0304] Four different test groups of cells were evaluated in this example: (1) a TC / THP-1 coculture test group, (2) a THP-1 monoculture test group, (3) a TC monoculture test group, and (4) a no treatment test group.5.4 Experimental Procedures

[0305] An overview of the timeline and steps for preparing the TC / THP-1 co-culture system described in this example is shown in FIG.21, and described hereinbelow.

[0306] DI TC cells were seeded in RGM (expansion media), and once cells were confluent, the media was switched to RMM (Day 0). On Day 2, co-culture was initiated with THP-1 cells.

[0307] THP-1 cells were counted and seeded on a 96-well plate with THP-1 media containing PMA (Day 0). On Day 1, media was replaced with THP-1 media without PMA. For the overnight priming test group, on Day 1 in the evening, LPS (100 ng / mL), IFN (0.5 ng / mL) in THP-1 media was added. For the 4 hour priming test group, on Day 2, 4 hours before co-culture was initiated, LPS (100 ng / mL) and IFN (25 ng / mL) was added.77#623862Attorney Docket No. 00009.008.1801

[0308] 4 hours later, TC and THP-1 cells were bought together and co-culture media ( / .<., RMM without EGF and NAC, and with A8301, LPS (100 ng / mL) and IFN (0.5 ng / mL) was added to all tested groups. Some of the groups received either Adalimumab (Ada) or Tofacitinib (Tofa) or both.

[0309] These steps are described in further detail below.5.4.1 Seeding, expansion, and differentiation of DI TC cells

[0310] DI TC cells were seeded on a 1% Matrigel coated 96-well plate transwell using RGM (expansion media). Daily monitoring of the cells and TEER measurements were performed. RGM (expansion media) was changed 48 hours post-plating. By day 3 post-plating cells were confluent and RMM media was added to cells. On Day 2 in RMM, co-culture with THP-1 cells was initiated.5.4.2 PMA differentiation of THP-1 cells

[0311] On the day that DI TC cells become confluent and RMM was added ( / .<., upon initiating differentiation of DI TC cells), THP-1 cells were also differentiated using PMA. THP-1 cells (passage 7) were collected in a 15 mL tube with a 10 mL pipette and were centrifuged at 600 rpm for 1 minute. THP-1 media without mercaptoethanol and without PMA was added to the cell pellet. Cells were counted and adjusted to 1.5X seeding density, where 1.5X corresponds to 2.1xl04cells / 100 pl. 100 pl of THP-1 cell suspension was added to each well of the basal compartment ( / .<., in the receiver plates) of a 96-well plate (without inserts) that was saved during seeding TC cells.

[0312] Cells were monitored visually under the microscope and bright field images were taken every day

[0313] Co-culture was initiated 48 hours after DI TC cells were differentiated (in RMM media for 48 hours) and THP-1 cells were differentiated in PMA for 48 hours.5.4.3 Priming

[0314] Two priming conditions were tested: (1) overnight priming with 100 ng / mL of LPS and 0.5 ng / mL of IFNy, and (2) 4 hour priming with 100 ng / mL LPS and 25 ng / mL of IFNy.For overnight priming, THP-1 cells were primed with LPS (100 ng / mL) and IFNy (0.5 ng / mL) in THP-1 media without mercaptoethanol on the day before co-culture.

[0315] For 4 hour priming, THP-1 cells were primed 4 hours in advance of co-culture, whereby LPS (100 ng / mL) and IFNy (25 ng / mL) were added to THP-1 media (without mercaptoethanol and PMA), and said media was then applied to the THP-1 cells at 100 pl per well.5.4.4 Co-culture of DI TC and THP-1 cells

[0316] Prior to co-culture, DI TC cells were monitored, and TEER was measured. THP-1 cells were monitored, and bright field images were taken. Media was aspirated from DI TC and THP-1 cells. THP-1 cells from the basal compartment and DI TC cells from the apical compartment were78#623862Attorney Docket No. 00009.008.1801 bought together. Co-culture media (RGM without EEC, without NAC, and with A8301, i. e., RGM-EEC-NAC+A8301) with 100 ng / mL of LPS and 0.5 ng / mL of IFNy were administered to cells. Some of the wells received either Ada or Tofa or both, which when added were added to both apical (100 pl) and basal (200 pl) compartments.

[0317] After co-culture, cells in apical and basal compartment were observed quickly to make sure everything went well and cells in the basal compartment were fine. Both THP-1 and TC cells were monitored every day. TEER measurements were taken for the co-culture (basal and apical compartments were kept together). After 48 hours of co-culture, TEER was measured, and cells were monitored for TC cells. For THP-1 cells, cells were monitored, and bright field images were taken. Both apical and basal supernatants were collected separately. Pooled supernatants were used to measure LDH activity (cytotoxicity). Remaining apical and basal supernatants were stored at -80 °C. A viability assay was performed on the TC and THP1 cells using a luminescent cell viability assay5.5 Results5.5.1 PMA differentiation of THP-1 cells

[0318] 24 hours after adding PMA to THP-1 cells, cells adhered and no visible debris was noticed.5.5.2 Co-culture barrier integrity (TEER measurements)

[0319] TEER curves for 4 hour priming / pretreatment in co-culture, overnight priming / pretreatment in co-culture and TC monocultures are shown in FIG. 22. The results demonstrate that with 4 hours of priming / pretreatment (100 ng / mL of LPS and 25 ng / mL of IFNy), LPS + IFNy (100+0.5 ng / mL) stimulation completely crashed TEER values at 48 hours. This drop was completely protected in either single or combination doses of Ada and Tofa. However, with overnight priming / pretreatment (100 ng / mL of LPS and 0.5 ng / mL IFNy), LPS + IFNy (100+0.5 ng / mL) stimulation did not negatively effect TEER values. In TC monocultures, LPS+IFN (100+0.5 ng / mL) stimulation caused a slight drop in TEER. This slight drop was prevented by Ada and Tofa treatments

[0320] The percent change in TEER, calculated as the co-culture TEER divided by the TC monoculture TEER at 48 hours (from FIG.22), for 4 hour pretreatment and overnight pretreatment of THP-1 cells is shown in FIG.23. The results demonstrate / show that for the 4 hour pretreatment condition, there was 100% reduction in TEER for combination dosing (LPS and IFNy), and that treatments with either single doses of Ada and Tofa or a combination dose of Ada and Tofa prevented barrier damage.79#623862Attorney Docket No. 00009.008.1801

[0321] The results of the luminescence viability assay for both apical (TC cells) and basal (THP-1 cells) compartments after 4 hour pretreatment with LPS and IFNy are shown in FIG. 24. The results show that on the apical side where the TC cells were present, only the combination dose treatments with LPS and IFNy showed decreased viability of TC cells in co-cultures compared to monocultures. For the individual treatments of LPS and IFNy, both co-cultures and monocultures had comparable values. On the basal side where THP-1 cells were present, viability of THP-1 cells in co-culture and monoculture was comparable in all groups.

[0322] The percentage of cytotoxicity as determined by colorimetric LDH cytotoxicity assay for cells that received a 4 hour pretreatment with LPS and IFNy is shown in FIG.25. The results show that LPS+IFNy treatment increased cytotoxicity in co-cultures and remaining groups had comparable values with monocultures.5.6 Conclusions

[0323] In this example, two priming / pretreatment conditions were tested in THP-1 cells. In both priming / pretreatment conditions, LPS concentration was kept constant (z.e., at 100 ng / mL) and IFNy concentration was varied (z.e., a concentration of 0.5 ng / mL IFNy was used for overnight priming, and a concentration of 25 ng / mL IFNy was used for 4 hour priming). The 4 hour pretreatment condition, with a higher concentration of IFNy, was the only condition where disruption in the barrier integrity lead to a drop in TEER value. The overnight pretreatment condition, with lower levels of IFNy, did not show a drop in TEER values and the TEER curve was like that of the vehicle groups.

[0324] Because no crash in TEER values was observed at 48 hours in the 4 hour pretreatment condition, cell viability and cell cytotoxicity assays were only performed for the 4 hour pretreatment condition. As was expected, cytotoxicity increased, and viability decreased in TC cells in co-culture compared to monocultures.

[0325] In conclusion, the 4 hour pretreatment condition worked effectively as disruption in barrier integrity was observed, as seen from the drop in TEER values when treated with LPS and IFNy. Overnight treatment with a lower concentration of IFNy did not cause damage to barrier integrity. Further, the inflammatory damage induced by stimulated macrophages was prevented by treatment with Ada and Tofa.Example 6: Evaluation of initiation of inflammatory conditions in a THP-l / epithelial cell coculture model, by priming and dosing with LPS and IFNY in the presence or absence of80#623862Attorney Docket No. 00009.008.1801 adalimumab (Ada) and tofacitinib (Tofa), in different media using different epithelial cell plate densities6.1 Objective

[0326] The objectives of the experiments described in this example were: (1) to evaluate the effects of 100 ng / mL LPS + 25 ng / mL IFNy priming and 100 ng / mL LPS + 0.5 ng / mL IFNy dosing on the initiation inflammatory conditions in a THP-l / epithelial cell co-culture model, with additional testing done in the presence of the TNFa inhibitor adalimumab and the IFNy inhibitor tofacitinib; (2) to assess the effects of this dosing in both co-culture media and standard RMM; and (3) to test this dosing in co-culture media with TC cells plated at 100% and 90% of normal density.6.2 Cell information

[0327] The following cell types were used in this example: (1) human intestinal transverse colon epithelial cells at passage 10, and (2) THP-1 cells at passage 5.6.3 Conditions Tested

[0328] Cells in this example were primed with 100 ng / mL LPS and 25 ng / mL IFNy 4 hours before initiation of co-culture.

[0329] At the time co-culture was initiated, cells were treated with 100 ng / mL LPS and 0.5 ng / mL IFNy.6.4 Experimental Procedures

[0330] The apical compartment of one 96 transwell plate with 0.4 pm membrane pores was coated with 1% Matrigel, and the plate was incubated overnight at 37° C.The following day, human intestinal epithelial cells were plated into the apical compartment of select wells in growth medium (z.e., in RGM). Two vials of cells were thawed, and the cell pellet was reconstituted in 11 mL of media. The 100% seeding density cells were plated. To plate the 90% seeding density group, 2 mL of the cell suspension was aliquoted, and then 220 pL of additional RGM was added.

[0331] TEER was measured and cells were monitored under a brightfield microscope daily throughout the experiment. The cells received one RGM media change on day 2 in culture before reaching confluence. On day 3 of culture, cells in both groups reached confluence. Media was changed to RMM media (z.e., a maturation medium) to drive differentiation of cells.6,4,1 Plating and Culturing of THP-1 Cells

[0332] On the same day that the epithelial cells reached confluence and switched to maturation media (z.e., RMM), THP-1 cells were plated onto a 96 well transwell receiver plate (basal compartment of transwells). This was done separately from the apical compartment containing the transverse colon epithelial cells until initiation of co-culture. THP-1 cells (passage 5) were81#623862Attorney Docket No. 00009.008.1801 collected into two 50 mL conical tubes from a T-225 flask where cells had been growing. Cells were spun down and resuspended, and then both cell suspensions were combined into one tube. The final volume of the cell suspension was 0.5 mL. Cells were counted using a hemocytometer. The final number of cells needed was 1,050,000, which allowed for 50 wells (5 extra, only 45 were plated) to be plated with 21,000 cells each. The cell suspension was brought to 50% of its final volume using THP-1 media with no PMA. The final volume was achieved by adding THP-1 media containing 200 mM PMA, for a final concentration of 100 mM PMA.

[0333] Cells were monitored daily throughout the experiment. On day 1 in culture, cells were observed to see if they had adhered to the bottom of the plate and differentiated. THP-1 media was then changed to remove the PMA.6.4.2 Dosing and Co-Culture

[0334] On day two in RMM for the epithelial cells and day two in culture for the THP-1 cells, priming, co-culture initiation, and dosing occurred.THP-1 cells alone were primed in THP-1 media (no PMA) with either vehicle, 100 ng / mL LPS + 25 ng / mL IFNy, or 100 ng / mL LPS + 25 ng / mL IFNy + 3 pg / mL Adalimumab + 100 pM Tofacitinib for four hours.After four hours, the apical compartment of the epithelial cell culture containing the epithelial cells was placed into the receiver plate containing the THP-1 cells, initiating the co-culture (T=0 hours). At this time, all media (apical and basal) was replaced with co-culture media with either vehicle, 100 ng / mL LPS + 25 ng / mL IFNy, or 100 ng / mL LPS + 25 ng / mL IFNy + 3 pg / mL Adalimumab + 100 pM Tofacitinib.

[0335] TEER was measured every 24 hours starting at T=0 hours until the final timepoint at T=48 hours6.4.3 Endpoints

[0336] At T=48 hours, apical and basal supernatants were collected individually in 96 well supernatant collection plates and stored at -80 °C.

[0337] A luminescent cell viability assay was performed. The assay reagent was mixed 1: 1 with cell culture media. 100 pL of this mixture was added to the apical and basal compartments of wells where cells were present (TC only wells did not have it added to basal compartment; THP-1 only wells did not have it added to apical compartments). This was allowed to incubate at room temperature in the dark for 10 minutes. At this time, 40 pL of the solution was then transferred out of each well in duplicate into white flat-bottom 96 well assay plates. Then luminescence was read on a plate reader82#623862Attorney Docket No. 00009.008.1801 6.5 Results

[0338] The percent change in TEER as compared to vehicle at 48 hours post-treatment for the test conditions described hereinabove is shown in FIG. 26. The results show that TEER dropped slightly in all transverse colon monoculture groups with treatment and with treatment and inhibitors. This small drop also occurred in the co-culture group that was dosed in RMM. A significant decrease in TEER occurred with LPS and IFNy treatment in the co-culture groups dosed in co-culture media. Treatment of LPS and IFNy and with the inhibitors adalimumab and tofacitinib brought the percentage change in TEER almost back to baseline.The results of the luminescence assay for cells from the apical compartment ( / .<., transverse colon (TC) epithelial cells) are shown in FIG. 27.

[0339] The results of the luminescence viability assay for cells from the basal compartment ( / .<., THP-1 cells) are shown in FIG. 28.6.6 Conclusions

[0340] THP-1 cell monocultures showed a slight decrease in cell activity with LPS and I Ny treatment compared to vehicle (FIG. 28). This occurred both in treatment and treatment with inhibitor groups. Through this, it appears that the 100 ng / mL LPS and 25 ng / mL IFNy four hour priming and 100 ng / mL LPS and 0.5 ng / mL IFNy dosing had some effect on the THP-1 cell activity. This was not mitigated with the addition of the inhibitors adalimumab and tofacitinib.

[0341] In transverse colon epithelial cell monocultures, treatment of cells with 100 ng / mL LPS and 0.5 ng / mL IFNy did not affect the barrier integrity or cell activity of the cells. No significant drop in TEER or luminescence in comparison to vehicle was observed in these groups (FIG. 26 and FIG. 27). These results indicate that the TC cells were not affected by the 100 ng / mL LPS and 0.5 ng / mL IFNy treatment at T=0 hours.

[0342] In THP-1 and TC co-cultures, the THP-1 cells were slightly affected by the addition of compounds and compounds with inhibitors, but this decrease in luminescence was the same as the decrease in the THP-1 only monocultures, which indicated that the TC cells did not affect the activity of the THP-1 cells in co-culture. However, in the co-cultures, the barrier integrity and cell activity of the TC cells was significantly affected by the LPS and IFNy treatment. Since they were not affected by this same treatment in monoculture, this was due to the release of cytokines such as TNFa by the THP-1 cells upon stimulation with the compounds. Additionally, this effect was not seen with compound treatment with the addition of inhibitors, likely due to the fact that tofacitinib mitigated the effect of IFNy on the THP-1 cells, and adalimumab mitigated the effect of TNFa that was released by the THP-1 cells upon inflammatory stimulation.83#623862Attorney Docket No. 00009.008.1801

[0343] This example was successful in causing inflammatory effects such as loss of barrier integrity and decreased cellular activity in transverse colon epithelial cells through immune cell signaling. Treatment with the priming and dosing compounds to cause immune cell stimulation did not influence the transverse colon cells alone; however, when paired with the immune cells, the downstream release of cytokines by the THP-1 cells caused inflammation of the epithelial cell layer. Additionally, in co-cultures treated only with vehicle, both cell types remained stable and healthy. Thus, this example demonstrated a successful attempt at culturing a stable immune-epithelial cell co-culture, and also at culturing an inflamed immune-epithelial cell co-culture through immune cell stimulation and downstream initiation of inflammationExample 7: Evaluation of response of transverse colon (TC) epithelial cells to stimulated macrophages and lower does of LPS7.1 Objective

[0344] The objectives of the experiments described in this example were: (1) to assess the response of Donor 1 (DI) transverse colon (TC) cells to stimulated (LPS+ IFNy) macrophages in one lot of TC cells (cell lot A) and to compare the results with data obtained from a separate lot of TC cells (cell lot B); (2) to evaluate the effects of lower doses of LPS (10 and 25 ng / mL), in addition to the previously used concentration of 100 ng / mL LPS, on both cell lots; and (3) to compare the response of TC cells to stimulated macrophages on Day 2 versus Day 3 in RMM.7.2 Cell information

[0345] The following cell types were used in this example: (1) human intestinal transverse colon epithelial cells, with cell lot A and cell lot B both at passage 10, and (2) THP-1 cells at passage 5.7.3 Conditions Tested

[0346] THP-1 cells (passage 5) from this example were pretreated with three combinations of concentrations of LPS and IFNy 4 hours prior to co-culture with TC cells. The three combinations of concentrations that were used were as follows: 100 ng / mL LPS and 25 ng / mL fFNy (Pretreatment 1), 10 ng / mL LPS and 25 ng / mL fFNy (Pretreatment 2), and 25 ng / mL LPS and 25 ng / mL IFNy (Pretreatment 3).7.4 Experimental Procedures7,4,1 Plating and Culturing of Intestinal Epithelial Cells

[0347] Two TC cell lots, cell lot A and cell lot B, were evaluated in this example. Time to reach confluency (TTC) for cell lot A was Day 4 whereas TTC for cell lot B was Day 3. There were two conditions for co-culture: Day 3 in RMM and Day 2 in RMM on the day of co-culture with stimulated THP-l’s. The timeline for the entire co-culture assay is provided in Table 7, and was84#623862Attorney Docket No. 00009.008.1801 implemented to achieve the conditions for TC cells. Two different cell lots were used (cell lot A and cell lot B), each reaching confluency on separate days. For each cell lot, there was two groups of TC cells: one group was in Day 2 RMM, and the other in Day 3 RMM, during co-culture with stimulated macrophages. This timeline ensured that all endpoint assays for both cell lots and groups were conducted on the same day.Table 7.Day Cell lot A Cell lot B1 Coat2 Seed lot A cells Day 3 Coat3 Seed lot A cells Day 2 Seed lot B cells Day 3 Coat4 Seed lot B cells Day 256 RMM media change Day 37 RMM media change Day 289 Pretreatment and Co-culture1011 Assay

[0348] The apical compartment of two 96 transwell plates with 0.4 pm membrane pores was coated with 1% Matrigel, and the plate was incubated overnight at 37 °C.

[0349] The following day, human intestinal epithelial cells (cell lot A / cell lot B) were plated into the apical compartment of select wells in growth medium (RGM). Two vials of cells were thawed for cell lot A and 1 vial of cells were thawed for cell lot B on the day of seeding cells, and the cell pellet was reconstituted. 100% seeding density cells were plated.

[0350] TEER was measured and cells were monitored under a brightfield microscope daily throughout the experiment. The cells received RGM media changes every 2 days till they reached confluence. Cell lot A took 5 days to reach confluence and cell lot B took 3 days to reach confluence. Media was changed to maturation medium (RMM) to drive differentiation of cells.7,4,2 Plating and Culturing of THP-1 Cells

[0351] On the same day that the epithelial cells reached confluence and switched to maturation media, THP-1 cells were plated onto two 96 well transwell receiver plates (basal compartment of transwells). This was done separately from the apical compartment containing the transverse colon epithelial cells until initiation of co-culture. THP-1 cells (passage 5) were collected into two 50 mL conical tubes from a T-225 flask where cells had been growing. Cells were spun down and resuspended, and then both cell suspensions were combined into one tube. The final volume of the cell suspension was 1 mL. Cells were counted using a hemocytometer. The final number of cells85#623862Attorney Docket No. 00009.008.1801 needed was 21,000 cells / well. The calculated cell suspension (100 pL / well) was added to THP-1 media without mercaptoethanol containing 100 nM PMA.

[0352] Cells were monitored daily throughout the experiment. On day 1 in culture, cells were observed to see if they had adhered to the bottom of the plate and differentiated. PMA containing THP-1 media without mercaptoethanol was replaced with fresh THP-1 media without mercaptoethanol without PMA.7.4.3 Dosing and Co-Culture

[0353] On either day two or day three in RMM for the epithelial cells and day two of PMA differentiated THP-1 cells, pretreatment, co-culture initiation, and dosing was performed.

[0354] THP-1 cells alone were pretreated in THP-1 media without mercaptoethanol (no PMA) with either vehicle, 100 ng / mL LPS + 25 ng / mL IFNy with or without 100 pM Tofacitinib or 10 ng / mL LPS + 25 ng / mL fFNy with or without 100 pM Tofacitinib or 25 ng / mL LPS + 25 ng / mL IFNy with or without 100 pM Tofacitinib.

[0355] After four hours, the media from the apical compartment of the epithelial cell culture containing the epithelial cells and media from the pretreated THP-1 cells was aspirated. Then, the apical compartment containing epithelial cells was placed into the receiver plate containing the pretreated PMA differentiated THP-1 cells, initiating the co-culture (T=0 hours). At this time, coculture media (RMM-EGF-NAC) media (apical and basal) with either vehicle, 100 ng / mL LPS + 0.5 ng / mL IFNy with or without 100 pM Tofacitinib or 10 ng / mL LPS + 0.5 ng / mL FFNy with or without 100 pM Tofacitinib or 25 ng / mL LPS + 0.5 ng / mL IFNy with or without 100 pM Tofacitinib.

[0356] TEER was measured every 24 hours starting at T=0 hours until the final timepoint at T=48 hours7.4.4 Endpoints

[0357] At T=48 hours, apical and basal supernatants were collected individually in 96 well supernatant collection plates and stored at -80 °C.

[0358] A luminescent cell viability assay was performed. The assay reagent was mixed 1: 1 with cell culture media. 100 pL of this mixture was added to the apical and basal compartments of wells where cells were present (TC only wells did not have it added to basal compartment; THP-1 only wells did not have it added to apical compartments). This was allowed to incubate at room temperature in the dark for 10 minutes. At this time, 40 pL of the solution was then transferred out of each well in duplicate into white flat-bottom 96 well assay plates. Then luminescence was read on a plate reader.86#623862Attorney Docket No. 00009.008.1801 7.5 Results

[0359] The TEER curves for the THP-l / TC cell lot A co-culture and TC cell lot A monoculture, where TC cells were in Day 2 of RMM at beginning of co-culture, are shown in FIG. 29. The TEER curves for the THP-l / TC cell lot B co-culture and TC cell lot B monoculture, where TC cells were in Day 2 of RMM at beginning of co-culture, are shown in FIG. 30. The TEER curves for the THP-l / TC cell lot B co-culture and TC cell lot B monoculture, where TC cells were in Day 3 of RMM at beginning of co-culture, are shown in FIG. 31. The percentage change in TEER for TC cell lot A (Day 2 in RMM) and TC cell lot B (Day 2 and Day 3 in RMM) from FIGs 29-30 is shown in FIG.32. For FIGs.29-32, co-culture with THP-1 cells were initiated when both TC cells lots were either in Day 2 or Day 3 in RMM. The results show that in both the cell lots, a slight decrease in TEER was observed across all transverse colon monoculture groups treated with or without inhibitors. However, a significant reduction in TEER occurred with all three combinations of LPS and IFNy treatments in co-culture groups dosed in co-culture media (RMM without EGF and without NAC, z.e., RMM-EGF-NAC). Treatment with LPS and IFNy, along with the Tofacitinib inhibitor, restored the TEER curve almost back to the baseline.

[0360] The results of the luminescence assay from the apical and basal compartments of TC cell lot B, where ATP activity was measured for cell lot B transverse colon (TC) epithelial cells in mono and co-cultures pretreated with one of three concentrations of LPS and IFNy (10+25, 25+25, 100+25 ng / mL) or with LPS and IFNy (100+25 ng / mL) together with Tofa, are shown in FIG.33.

[0361] The results of the luminescence assay from the apical and basal compartments of TC cell lot A, where ATP activity was measured for cell lot A transverse colon (TC) epithelial cells in mono and co-cultures pretreated with one of three concentrations of LPS and IFNy (10+25, 25+25, 100+25 ng / mL), are shown in FIG. 34.7.6 Conclusions

[0362] One of the objectives of this experiment was to evaluate two different DI TC cell lots and examine two differentiation points (Day 2 and Day 3 in RMM) at the time of co-culture. However, cell lot A reached confluency later than expected, so the experiment was done with only Day 2 in RMM to stay within the proposed timeline. On the other hand, cell lot B reached confluency as planned, allowing for testing on both Day 2 and Day 3 in RMM.

[0363] For both cell lots, across both days of RMM, TEER values dropped significantly at 48 hours with three combinations of concentrations of LPS and IFNy. This drop was prevented by the addition of Tofacitinib treatment, indicating that the cells responsiveness to THP-1 stimulated macrophages was not specific to one cell lot, which suggests that other cell lots may respond similarly to stimulated macrophages. Furthermore, TC cells on either Day 2 or Day 3 in RMM87#623862Attorney Docket No. 00009.008.1801 responded similarly to co-culture stimulation, indicating that working with cell lots at either differentiation time point is feasible.

[0364] In transverse colon epithelial cell monocultures, treating cells with 100, 25, or 10 ng / mL LPS and 0.5 ng / mL IFNy did not affect barrier integrity or cell activity. No significant drop in TEER or decrease in luminescence was observed compared to vehicle control (FIGs. 33-34), indicating that these concentrations of LPS and IFNy did not impact the TC cells.

[0365] THP-1 monocultures showed a slight decrease in cell activity with LPS and IFNy treatment compared to vehicle control in the cell lot A (FIG. 34), though this decrease occurred only in treatment groups, not in inhibitor-treated groups. The reduction in viable THP-1 cells in the treatment groups aligned with previous experiments.

[0366] In the cell lot B co-culture with THP-1 and TC cells, the number of viable TC cells decreased with treatment, but this was mitigated by Tofacitinib (FIG.33). Day 2 RMM cells were more sensitive than Day 3 cells, as shown by a greater decrease in viability on Day 2. Despite this, TEER values dropped equally on both days, indicating similar responsiveness to THP-1 stimulation at both time points.

[0367] For the cell lot A co-culture, the number of viable TC cells decreased with treatment (FIG. 34) compared to monocultures. Tofacitinib protected TC cells from the co-culture stimulation as the number of viable cells in the inhibitor-treated groups were comparable to vehicle-treated groups.

[0368] This examples demonstrates the successful induction of inflammatory effects, such as loss of barrier integrity and decreased cellular activity in transverse colon epithelial cells, through immune cell signaling in both cell lots (cell lot A and cell lot B). TC cells in both Day 2 and Day 3 RMM responded similarly to THP-1 stimulation. Lower LPS concentrations affected barrier integrity, causing TEER values to drop, suggesting that lower LPS doses may also be suitable.

[0369] The foregoing examples are illustrative of the present disclosure and are not to be construed as limiting thereof. Although the disclosure has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the disclosure as described and defined in the following claims.88#623862

Claims

Attorney Docket No. 00009.008.1801CLAIMS1. A method for co-culturing intestinal epithelial cells and immune cells, comprising:(a) culturing the intestinal epithelial cells in an expansion medium followed by a differentiation medium;(b) culturing the immune cells in a first cell culture medium comprising phorbol 12- myri state- 13 -acetate (PMA); and(c) combining the intestinal epithelial cells from (a) and the immune cells from (b) in a co-culture cell construct comprising a first co-culture medium.

2. The method of claim 1, wherein the intestinal epithelial cells are seeded on a hydrogel layer.

3. The method of claim 2, wherein the hydrogel layer is Matrigel, optionally about 1% Matrigel.

4. The method of any one of claims 1-3, wherein the intestinal epithelial cells are grown to confluence prior to being combined with the immune cells in the co-culture cell construct.

5. The method of any one of claims 1-4, wherein the intestinal epithelial cells are grown to confluence in the expansion medium prior to being combined with the immune cells in the coculture cell construct.

6. The method of any one of claims 1-5, wherein the expansion medium is exchanged for the differentiation medium once the intestinal epithelial cells reach confluence.

7. The method of any one of claims 1-6, wherein the intestinal epithelial cells are primary intestinal epithelial cells.

8. The method of any one of claims 1-7, wherein the immune cells are cultured in suspension prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

9. The method of any one of claims 1-8, wherein the immune cells are seeded at a density from up to a range of about 7xl03cells / 12.6 cm2to about 1.12xl05cells / 12.6 cm2in the first cell culture medium.

10. The method of any one of claims 1-9, wherein the first cell culture medium does not comprise mercaptoethanol.

11. The method of any one of claims 1-10, wherein the first cell culture medium comprises Roswell Park Memorial Institute 1640 (RPMI-1640) medium.

12. The method of any one of claims 1-11, wherein the first cell culture medium comprises from up to a range of about 1 nM to 10 pM of the PMA.

13. The method of any one of claims 1-12, wherein the immune cells are cultured in the first cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.89#623862Attorney Docket No. 00009.008.1801 14. The method of any one of claims 1-13, further comprising removing the first cell culture medium and providing a second cell culture medium that does not contain PMA.

15. The method of claim 14, wherein the second cell culture medium does not contain mercaptoethanol.

16. The method of any one of claims 14-15, wherein the immune cells are cultured in the second cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

17. The method of any one of claims 1-16, wherein the immune cells are exposed to one or more immune cell-differentiating and / or stimulating agents prior to being combined with the intestinal epithelial cells in the co-culture cell construct, wherein the one or more immune celldifferentiating and / or stimulating agents is selected from the group consisting of: lipopolysaccharide (LPS), interferon gamma (IFN-y), interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT).

18. The method of any one of claims 1-16, wherein the immune cells are exposed to lipopolysaccharide (LPS) and / or interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

19. The method of claim 18, wherein the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

20. The method of any one of claims 18-19, wherein the immune cells are exposed to from up to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS) and / or from up to a range of about 0.5 ng / mL to about 25 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

21. The method of any one of claims 1-20, wherein the immune cells are exposed to interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM- CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

22. The method of claim 21, wherein the immune cells are exposed to IL-4, CRP, GM-CSF, Zymosan, TNFa, Nigericin, and / or CT for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

23. The method of any one of claims 21-22, wherein the immune cells are exposed to from up to a range of about 5 ng / mL to about 100 ng / mL IL-4, from up to a range of about 5 pg / mL to about 100 pg / mL CRP, from up to a range of about 1 ng / mL to about 1000 ng / mL GM-CSF, from90#623862Attorney Docket No. 00009.008.1801 up to a range of about 1 pg / mL to about 1000 pg / mL Zymosan, from up to a range of about 0.25 ng / mL to about 100 ng / mL of TNFa, from up to a range of about 1 pM to about 100 pM Nigericin, and / or from up to a range of about 1 ng / mL to about 1000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

24. The method of any one of claims 18-23, wherein the immune cells are treated with one or more inhibitors.

25. The method of claim 24, wherein the one or more inhibitors is one or more cytokine inhibitors.

26. The method of claim 25, wherein the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X- C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G- CSF) inhibitor.

27. The method of claim 25, wherein the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor.

28. The method of any one of claims 26-27, wherein the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab.

29. The method of any one of claims 26-27, wherein the IFNY inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib.

30. The method of claim 26, wherein the IL-8 inhibitor is selected from the group consisting of:MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756).

31. The method of claim 26, wherein the IL-ip inhibitor is selected from the group consisting of:Rilonacept (Arcalyst) and Canakinumab (Haris).

32. The method of claim 26, wherein the IL-6 inhibitor is selected from the group consisting of:Tocilizumab (Actemra) and Sarilumab (Kevzara).

33. The method of claim 26, wherein the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi).

34. The method of claim 26, wherein the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418).

35. The method of any one of claims 26-34, wherein the TNFa inhibitor is adalimumab.

36. The method of any one of claims 26-35, wherein the IFNy inhibitor is tofacitinib.

37. The method of any one of claims 1-36, wherein the immune cells are Tohoku Hospital Pediatrics-1 (THP-1) cells.91#623862Attorney Docket No. 00009.008.1801 38. A method for co-culturing intestinal epithelial cells and immune cells comprising:(a) culturing the intestinal epithelial cells in an expansion medium followed by a differentiation medium;(b) culturing the immune cells in a first cell culture medium comprising phorbol 12- myri state- 13 -acetate (PMA); and(c) combining the intestinal epithelial cells from (a) and the immune cells from (b) in a co-culture cell construct comprising a first co-culture medium that does not contain growth factors.

39. The method of claim 38, wherein the first co-culture medium does not contain epidermal growth factor (EGF).

40. The method of any one of claims 38-39, wherein the first co-culture medium does not contain N-acetylcysteine (NAC).

41. The method of any one of claims 38-40, wherein the first co-culture medium comprises lipopolysaccharide (LPS) and / or interferon gamma (IFN-y).

42. The method of claim 41, wherein the first co-culture medium comprises from up to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS).

43. The method of any one of claim 41-42, wherein the first co-culture medium comprises about 10 ng / mL lipopolysaccharide (LPS).

44. The method of any one of claims 41-43, wherein the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 1 ng / mL interferon gamma (IFN-y).

45. The method of any one of claims 41-44, wherein the first co-culture medium comprises about 0.5 ng / mL interferon gamma (IFN-y).

46. The method of any one of claims 38-45, wherein the first co-culture medium comprises one or more transforming growth factor-beta (TGF-beta) / Smad inhibitors.

47. The method of claim 46, wherein the one or more TGF-beta / Smad inhibitors is selected from the group consisting of 3,3-dimethyl-l-butanol, A8301, AUDA, BIBF-0775, DMH1, dorsomorphin, dorsomorphin 2HC1, Galunisertib (LY2157299), ginsenoside Rh4, GW788388, halofuginone, ITD-1, K02288, LDN-193189, LDN-193189 2HC1, LDN-212854, LDN- 214117, LY2109761, LY 3200882, LY364947, lycopus extract, ML347, PD 169316, PF- 06952229, Pirfenidone, R-268712, RepSox (E-616452), SB431542, SB505124, SB525334, SD-208, SIS3, SIS3 HC1, sulfasalazine, TA-02, TGFpRI-IN-3, TP0427736 HC1, and Vactosertib (TEW-7197).

48. The method of claim 46 or 47, wherein the one or more TGF-beta / Smad inhibitors is A8301.92#623862Attorney Docket No. 00009.008.1801 49. The method of any one of claims 38-48, wherein the first co-culture medium comprises one or more inhibitors.

50. The method of claim 49, wherein the one or more inhibitors is one or more cytokine inhibitors.

51. The method of claim 50, wherein the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X- C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G- CSF) inhibitor.

52. The method of claim 50, wherein the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor.

53. The method of any one of claims 51-52, wherein the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab.

54. The method of any one of claims 51-52, wherein the IFNY inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib.

55. The method of claim 51 wherein the IL-8 inhibitor is selected from the group consisting of:MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756).

56. The method of claim 51 wherein the IL-ip inhibitor is selected from the group consisting of:Rilonacept (Arcalyst) and Canakinumab (Haris).

57. The method of claim 51 wherein the IL-6 inhibitor is selected from the group consisting of:Tocilizumab (Actemra) and Sarilumab (Kevzara).

58. The method of claim 51 wherein the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi).

59. The method of claim 51 wherein the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418).

60. The method of any one of claims 51-59, wherein the TNFa inhibitor is adalimumab.

61. The method of any one of claims 51-60, wherein the IFNy inhibitor is tofacitinib.

62. A co-culture cell construct produced by the method of any one of the preceding claims, the coculture cell construct comprising:(a) a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well, the first well containing the first co-culture medium;(b) a luminal container held within the first well of the basal container and comprising:93#623862Attorney Docket No. 00009.008.1801 (i) a second porous wall; and(ii) at least one side wall extending upwardly from the second wall to define a second well, the second well also containing the first co-culture medium; andwherein the immune cells are cultured in the first well of the basal container and the intestinal epithelial cells are cultured in the second well of the luminal container.

63. A method for producing a co-culture cell construct comprising a co-culture of intestinal epithelial cells and immune cells, the method comprising:(a) providing an apparatus, the apparatus comprising a basal container comprising a first wall and at least one side wall extending upwardly from the first wall to define a first well; and a luminal container capable of being held within the first well of the basal container;(b) culturing the intestinal epithelial cells and the immune cells separately, wherein:(i) the intestinal epithelial cells are cultured in the luminal container and in an expansion medium followed by a differentiation medium; and(ii) the immune cells are cultured in the basal container and in a first cell culture medium comprising phorbol 12-myri state- 13 -acetate (PMA) followed by a second cell culture medium that does not contain PMA; (c) replacing the differentiation medium in the luminal container and the second cell culture medium in the basal container with a first co-culture medium; and(d) providing the luminal container into the basal container so that the luminal container is held within the first well of the basal container and the intestinal epithelial cells and the immune cells are combined in the co-culture;thereby generating the co-culture cell construct comprising the co-culture of the intestinal epithelial cells and immune cells.

64. The method of claim 63, wherein the intestinal epithelial cells are seeded on a porous hydrogel layer in the luminal container, optionally wherein the hydrogel layer is Matrigel, further optionally about 1% Matrigel.

65. The method of any one of claims 63-64, wherein the intestinal epithelial cells are grown to confluence in expansion medium prior to being combined with the immune cells in the coculture cell construct.

66. The method of any one of claims 63-65, wherein the expansion medium is exchanged for the differentiation medium once the intestinal epithelial cells reach confluence.94#623862Attorney Docket No. 00009.008.1801 67. The method of any one of claims 63-66, wherein the intestinal epithelial cells are primary intestinal epithelial cells.

68. The method of any one of claims 63-67, wherein the immune cells are cultured in suspension in the first cell culture medium and / or in the second cell culture medium prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

69. The method of any one of claims 63-68, wherein the immune cells are seeded at a density from up to a range of about 7xl03cells / 12.6 cm2to about 1.12xl05cells / 12.6 cm2in the first cell culture medium.

70. The method of any one of claims 63-69, wherein the first cell culture medium does not comprise mercaptoethanol.

71. The method of any one of claims 63-70, wherein the first cell culture medium comprises Roswell Park Memorial Institute 1640 (RPMI-1640) medium.

72. The method of any one of claims 63-71, wherein the first cell culture medium comprises about 1 nM to 10 pM of the PMA.

73. The method of any one of claims 63-72, wherein the immune cells are cultured in the first cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

74. The method of any one of claims 63-73, wherein the second cell culture medium does not contain mercaptoethanol.

75. The method of any one of claims 73-74, wherein the immune cells are cultured in the second cell culture medium for a period of up to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

76. The method of any one of claims 63-75, wherein the immune cells are exposed to one or more immune cell-differentiating and / or stimulating agents prior to being combined with the intestinal epithelial cells in the co-culture cell construct, wherein the one or more immune celldifferentiating and / or stimulating agents is selected from the group consisting of: lipopolysaccharide (LPS), interferon gamma (IFN-y), interleukin-4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM-CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT).

77. The method of any one of claims 63-76, wherein the immune cells are exposed to lipopolysaccharide (LPS) and / or interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.95#623862Attorney Docket No. 00009.008.1801 78. The method of claim 77, wherein the immune cells are exposed to lipopolysaccharide (LPS) and interferon gamma (IFN-y) for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

79. The method of any one of claims 77-78, wherein the immune cells are exposed to from up to a range of about 10 ng / ml to about 1000 ng / mL lipopolysaccharide (LPS) and / or from up to a range of about 0.5 ng / mL to about 25 ng / mL interferon gamma (IFN-y) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

80. The method of any one of claims 63-79, wherein the immune cells are exposed to interleukin- 4 (IL-4), C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM- CSF), Zymosan, Tumor Necrosis Factor-alpha (TNFa), Nigericin, and Cholera toxin (CT) prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

81. The method of claim 80, wherein the immune cells are exposed to IL-4, CRP, GM-CSF, Zymosan, TNFa, Nigericin, and / or CT for a period of from about 4 hours to about 24 hours prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

82. The method of any one of claims 80-81, wherein the immune cells are exposed to from up to a range of about 5 ng / mL to about 100 ng / mL IL-4, from up to a range of about 5 pg / mL to about 100 pg / mL CRP, from up to a range of about 1 ng / mL to about 1000 ng / mL GM-CSF, from up to a range of about 1 pg / mL to about 1000 pg / mL Zymosan, from up to a range of about 0.25 ng / mL to about 100 ng / mL of TNFa, from up to a range of about 1 pM to about 100 pM Nigericin, and / or from up to a range of about 1 ng / mL to about 1000 ng / mL CT prior to being combined with the intestinal epithelial cells in the co-culture cell construct.

83. The method of any one of claims 77-82, wherein the immune cells are treated with one or more inhibitors.

84. The method of claim 83, wherein the one or more inhibitors is one or more cytokine inhibitors.

85. The method of claim 84, wherein the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X- C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G- CSF) inhibitor.

86. The method of claim 84, wherein the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor.

87. The method of any one of claims 85-86, wherein the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab.96#623862Attorney Docket No. 00009.008.1801 88. The method of any one of claims 85-86, wherein the IFNy inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib.

89. The method of claim 85, wherein the IL-8 inhibitor is selected from the group consisting of:MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756).

90. The method of claim 85, wherein the IL-ip inhibitor is selected from the group consisting of:Rilonacept (Arcalyst) and Canakinumab (Haris).

91. The method of claim 85, wherein the IL-6 inhibitor is selected from the group consisting of:Tocilizumab (Actemra) and Sarilumab (Kevzara).

92. The method of claim 85, wherein the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi).

93. The method of claim 85, wherein the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercimon (TRA-418).

94. The method of any one of claims 83-93, wherein the TNFa inhibitor is adalimumab.

95. The method of any one of claims 83-94, wherein the IFNy inhibitor is tofacitinib.

96. The method of any one of claims 63-95, wherein the immune cells are Tohoku Hospital Pediatrics-1 (THP-1) cells.

97. The method of any one of claims 63-96, wherein the first co-culture medium does not contain any growth factors.

98. The method of any one of claims 63-97, wherein the first co-culture medium does not contain epidermal growth factor (EGF).

99. The method of any one of claims 63-98, wherein the first co-culture medium does not contain N-acetylcysteine (NAC).

100. The method of any one of claims 63-99, wherein the first co-culture medium comprises lipopolysaccharide (LPS) and / or interferon gamma (IFN-y).

101. The method of claim 100, wherein the first co-culture medium comprises from up to a range of about 10 ng / mL to about 1000 ng / mL lipopolysaccharide (LPS).

102. The method of any one of claim 100-101, wherein the first co-culture medium comprises about 10 ng / mL lipopolysaccharide (LPS).

103. The method of any one of claims 100-102, wherein the first co-culture medium comprises from up to a range of about 0.1 ng / mL to about 1 ng / mL interferon gamma (IFN-y).

104. The method of any one of claims 100-103, wherein the first co-culture medium comprises about 0.5 ng / mL interferon gamma (IFN-y).97#623862Attorney Docket No. 00009.008.1801 105. The method of any one of claims 100-104, wherein the first co-culture medium comprises one or more TGF-beta / Smad inhibitors.

106. The method of claim 105, wherein the one or more TGF-beta / Smad inhibitors is selected from the group consisting of 3,3-dimethyl-l-butanol, A8301, AUDA, BIBF-0775, DMH1, dorsomorphin, dorsomorphin 2HC1, Galunisertib (LY2157299), ginsenoside Rh4, GW788388, halofuginone, ITD-1, K02288, LDN-193189, LDN-193189 2HC1, LDN-212854, LDN- 214117, LY2109761, LY 3200882, LY364947, lycopus extract, ML347, PD 169316, PF- 06952229, Pirfenidone, R-268712, RepSox (E-616452), SB431542, SB505124, SB525334, SD-208, SIS3, SIS3 HC1, sulfasalazine, TA-02, TGFpRI-IN-3, TP0427736 HC1, and Vactosertib (TEW-7197).

107. The method of any one of claim 106, wherein the one or more TGF-beta / Smad inhibitors is A8301.

108. The method of any one of claims 63-107, wherein the first co-culture medium comprises one or more inhibitors.

109. The method of claim 108, wherein the one or more inhibitors is one or more cytokine inhibitors.

110. The method of claim 109, wherein the one or more cytokine inhibitors is selected from the group consisting of: a Tumor Necrosis Factor-alpha (TNFa) inhibitor, an interferon-gamma (IFNy) inhibitor, an interleukin-8 (IL-8) inhibitor, an interleukin- 1 beta (IL-ip) inhibitor, an interleukin-6 (IL-6) inhibitor, an interleukin- 12 / interleukin-23 (IL-12 / IL-23) inhibitor, a C-X- C motif chemokine 11 (CXCL11) inhibitor, and a Granulocyte Colony-Stimulating Factor (G- CSF) inhibitor.

111. The method of claim 109, wherein the one or more cytokine inhibitors is selected from the group consisting of: a TNFa inhibitor and a IFNy inhibitor.

112. The method of any one of claims 110-111, wherein the TNFa inhibitor is selected from the group consisting of: adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab.

113. The method of any one of claims 110-111, wherein the IFNY inhibitor is selected from the group consisting of: AMG 811, baricitinib, filgotinib, fontolizumab, oclacitinib, ruxolitinib, tofacitinib, and upadacitinib.

114. The method of claim 110, wherein the IL-8 inhibitor is selected from the group consisting of: MS-986253, Ab-8 (GSK2140935), and Danirixin (GSK1325756).

115. The method of claim 110, wherein the IL- 1 P inhibitor is selected from the group consisting of: Rilonacept (Arcalyst) and Canakinumab (Haris).98#623862Attorney Docket No. 00009.008.1801 116. The method of claim 110, wherein the IL-6 inhibitor is selected from the group consisting of: Tocilizumab (Actemra) and Sarilumab (Kevzara).

117. The method of claim 110, wherein the IL-12 / IL-23 inhibitor is selected from the group consisting of: Ustekinumab (Stelara) and Risankizumab (Skyrizi).

118. The method of claim 110, wherein the CXCL11 inhibitor is selected from the group consisting of: BMS-936559 (MDX-1106) and Vercirnon (TRA-418).

119. The method of any one of claims 110-118, wherein the TNFa inhibitor is adalimumab.

120. The method of any one of claims 110-119, wherein the IFNy inhibitor is tofacitinib.99#623862

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