Co-culture models and related methods

Abstract

The present disclosure relates to in vitro co-culture models and methods for generating such co-culture models. Co-culture models of this disclosure, and related methods, may include co- culturing or maintaining different cell types in a culture receptacle in a common culture medium. In a specific example, a co-culture model (and related methods) may include co- culturing or maintaining one, two, or more mesoderm lineage cell-types and at least one endoderm lineage cell-type in a culture receptacle in a common culture medium.

Description

CO-CULTURE MODELS AND RELATED METHODSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Provisional Patent Application No. 63 / 567,772 filed March 20, 2024, the entire content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to cell culture applications, and more specifically to in vitro cell culture applications combining different types of cells, as may derive or originate from different germ layer lineages.BACKGROUND

[0003] Biological tissues are complex, and comprise a diversity of cell types, including cells from different germ layers. However, in vitro or ex vivo culture models, such as for the purpose of drug discovery, toxicity testing, or disease modeling, typically do not account for the complexity of tissues. Rather, in vitro or ex vivo culture models employed for the foregoing purposes typically assay cells in isolation from the context of their native tissue. Indeed, culture conditions tend to be specific to the type of cell being cultured, raising issues where various cell types are intended to be co-cultured in a common culture condition.

[0004] Organoids represent one model where different cell types may be cultured in the same environment; however, this model is limited by the stochastic development of different cell types and an inability to incorporate relevant cell types of certain germ layers, such as immune cells. In addition, various engineering approaches (e.g. organ on a chip) endeavour to culture a cell type of interest while providing exposure to paracrine signals of different cell types. The foregoing systems are limited by an inability to predictably, simply, and / or modularly assemble different cell types of interest in a common culture condition.

[0005] Complex co-culture models are increasingly needed, such as to perform assays in a more physiologically relevant setting. However, the assembly and culture conditions of a simple, accessible, reproducible, modular, and relevant model remain as unresolved challenges. Therefore, there is a need for improved co-culture models, including how to assemble such models and the culture conditions for sustaining and assaying such models.SUMMARY

[0006] The present disclosure relates to co-culture models, such as in vitro co-culture models, and to methods of generating same.

[0007] In one aspect of this disclosure are provided methods of generating (in vitro) co-culture models. Methods of generating a co-culture model may comprise providing a substrate in a culture receptacle that separates the culture receptacle (or the substrate) into a first (e.g. apical) compartment and a second (e.g. basolateral) compartment, and co-culturing different cell types in a common culture medium.

[0008] Methods of this disclosure may comprise forming a cell barrier, such as by layering a first mesoderm lineage cell-type and at least one endoderm lineage cell-type in a first / apical compartment (as formed by a substrate). Relatedly, methods of this disclosure may comprise seeding a first mesoderm lineage cell-type in a first / apical compartment (as formed by a substrate), and culturing for a first culture period, then overlaying at least one endoderm lineage cell type in the first / apical compartment, and culturing for a second culture period.

[0009] Methods of this disclosure may comprise seeding a second mesoderm lineage cell type in a second / basolateral compartment (as formed by a substrate), and culturing for a third culture period.

[0010] Methods of this aspect may comprise culturing all or certain cell types of a co-culture model submerged in a common culture medium or at an air liquid interface exposed to a common culture medium. In one embodiment, a first mesoderm lineage cell-type and / or at least one endoderm lineage cell-type are submerged in a common culture medium in an apical compartment (as formed by a substrate). In one embodiment, a first mesoderm lineage celltype and / or at least one endoderm lineage cell-type are exposed to a common culture medium at an air liquid interface. In such an embodiment, the first mesoderm lineage cell-type and / or the at least one endoderm lineage cell-type may be adhered to a substrate, such as in the apical compartment.

[0011] Methods of generating a co-culture model may be performed in the absence of an exogenously added extracellular matrix or extracellular matrix protein(s). Thus, the different cell types of a co-culture model may be cultured or incubated in the absence of an exogenously added extracellular matrix or extracellular matrix protein(s). A cell type included in a co-culture model may produce / secrete an extracellular matrix and / or one or more extracellular matrix protein(s), which may support the attachment and / or culture of different cell types of the coculture model.

[0012] Methods of generating a co-culture model may comprise co-culturing or maintaining a co-culture for a period of time elapsing between 1 and 42 days.

[0013] In another aspect of this disclosure are provided in vitro co-culture models. A coculture model of this disclosure may comprise different cell types in a culture receptacle / well, which cells may be exposed to a common culture medium.

[0014] In any aspect, different cell types that may be co-cultured or comprised in a co-culture model may correspond to cell types derived or originating from different germ layers, such as from at least two germ layers. Different cell types may correspond to a first mesoderm lineage cell-type (as may be comprised in a monolayer) and at least one endoderm lineage cell-type (as may also be comprised in a monolayer). Different cell types may correspond to a first mesoderm lineage cell-type, a second mesoderm lineage cell-type, and at least one endoderm lineage cell-type.

[0015] A first mesoderm lineage cell-type of at least two mesoderm lineage cell-types in a coculture model may be a mesenchymal cell, such as a fibroblast. A second mesoderm lineage cell-type of at least two mesoderm lineage cell-types in a co-culture model may be an immune cell, such as an antigen-presenting cell. An endoderm lineage cell-type of a co-culture model may be an epithelial cell, such as an intestinal or airway epithelial cell. Epithelial cells may be derived or obtained from a primary tissue, from an organoid culture, or from a monolayer culture. If an intestinal epithelial cell, the intestinal cell may be derived or obtained from a small intestine or a colon, or from a culture of small intestine or colon cells (e.g. organoid or monolayer culture).

[0016] In one embodiment of a co-culture model, a first mesoderm cell type and / or at least one endoderm lineage cell-type are physically separated from a second mesoderm lineage cell type, such as by a substrate that separates a culture receptacle into a first / apical compartment and a second / basolateral compartment. A substrate may separate a culture receptacle / well into a first / apical compartment and a second / basolateral compartment, the first and second compartments separated by a porous member (of the substrate).

[0017] One or more cell types of a co-culture model may be submerged in a common culture medium, such as within a first / apical or a second / basolateral compartment. One or more cell types of a co-culture model may be submerged in a common culture medium in a second / basolateral compartment, and one or more cell types of a co-culture model may either be i) submerged in a common culture medium in a first / apical compartment, or ii) exposed to a common culture medium at an air liquid interface (in the first / apical compartment). In one embodiment, a first mesoderm lineage cell type and at least one endoderm lineage cell type may be comprised in a first / apical compartment (as formed by a substrate) and exposed to a common culture medium at an air liquid interface, and a second mesoderm lineage cell-type may be comprised in a second / basolateral compartment (as formed by a substrate) and submerged in the common culture medium.

[0018] An in vitro co-culture model may be free of an exogenously added extracellular matrix or extracellular matrix protein(s). Nevertheless, an in vitro co-culture model may comprise anextracellular matrix or extracellular matrix protein(s) as produced / secreted by one or more cell types of the in vitro co-culture model (e.g. a mesenchymal cell, such as a fibroblast).

[0019] In any aspect, a substrate may comprise a porous member, such as to permit exchange of signals between apical and basolateral compartments. Substrates may correspond to a cell culture insert (e.g. a Transwell™ insert). A substrate may be removable from a culture well or a culture receptacle, or transferable from one culture well / receptacle to another.

[0020] In any aspect, common culture media may comprise one or more of an agonist of Wnt signaling, an inhibitor of BMP signaling, and a mitogen.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0022] Figure 1 shows the establishment of a co-culture model comprising epithelial lineage cells (ECs) and mesenchymal lineage cells (IFs), and the characterization of the structure and function of an arising EC-IF barrier. Panel A) shows a line graph of mean (n=3) trans-epithelial electrical resistance (TEER) measurements performed on 4 independent (gut mucosa) models, represented by the pairwise combinations of EC and IF from two donors. TEER measurements were normalized to the surface area of the cell culture inserts (0.332 cm2) and plotted as Q cm2over time (days). Panel B) shows representative immunofluorescence images from 4 independent models of Zonula Occludens-1 (ZO-1) expression on day 14, highlighting a continuous and uniform network of tight junctions (indicated by arrows). Scale bars = 100 pm. Panel C) shows a graph summarizing the results of a FITC-dextran 4 kDa (FD4) permeability assay of a day- 14 model (n = 8). Error bars represent the mean ± SD. Panel D) shows representative immunofluorescence images of Collagen Type I and Collagen Type III expression (indicated by arrows) in two different models. Scale bars = 500 pm.

[0023] Figure 2 shows the establishment of a co-culture model comprising epithelial lineage cells, mesenchymal lineage cells, and immune cells, and the characterization of its structure and function. Viability of day-14 EPCAM+EC and CD90+IF cells was assessed by flow cytometry (A), n = 4; error bars represent the mean ± SD. Viability of day-14 immune cells cultured in the presence or absence of maturation conditions (±MAT) was assessed by acridin orange (AO) and DAPI staining (B), n = 8; error bars represent mean ± SD. Viability of day 14 macrophages (M<t>) of the co-culture - either control (M0), M1-like, or M2-like - wasassessed by acridin orange (AO) and DAPI staining (C), n = 2; error bars represent the mean ± SD.

[0024] Figure 3 shows the establishment of a co-culture model comprising epithelial lineage cells, mesenchymal lineage cells, and immune cells, and the characterization of its structure and function. Representative immunofluorescence images of day-14 CD45 expression (indicated by arrows) among DCs of 4 independent models (A). Scale bars = 250 pm. Representative immunofluorescence images of day-14 CD45, CD86 or CD206 expression levels (indicated by arrows) among cultured macrophages (M<t>) of a co-culture model (B). Scale bars = 50 pm. Flow cytometry analysis of CD80, CD83, CD86, and CD108 expression levels among cultured DCs of a co-culture model (C), n = 8; error bars represent the mean ± SD. Secretion of IL-12p40 by DCs of the co-culture model in the presence or absence of maturation conditions (±MAT), as assessed by ELISA (D), n = 8; error bars represent the mean ± SD. Flow cytometry analysis of CD45, CD80, CD86, CD206, and CD209 expression levels among MO, M1-like, and M2-like M<t> of the co-culture model (E), n = 4; error bars represent the mean ± SD.DETAILED DESCRIPTION

[0025] This disclosure relates to in vitro co-culture models, and to methods of generating in vitro co-culture models. Co-culture models of this disclosure, and related methods, may comprise cell types derived or originating from different germ layers, such as from at least two of the three germ layers. Co-culture models of this disclosure, and related methods, may comprise at least two mesoderm lineage cell-types and at least one endoderm lineage celltype.

[0026] Where used in this disclosure, the terms “co-culture model” or “co-culture system”, used interchangeably herein, refer to a culture of more than one cell type, in a common culture condition (e.g. medium) and / or in communication with one another. In vitro co-culture models may more closely mimic or replicate an in vivo (micro)environment specific to an individual or a patient (relative to traditional monoculture conditions, or organoid culture systems). In an in vitro co-culture model, different cell types, such as of different germ layer origins, are cultured or incubated in close proximity (e.g. direct or indirect contact) while remaining viable and functional, and retaining identity. For indirect approaches, cells are physically separated in two different compartments, such as via cell culture inserts (e.g. Transwell™ inserts) and / or overflow culture chambers that allow communication only via paracrine signaling (e.g. secretory factors). For direct approaches, cell-cell contact or interactions are unimpeded, but may be achieved by spatially controlling the positions of adherent cells within a culture dish. A co-culture model of this disclosure may comprise a direct approach, or a hybrid betweendirect and indirect approaches. A co-culture model may comprise a first cell type of a first germ layer and a second cell type of a second germ layer. A co-culture model of this disclosure may further comprise a third cell type, whether of the first or second germ layer, or otherwise. More specifically, a co-culture model of this disclosure may comprise a first cell type of mesoderm origin and a second cell type of endoderm-origin, and may further comprise a second cell type of mesoderm origin.

[0027] Where used in this disclosure, the term “gut mucosa model” refers to a co-culture model comprising more than one cell type typically found in the gut, which may closely mimic the structure of the in vivo intestinal mucosa in terms of three-dimensional architecture, apical- basolateral polarity, well-formed tight / adherent junctions, mucous secretion, multiple epithelial cell types, and / or functional antigen-presenting cells. Cell types typically found in the gut include, but are not limited to, various epithelial cell types, fibroblasts and / or other mesenchymal cell types, and one or more immune cell types. A gut mucosa model may be formed / generated by establishing endoderm origin cells (e.g. intestinal epithelial cells) on a surface of a sub-epithelial-like tissue construct, such as a monolayer of mesoderm origin cells (e.g. mesenchymal cells and / or (intestinal) fibroblasts). In such conditions, endogenous production of an extracellular matrix or extracellular matrix proteins by mesenchymal cells (e.g. fibroblasts) may provide signals supportive of intestinal epithelium. In one embodiment, a gut mucosa model may comprise a first cell type of mesoderm origin (e.g. mesenchymal cells or intestinal fibroblasts) and a second cell type of endoderm-origin (e.g. intestinal epithelial cells), and may further comprise a second cell type of mesoderm origin (e.g. immune cells). Likewise, a co-culture model may be an airway mucosa model when comprising airwayrelevant cells.

[0028] Where used in this disclosure, the term “culture media” or “organoid media” refers to cell culture media that can support (e.g., maintain, expand and / or differentiate) at least one cell type of an assembled co-culture model, and preferably more than one cell type of an assembled co-culture model. Culture media may comprise a basal medium. Basal media are well known in the art and are routinely formulated to include one or more of salt(s), amino acid(s), carbohydrate(s), buffer(s), trace elements, etc. Examples of commercially available basal media include DMEM, Adv-DMEM, DMEM / F-12, RPMI, Iscoves, and numerous others. Basal media may require appropriate supplementation, such as with cytokines / growth factors, small molecules, serum / albumin, and / or lipids, depending on the application and type of cell(s) to be cultured. In a specific embodiment, culture media of this disclosure may comprise one or more of, two or more of, or each of: a mitogen (e.g. an FGF and / or an EGF); a protein or small molecule inhibitor of signaling through a bone morphogenic protein (e.g. Noggin, LDN193189, Dorsomorphin); and a protein or small molecule agonist of Wnt signaling (e.g.an R-Spondin, CHIR99021). In the context of a co-culture model of this disclosure, culture media may support an assembled co-culture model comprising a first mesoderm lineage celltype (e.g. mesenchymal cells, fibroblasts) and at least one endoderm lineage cell-type (e.g. epithelial cells), and preferably a second mesoderm lineage cell-type (e.g. immune cells)Methods

[0029] In one aspect of this disclosure are provided methods for generating in vitro co-culture models comprising component cell types, to model tissues or biological structures. Co-culture models of this disclosure may comprise cell types derived or originating from different germ layers. Upon generating an in vitro co-culture model in accordance with the methods of this disclosure, methods may further comprise a downstream application / assay step, including assessing responsiveness to a chemical or biological insult, or a preventative or a therapeutic modality.

[0030] Methods of generating an in vitro co-culture model may comprise providing a substrate in a culture well / receptacle, and co-culturing at least two different cell types in a common culture medium.

[0031] Receptacles that may be used in methods of generating in-vitro co-cultures are not particularly limited provided they may contain a liquid, support a cell culture, and be divided into different sub-compartments, such as by a substrate disposed therein. Such receptacles are well known and are commercially available. For example, an appropriate receptacle may correspond to a dish, (micro)plate, flask, or the like. Where used in this disclosure, the term culture receptacle may be used interchangeably with the term culture well (such as of a microplate or dish).

[0032] When disposed in a culture receptacle, a substrate may separate the receptacle into at least two distinct sub-compartments. Preferably, a substrate comprises a porous member, to facilitate communication (e.g. paracrine signaling) between the distinct sub-compartments formed by the substrate, such as a membrane or filter. An exemplary substrate may include, but is not limited to a cell culture insert (e.g. a Transwell™ insert).

[0033] A porous member comprised in a substrate may support the adhesion / growth of cells, while permitting the flow of signaling and other molecules / factors therethrough. Thus, a substrate (comprising a porous member) may be well suited to separating a culture receptacle into two compartments (e.g. an apical / top compartment and a basal / bottom compartment).

[0034] In some cases, a substrate (e.g. a cell culture insert) may be removable from a culture receptacle, and / or transferable between different culture receptacles. A first (e.g. apical) compartment, as formed by a substrate in a culture receptacle, may be accessible from theexternal environment, and a second (e.g. basal) compartment may be accessible by removing the substrate from the receptacle.

[0035] Methods of this disclosure may comprise co-culturing different cell-types, either in the presence or in the absence of an exogenously added extracellular matrix (ECM) or ECM protein(s). Preferably, methods of this disclosure do not comprise an exogenously added extracellular matrix (ECM) or ECM protein(s). More specifically, methods of this disclosure preferably do not comprise adding or coating an ECM or ECM protein(s) to or on a culture receptacle and / or culture substrate (disposed in the receptacle).

[0036] When no exogenous ECM or ECM protein(s) are used in the methods of this disclosure, cell type may be selected for the co-culture that contributes (e.g. produces / secretes) an ECM or ECM protein(s) within the co-culture model. Many types of cells, particularly mesenchymal cells, are known to produce / secrete an ECM or ECM protein(s). Thus, including such cells may provide necessary or beneficial signals that support the generation and / or integrity of a co-culture model comprising different cell types (e.g. those that benefit from or rely upon and ECM or ECM protein(s)). Examples of ECM proteins that may be secreted by one or more cells of the co-culture model include one or any combination of a vitronectin, a laminin, a fibronectin, or a collagen. In the specific context of mesenchymal cells (e.g. fibroblasts), such cells may secrete or produce at least Collagen I and / or Collagen III. In a method of this disclosure, at least one mesoderm lineage cell-type and the at least one endoderm lineage cell-type are co-cultured in the absence of an exogenously added extracellular matrix protein, which co-culture may further comprise a second mesoderm lineage cell-type.

[0037] If an ECM or ECM protein(s) is exogenously added, such ECM or ECM protein(s) may be selected to correspond with what is naturally (in the tissue context) present, such as one of or any combination of a collagen, a vitronectin, a laminin, or a fibronectin. In one embodiment, an exogenously added ECM is or comprises Matrigel™ (e.g. a basement membrane extract).

[0038] Methods of this disclosure may generate a co-culture comprising different cell types, such as may derive or originate from one of the three germ layers, two of the three germ layers, or each of the three germ layers. Cells of this disclosure comprised in a co-culture model, may comprise one or more disease-associated mutations. Regardless of whether normal or diseased cells are comprised in an in vitro co-culture model comprising different cell types, it may be important for each cell type to be obtained from the same subject or from different but HLA-matched subjects.

[0039] A method of this disclosure may comprise co-culturing at least one mesoderm-origin cell type and at least one endoderm-origin cell type. A method of this disclosure may comprise co-culturing a first mesoderm-lineage cell type, a second mesoderm-lineage cell type, and at least one endoderm-lineage cell type.

[0040] Where a mesoderm-origin cell type is comprised in a co-culture model, it may correspond to a mesenchymal cell or an immune cell. Where an endoderm-origin cell type is comprised in a co-culture model, it may correspond to an epithelial cell. Where a co-culture model comprises a first mesoderm lineage cell-type and at least one endoderm lineage celltype, the first mesoderm lineage cell-type may correspond to a mesenchymal cell and the at least one endoderm lineage cell-type may correspond to an epithelial cell. Where a co-culture model comprises a first mesoderm lineage cell-type, a second mesoderm lineage cell-type, and at least one endoderm lineage cell-type, the first mesoderm lineage cell-type may correspond to a mesenchymal cell, the second mesoderm lineage cell-type may correspond to an immune cell, and the at least one endoderm lineage cell-type may correspond to an epithelial cell.

[0041] Where a mesenchymal cell, or a plurality thereof, is comprised in a co-culture model of this disclosure, the mesenchymal cell may be a fibroblast (e.g. a tissue-resident fibroblast). Other exemplary mesenchymal cell types may include, but are not limited to, mesenchymal stem cells, chondrocytes, adipocytes, tenocytes, and osteocytes. In the context of a gut mucosa co-culture model, a component mesenchymal cell may be an intestinal fibroblast. In the context of an airway mucosa co-culture model, a component mesenchymal cell may be an airway fibroblast. Such cell types may be isolated from a subject, purchased from a commercial vendor, or differentiated from a parent cell population.

[0042] Where an immune cell, or a plurality thereof, is comprised in a co-culture model of this disclosure, the immune cell may be a lymphoid cell or a myeloid cell, or a progenitor thereof (e.g. an antigen-presenting cell or a cell that may be differentiated to an antigen-presenting cell). In the context of a gut mucosa co-culture model, a component immune cell may be a dendritic cell or a (M0, M1 , or M2) macrophage. In the context of an airway mucosa co-culture model, a component immune cell may be a dendritic cell or a (M0, M1 , or M2) macrophage. Such cell types may be isolated from a subject (e.g. whole blood or PBMCs), purchased from a commercial vendor, or differentiated from a parent cell population.

[0043] Where an epithelial cell, or a plurality thereof, is comprised in a co-culture model of this disclosure, the epithelial cell may be or comprise an epithelial stem cell. An epithelial (stem / progenitor) cell may derive or originate from a mammalian (e.g. human) organ, such as a liver, lung, pancreas, small intestine, large intestine, colon, stomach, prostrate or breast. Inthe context of a gut mucosa co-culture model, a component epithelial cell may be an intestinal epithelial cell, such as originating from a small intestine or a colon. In the context of an airway mucosa co-culture model, a component epithelial cell may be an airway epithelial cell. Such cell types may be isolated from a subject, purchased from a commercial vendor, or differentiated from a parent cell population.

[0044] Co-culturing different cell types in a common culture medium may mean that such medium at minimum supports or maintains, for each cell type of the co-culture, one or more of a viability, function, and identity. In addition, a common culture medium may promote the growth / expansion of one or more cell type(s) of the co-culture model, and / or may promote the differentiation of the same or other cell type(s) of the co-culture model.

[0045] In a method of this disclosure, a first cell type of mesoderm origin and at least one cell type of endoderm origin may be co-cultured in a common culture medium, and (optionally) a second cell type of mesoderm origin may also be co-cultured in the common culture medium.

[0046] In one embodiment, the common culture medium is a medium that supports the generation of an organoid (e.g. an organoid medium), such as an epithelial organoid medium. Such a medium may support the formation / maintenance of a barrier comprising layered mesenchymal (e.g. intestinal fibroblast) and epithelial (e.g. intestinal epithelial) cells, and also the formation / maintenance of a barrier comprising layered mesenchymal (e.g. intestinal fibroblast) and epithelial (e.g. intestinal epithelial) cells in direct or indirect contact with relevant immune cells (e.g. DCs, T cells or macrophages). Exemplary organoid media have been commercialized under the following brands: IntestiCult™, PancreaCult™, HepatiCult™, ProstaCult™, PneumaCult™, and STEMdiff™.

[0047] In the context of a gut mucosa model, an lntestiCultTM-branded medium may be an appropriate common culture medium. In the context of an airway mucosa model, a PneumaCultTM-branded medium may be an appropriate common culture medium. Regardless, a common culture medium (or the basal medium thereof) may comprise one or more of, two or more of, three or more of, or each of: a mitogen; an inhibitor of signaling through a TGF; an agonist of signaling through a WNT; and an inhibitor of signaling through a BMP.

[0048] If present, a common culture medium may comprise one or more agonists of signaling though a WNT, and such agonist may be a protein or small molecule modulator that activates or perpetuates the Wnt-beta-catenin pathway. Exemplary protein agonists include, but are not limited to, WNT3A or an R-Spondin (e.g. R-Spondin 1 , 2, 3, or 4). A concentration of a protein WNT agonist may range between about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 pg / mL, or about 3 ng / mL to 150 ng / mL or about 5 ng / mL to 100 ng / mL or about 10 ng / mL to50 ng / mL. An exemplary small molecule agonist includes, but is not limited to, CHIR99021. A concentration of a small molecule WNT agonist may range between about 1 nM and 1 mM, between about 5 nM and 500 pM, between about 10 nM and 200 pM, between about 50 nM and 100 pM, between about 100 nM and 50 pM, or between about 0.5 pM and 25 pM.

[0049] If present, a common culture medium may comprise one or more mitogens, which are typically proteins that stimulate cell growth / proliferation. Exemplary mitogens include, but are not limited to, an FGF, or members of the Epidermal growth factor (EGF) family, such as EGF, TGF-alpha, amphiregulin, betacellulin, epiregulin, heparin-binding EGF-like growth factor, epigen and neuregulin-1 , -2, -3 and -4. A concentration of a protein WNT agonist may range between about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 pg / mL, or about 3 ng / mL to 150 ng / mL or about 5 ng / mL to 100 ng / mL or about 10 ng / mL to 50 ng / mL.

[0050] If present, a common culture medium may comprise one or more inhibitors of signaling though a BMP, and such inhibitor may be a protein or small molecule modulator that reduces or blocks the BMP pathway. Exemplary protein agonists include, but are not limited to, Noggin, Chordin, or Follistatin. A concentration of a protein BMP signaling antagonist may range between about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 pg / mL, or about 3 ng / mL to 150 ng / mL or about 5 ng / mL to 100 ng / mL or about 10 ng / mL to 50 ng / mL. Exemplary small molecule antagonists include, but are not limited to, LDN193189 or dorsomorphin. A concentration of a small molecule BMP signaling antagonist may range between about 1 nM and 1 mM, between about 5 nM and 500 pM, between about 10 nM and 200 pM, between about 50 nM and 100 pM, between about 100 nM and 50 pM, or between about 0.5 pM and 25 pM.

[0051] If present, a common culture medium may comprise one or more inhibitors of signaling through a TGF (e.g. TGFalpha or TGF TGFbeta), and such inhibitor may be a natural inhibitor (e.g. a protein, such as a SMAD family member) or small molecule. A concentration of a protein TGF signaling antagonist may range between about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 pg / mL, or about 3 ng / mL to 150 ng / mL or about 5 ng / mL to 100 ng / mL or about 10 ng / mL to 50 ng / mL. Exemplary small molecule inhibitors of signaling through a TGF include, but are not limited to, A83-01 , A77-01 , SB 431542, LY364947, and LDN 214117. A concentration of a small molecule TGF signaling antagonist may range between about 1 nM and 1 mM, between about 5 nM and 500 pM, between about 10 nM and 200 pM, between about 50 nM and 100 pM, between about 100 nM and 50 pM, or between about 0.5 pM and 25 pM.

[0052] In one embodiment, the common culture medium comprises B27 or one or more components thereof. In the same or different embodiment, the common culture mediumcomprises N2 or one or more components thereof. In the same or different embodiment, the common culture medium comprises N-Acetylcysteine.

[0053] As previously described, methods of generating an in vitro co-culture model may comprise providing a substrate in a culture well / receptacle, and co-culturing at least two different cell types in a common culture medium.

[0054] Methods of this disclosure may comprise layering different cell types, such as in a first (e.g. apical) compartment of a culture receptacle (as divided by a substrate). By way of example, a first mesoderm lineage cell-type (of the at least two mesoderm lineage cell types) and the at least one endoderm lineage cell-type may be layered in the first / apical compartment (as formed by a substrate). A first mesoderm lineage cell-type (e.g. mesenchymal cell, such as fibroblast) may be seeded in a first / apical compartment, such as on a bottom wall thereof, and at least one endoderm lineage cell-type (e.g. an epithelial cell, such as an intestinal or airway epithelial cell) may be layered thereover. Alternatively, at least one endoderm lineage cell-type (e.g. an epithelial cell, such as an intestinal or airway epithelial cell) may be seeded in a first / apical compartment, such as on a bottom wall thereof, and a first mesoderm lineage cell-type (e.g. mesenchymal cell, such as fibroblast) may be layered thereover. Thus, establishing a co-culture of a first mesoderm lineage cell-type and at least one endoderm lineage cell-type may be performed sequentially by: initially seeding a first cell type and culturing for a first culture period (such as in a common culture medium) to establish a monolayer in a first / apical compartment, such as on an upper (apical compartment facing) side of a substrate (e.g. porous member); and then overlaying a second cell type and culturing for a second culture period (such as in a common culture medium) to establish a monolayer over the first cell type (monolayer). Accordingly, a stratified barrier layer comprising different (layered) cell types may be generated / formed.

[0055] Such a layered co-culture of a first mesoderm lineage cell-type (e.g. mesenchymal cell, such as fibroblast) and at least one endoderm lineage cell-type (e.g. an epithelial cell, such as an intestinal or airway epithelial cell) may similarly be formed in a second / basolateral compartment (as formed by a substrate) by seeding / layering the cells on an under (basal compartment facing) side of a substrate (e.g. porous member).

[0056] After establishing a barrier layer of a first mesoderm lineage cell-type (e.g. mesenchymal cell, such as a fibroblast) and at least one endoderm lineage cell-type (e.g. an epithelial cell, such as an intestinal or airway epithelial cell) on a surface of a substrate, a second mesoderm lineage cell-type, may be added to the co-culture model (such as in a common culture medium). A second mesoderm lineage cell-type (e.g. immune cells) may be added to either compartment of a culture receptacle as divided by a substrate disposedtherein. If a barrier layer (as descried above) is formed in a first / apical compartment, then a second mesoderm lineage cell-type is preferably added to a second / basolateral compartment, or if a barrier layer (as descried above) is formed in a second / basolateral compartment, then a second mesoderm lineage cell-type is preferably added to a first / apical compartment.

[0057] In the specific context of a gut mucosa model, an intestinal fibroblast:intestinal epithelial cell barrier may be established in or on a substrate (e.g. porous member) of a first / apical compartment of a culture receptacle, such that intestinal epithelial cells are supported by mesenchyme and still accessible apically by air (if cultured at an air liquid interface) and / or environmental or pathogen insults. Addition of intestine-relevant immune cells (e.g. antigen presenting cells, such as DCs or macrophage) in a second / basolateral compartment of a culture receptacle may potentiate immune responses in response to environmental or pathogen insults.

[0058] In the specific context of an airway mucosa model, an airway fibroblastairway epithelial cell barrier may be established in or on a substrate (e.g. porous member) of a first / apical compartment of a culture receptacle, such that airway epithelial cells are supported by mesenchyme and still accessible apically by air (if cultured at an air liquid interface) and / or environmental or pathogen insults. Addition of airway-relevant immune cells (e.g. antigen presenting cells, such as DCs or macrophage) in a second / basolateral compartment of a culture receptacle may potentiate immune responses in response to environmental or pathogen insults.

[0059] As described herein, a complex co-culture model may be formed and supported by a common culture medium, wherein cell types of the co-culture model maintain one or more of their identity, viability, and function. It is striking and unexpected that a complex co-culture model may be formed in and supported by a common culture medium in the absence of an exogenously added extracellular matrix (ECM) or ECM protein(s). Nevertheless, depending on the nature of the co-culture model, such as when cell types are included that do not naturally produce / secrete ECM or ECM protein(s), it may be desirable to add ECM or ECM protein(s) to the model. As described above, ECM or ECM protein(s) that help support an in vitro co-culture model may correspond to one or more of a collagen, a vitronectin, a laminin, a fibronectin, or any combination thereof (e.g. Matrigel™ or the like).

[0060] Methods of forming / generating an in vitro co-culture model may comprise culturing at least some of the component cells submerged in a common culture medium. Typically, cells comprised within a second / basolateral compartment of a culture receptacle (as divided by a substrate disposed therein) are submerged in a culture medium. However, cells comprised within a first / apical compartment of a culture receptacle (as divided by a substrate disposedtherein) may either be submerged in a culture medium or exposed to a culture medium from a basolateral compartment (e.g. cultured at air-liquid interface). Where both the apical and basolateral compartments comprise medium, the medium may be the same (and thus a common culture medium), or may be different depending on the nature of the cells being cultured or the assay / experiment being performed. In the context of certain tissues, such as intestine or airway, culturing at air-liquid interface may be of greater physiological relevance in comparison to a submerged culture condition, and since only the basolateral compartment comprises culture medium all cell types of the co-culture model are exposed to a common culture medium.

[0061] An in vitro co-culture model formed / generated in accordance with this disclosure may be stable (e.g. maintained architecture and / or viability, retained cell identity and / or function) for a prolonged period in a common culture medium. Co-culturing may elapse numerous days or weeks, such as between 1 and 42 days, or more. If different cell types of the in vitro coculture are incorporated sequentially, a first culture period (to establish a monolayer of a mesoderm lineage cell-type and / or an endoderm lineage cell-type), such as in a common culture medium, may elapse between about 1 and 14 days, between about 5 and 12 days, or between about 1 and 7 days. If after a first culture period a second cell type is layered with an earlier established monolayer (e.g. to establish a monolayer over the earlier established monolayer), such as in a common culture medium, a second culture period may elapse between about 1 and 14 days, between about 5 and 12 days, or between about 1 and 7 days. Concurrent with or after establishing a barrier layer (as described) a third cell type may be added to the system (e.g. to facilitate an immune response), such as in a common culture medium, for a third culture period, which may elapse between about 1 and 35 days, between about 5 and 28 days, or between about 7 and 21 days. In one embodiment, a first, a second and / or a third culture period respectively elapse between about 3 and 28 days; between about 5 and 21 days; or between about 7 and 14 days.

[0062] Methods of generating / forming an in vitro co-culture model may comprise pre-culturing any component cell type prior to incorporating such component cell type(s) in the model. By way of example any, or any combination, of a first mesoderm lineage cell-type, a second mesoderm lineage cell-type, and an endoderm origin cell type may respectively be maintained or pre-differentiated in separate cultures before generating / forming an in vitro co-culture model of this disclosure. Or, any, or any combination, of a first mesoderm lineage cell-type, a second mesoderm lineage cell-type, and an endoderm origin cell-type may respectively be freshly isolated from a subject before generating / forming an in vitro co-culture model of this disclosure.

[0063] Particularly in the context of immune cells, if incorporated in an in vitro co-culture model, it may be important to pre-differentiate / mature such cells before generating / forming an in vitro co-culture model of this disclosure. For example, if incorporating dendritic cells in an in vitro co-culture model of this disclosure it may be desirable to pre-differentiate such cells, such as from a population of maintained or freshly isolated monocytes, using known methodologies. Indeed, monocytes may be differentiated to dendritic cells in a matter of days (e.g. 1-14 days) in the presence of one or both of GM-CSF (~5-100 ng / mL) and IL-4 (~5-100 ng / mL).

[0064] As another example, if incorporating macrophages in an in vitro co-culture model of this disclosure it may be desirable to pre-differentiate such cells, such as from a population of maintained or freshly isolated monocytes, using known methodologies. Indeed, monocytes may be differentiated to MO-like macrophages using a published or commercially available medium (E.g. ImmunoCultTM SF Macrophage Medium, STEMCELL Technologies). M1-like or M2-like macrophages may be further differentiated from MO-like macrophages in a matter of days (e.g. 1-14 days)in the presence of either LPS (~5-100 ng / mL) and / or IFN-y (~5-100 ng / mL), or IL-4 (~5-100 ng / mL), respectively.

[0065] As another example, if incorporating T cells in an in vitro co-culture model of this disclosure it may be desirable to pre-activate such cells, such as from a population of maintained or freshly isolated unactivated T cells, using known methodologies. Indeed, T cells may be activated using commercially available reagents (e.g. STEMCELL Technologies) that cross-link T cell receptors.

[0066] Immune cells may be further matured before or after they are added to an in vitro coculture model. In the context of dendritic cells, exposure to one or more cytokines may model an inflammatory response, such as to a pathogen. Exemplary cytokines include, but are not limited to, TNFa, IFNy, IL-1 p, IL-4, and anti-CD40 either alone or any combination thereof, such as at a concentration ranging between 20-50 ng / mL. When matured through exposure to one or more cytokines, arising cells may exhibit increased marker expression (e.g. CD80, CD83, CD86, CD108, CD206, CD209, etc) and / or cytokine secretion (e.g. one or more interleukins, such as I L- 12p40) in comparison to when cells are not matured through exposure to the one or more cytokines.

[0067] Thus, a common culture medium, that is supportive of generating / forming / maintaining an in vitro co-culture model, may also be supportive of maintaining the viability and / or identity of certain cell types of the model while also differentiating and / or functionally maturing other cell types of the model.

[0068] Methods of generating / forming an in vitro co-culture model may further comprise assessing environmental and pathogenic insults in a normal or diseased / challenged genetic background, or assessing / screening drugs and other compounds for toxicity and / or efficacy in a normal or diseased / challenged genetic background.

[0069] Methods of generating an in vitro co-culture model as described in the foregoing provide many advantages and / or address limitations in relation to existing co-culture models. For example, performing the methods may generate / form a physiologically relevant model that mimics an in vivo environment, in terms of component cell types (e.g. epithelial, mesenchymal, immune, etc), architecture (e.g. apical-basolateral polarity, well-formed tight / adherent junctions), function (e.g. selective permeability, mucous secretion, cytokine secretion, marker expression), etc. In the context of a gut or airway mucosa model, the methods may yield a stratified, selectively permeable barrier having a surface exposed to air and an opposed surface exposed to liquid.

[0070] An arising co-culture model may also advantageously use cells (e.g. primary or otherwise) that originate from a single or different mammalian source (e.g., human). Where cells are single sourced, co-culture models of this disclosure may be immune compatible or histocompatible. Where cells of the model may be sourced from different subjects / individuals, it may be possible to investigate mechanisms of immune rejection or therapies relevant to blood transfusion or tissue / organ transplantation.

[0071] Further advantages of the described models may relate to simplicity and accessibility. The described in vitro models are straightforward to generate / form, due to a defined set of cell types and assembly steps without the need to use different culture media or exogenously add an extracellular matrix or extracellular matrix protein(s). Thus, the described in vitro models are also accessible, for the foregoing reasons and also due to the use of low-cost and widely commercialized cultureware and reagents.

[0072] A still further advantage may relate to the modularity of the described in vitro co-culture models. For example, barrier layers may be combined with different immune cell types or combinations of different immune cell types dependent on the nature of the experiment / assay. In addition, barrier layers may be formed using cell types from different sources or subjects, having a desired genotype and / or phenotype.Co-culture model

[0073] In one aspect of this disclosure are provided in vitro co-culture models comprising component cell types, to model tissues or biological structures. Co-culture models of this disclosure may comprise cell types derived or originating from different germ layers. In vitro co-culture models of this disclosure may be used in downstream biological applications and / orassays, including: assessing responsiveness to a chemical or biological insult, or a preventative or a therapeutic modality.

[0074] In vitro co-culture models of this disclosure may comprise at least two different cell types (e.g. a first mesoderm lineage cell-type and at least one endoderm lineage cell-type) in a culture receptacle. In vitro co-culture models of this disclosure may further comprise in a culture receptacle a third cell type (e.g. a second mesoderm lineage cell-type).

[0075] A culture receptacle, dividable by a substrate into physically separated subcompartments, may be as described herein. Thus, an in vitro co-culture model of this disclosure may comprise a first sub-compartment of a culture receptacle (as formed by a substrate) that is physically separated from a second sub-compartment of the culture receptacle. Where one or more cell types are comprised in a first sub-compartment of a culture receptacle (as formed by a substrate), they may be physically separated from a second sub-compartment of the culture receptacle. Or, where one or more cell types are comprised in a first sub-compartment of a culture receptacle (as formed by a substrate), they may be physically separated from one or more cell types comprised in a second sub-compartment of the culture receptacle.

[0076] A substrate that subdivides a culture receptacle into physically separated subcompartments may nevertheless permit “cross-talk” between the sub-compartments, such as via a porous member. Thus, a substrate comprising a porous member may permit the exchange of paracrine factors or signals between sub-compartments of the in vitro co-culture model. An exemplary substrate comprising a porous member may correspond to a cell culture insert (e.g. a Transwell™ insert).

[0077] Cell types comprised in an in vitro co-culture model are not particularly limited, and may be as described herein. In most cases, cell types included in an in vitro co-culture model are ordinarily present in the tissue being modeled; however, in some cases an in vitro coculture model may be amenable to inclusion of out-of-context cell type(s), such as to study the effect(s) on the model system.

[0078] In vitro co-culture models may comprise two or more different cell types, or three or more different cell types. If the cell types are different, they may either derive or originate from the same germ layer - whether mesoderm, endoderm, or ectoderm - or from different germ layers. In one embodiment, an in vitro co-culture model comprises cell types derived or originating from at least two different germ layers.

[0079] An in vitro co-culture model may comprise one or more than one mesoderm lineage cell-type. Stated in another way, an in vitro co-culture model may comprise a first mesodermlineage cell-type and, if present, a second mesoderm lineage cell-type (different from the first mesoderm lineage cell-type).

[0080] An in vitro co-culture model may comprise one or more than one endoderm lineage cell-types. Stated in another way, an in vitro co-culture model may comprise a first endoderm lineage cell-type and, if present, a second endoderm lineage cell-type (different from the first endoderm lineage cell-type). In one embodiment, such as when an endoderm lineage celltype corresponds to an immune cell, different cell types may co-exist due to impurities and / or the presence of progenitors (due to incomplete differentiation).

[0081] As described above, and incorporated herein, where at least two different mesoderm lineage cell-types are comprised in an in vitro co-culture model, a first mesoderm lineage celltype may be mesenchymal in nature (as described herein) and a second mesoderm lineage cell-type may be one or more immune cell types (as described herein.

[0082] In one embodiment, an in vitro co-culture model comprises at least one mesoderm lineage cell-type and at least one endoderm lineage cell-type. In one embodiment, an in vitro co-culture model comprises two or more than two mesoderm lineage cell-types and one or more than one endoderm lineage cell-type. In one embodiment, an in vitro co-culture model comprises a plurality of mesoderm lineage cell-types and a plurality of endoderm lineage celltypes.

[0083] In a specific embodiment, an in vitro co-culture model comprises at least two mesoderm lineage cell-types and at least one endoderm lineage cell-type in a culture receptacle, wherein the cell types in the culture receptacle are exposed to a common culture medium. Details regarding common culture media are described herein, and may be incorporated in the context of in vitro co-culture models.

[0084] Where a culture receptacle is divided into sub-compartments by a substrate (as described above), two or more cell types of an in vitro co-culture model may be physically separated by a substrate (as described herein). In one embodiment, a first mesoderm lineage cell-type and / or at least one endoderm lineage cell-type may be physically separated from a second mesoderm lineage cell-type by a substrate disposed in a culture receptacle.

[0085] A substrate, such as a porous member of a cell culture insert, may separate a culture receptacle / well into a first (e.g. apical) compartment and a second (e.g. basolateral, or basal) compartment. Thus, cells comprised in one compartment may communicate with cells and / or culture medium and / or environmental / pathogenic conditions in another compartment, if the substrate is porous.

[0086] Cells of the co-culture model may be comprised in one sub-compartment or multiple (e.g. two or each) sub-compartments of a culture receptacle (as formed by a substrate). Preferably, cells of the co-culture model are comprised in a first (e.g. apical) sub-compartment and in a second (e.g. basolateral) sub-compartment. When cells are comprised in a second / basolateral sub-compartment they are typically submerged in a (common) culture medium, regardless of if they are adhered (as a monolayer) to a bottom wall of or suspended in such sub-compartment, or adhered (as a monolayer) to an underside (e.g. basolateral compartment facing surface) of a substrate, such as a cell culture insert. However, when cells are comprised in a first / apical sub-compartment they may either be submerged in a (common) culture medium or cultured at an air-liquid interface (the liquid in contact with a porous member at the interface of the sub-compartments). While cells in a first / apical sub-compartment may be suspended in a (common) culture environment, typically endoderm-origin (e.g. epithelial) and mesoderm-origin (e.g. mesenchymal) cells tend to adhere to a substrate.

[0087] An in vitro co-culture model may comprise one or more cell types in a first (e.g. apical) sub-compartment of a culture receptacle physically subdivided by a substrate into first / apical and second (e.g. basal) sub-compartments. An in vitro co-culture model may comprise one or more cell types in a second (e.g. basal) sub-compartment of a culture receptacle physically subdivided by a substrate into first / apical and second / basal sub-compartments. An in vitro co-culture model may comprise one or more cell types in a first / apical sub-compartment of a culture receptacle physically subdivided by a substrate into first / apical and second / basal subcompartments and one or more cell types in the second / basal sub-compartment.

[0088] In one embodiment, a first mesoderm lineage cell-type is comprised in (or on a surface of) a first / apical sub-compartment. In one embodiment, a first mesoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment. In one embodiment, a first mesoderm lineage cell-type is comprised in (or on a surface of) a first / apical sub-compartment, and a first mesoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment.

[0089] In one embodiment, at least one endoderm lineage cell-type is comprised in (or on a surface of) a first / apical sub-compartment. In one embodiment, at least one endoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a second / basolateral subcompartment. In one embodiment, at least one endoderm lineage cell-type is comprised in (or on a surface of) a first / apical sub-compartment, and at least one endoderm lineage celltype is comprised in (or on a surface of a substrate facing) a second / basolateral subcompartment.

[0090] In one embodiment, a first mesoderm lineage cell-type and at least one endoderm lineage cell-type are comprised in (or on a surface of) a first / apical sub-compartment. In one embodiment, a first mesoderm lineage cell-type and at least one endoderm lineage cell-type are comprised in (or on a surface of) a second / basolateral sub-compartment. In one embodiment, a first mesoderm lineage cell-type and at least one endoderm lineage cell-type are comprised in (or on a surface of) a first / apical sub-compartment, and a first mesoderm lineage cell-type and at least one endoderm lineage cell-type are comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment.

[0091] In one embodiment, a first mesoderm lineage cell-type is comprised in (or on a surface of) a first / apical sub-compartment, and a second mesoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment. In one embodiment, at least one endoderm lineage cell-type is comprised in (or on a surface of) a first / apical sub-compartment, and at least one mesoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment. In one embodiment, a first mesoderm lineage cell-type and at least one endoderm-lineage cell type are comprised in (or on a surface of) a first / apical sub-compartment, and a second mesoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment. In one embodiment, a first mesoderm lineage cell-type and at least one endoderm-lineage cell type are comprised in (or on a surface of a substrate facing) a second / basolateral sub-compartment, and second mesoderm lineage cell-type is comprised in (or on a surface of a substrate facing) a first / apical sub-compartment.

[0092] In the foregoing, a first mesoderm lineage cell-type may correspond to a mesenchymal cell, such as a (tissue-resident) fibroblast or a mesenchymal stromal cell. In the foregoing, a second mesoderm lineage cell-type may correspond to an immune cell, such as a lymphoid (e.g. T cells) or a myeloid cell (e.g. antigen presenting cells, such as dendritic cells or macrophages). In the foregoing, at least one endoderm lineage cell-type may correspond to an epithelial cell, such as a tissue-resident epithelial cell or a tissue-resident epithelial stem or progenitor cell.

[0093] Cells in either compartment may be adhered to a surface of a substrate that separates a culture receptacle into distinct sub-compartments. In such cases, the cells may be adhered to and establish a monolayer over the surface of the substrate. If multiple adhered cell types are comprised in either a first (e.g. apical) and / or a second (e.g. basolateral) subcompartments, they may be stratified / layered. For example, a first cell layer (e.g. a first mesoderm lineage cell-type) may be layered over a second cell layer (e.g. at least one endoderm lineage cell-type), or vice versa.

[0094] Also as described herein, co-culture models may be established / assembled in the absence of exogenously added extracellular matrix (ECM) or ECM protein(s). Thus, in vitro co-culture models may lack exogenously added ECM or ECM protein(s) on any surface of a culture receptacle, substrate (and porous member), culture of cells, or in a common culture medium

[0095] Cells comprised in an in vitro co-culture model may be pre-differentiated or -treated, particularly for certain types of immune cells if comprised in an in vitro co-culture. As described herein, dendritic cells and different types of macrophages may be pre-differentiated under known conditions. Also as described herein, T cells may be pre-activated under known conditions. As further described herein, epithelial cells may be isolated or derived by dissociating an established monolayer culture or an epithelial organoid. Thus, an in vitro coculture model may comprise pre-differentiated and / or pre-treated cells. Further, a common culture media of an in in vitro co-culture model may comprise maturation factors that stimulate certain cells of the model, while not (markedly or noticeably or otherwise) affecting the identify and / or function of other cells of the model.

[0096] In vitro co-culture model may be used to assess environmental and pathogenic insults in a normal or diseased / challenged genetic background, or assess / screen drugs and other compounds for toxicity and / or efficacy in a normal or diseased / challenged genetic background.

[0097] In vitro co-culture models as described in the foregoing provide many advantages and / or address limitations in relation to existing co-culture models. For example, the models may be physiologically relevant by mimicking an in vivo environment, in terms of component cell types (e.g. epithelial, mesenchymal, immune, etc), architecture (e.g. apical-basolateral polarity, well-formed tight / adherent junctions), function (e.g. selective permeability, mucous secretion, cytokine secretion, marker expression), etc. In the context of a gut or airway mucosa model, the models may yield a stratified, selectively permeable barrier having a surface exposed to air and an opposed surface exposed to liquid.

[0098] A co-culture model may also advantageously comprise cells (e.g. primary or otherwise) that originate from a single or different mammalian source (e.g., human). Where cells are single sourced, co-culture models of this disclosure may be immune compatible or histocompatible. Where cells of the model may be sourced from different subjects / individuals, it may be possible to investigate mechanisms of immune rejection or therapies relevant to blood transfusion or tissue / organ transplantation.

[0099] Further advantages of the described models may relate to simplicity and accessibility. The described in vitro models are straightforward to generat e / form, due to a defined set of celltypes and assembly steps without the need to use different culture media or exogenously add an extracellular matrix or extracellular matrix protein(s). Thus, the described in vitro models are also accessible, for the foregoing reasons and also due to the use of low-cost and widely commercialized cultureware and reagents.

[0100] A still further advantage may relate to the modularity of the described in vitro co-culture models. For example, barrier layers may be combined with different immune cell types or combinations of different immune cell types dependent on the nature of the experiment / assay. In addition, barrier layers may be formed using cell types from different sources or subjects, having a desired genotype and / or phenotype.

[0101] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: Establishing an in vitro co-culture model

[0102] An exemplary in vitro co-culture model was established by seeding ~1 x 105commercially sourced mesoderm-origin cells / cm2(e.g. intestinal fibroblasts, “IFs”) in the apical compartment of a cell culture insert (e.g. Transwell™ insert (Corning)), and expanding a monolayer of cells in a suitable medium, such as in IntestiCult™ Organoid Differentiation Medium (Human) (STEMCELL Technologies, IntestiCult™ ODMh) or a MesenCult™ branded medium (e.g. MesenCult™ Proliferation Kit, STEMCELL Technologies), as per the manufacturer’s instructions. Once the cells reached confluence, typically after 3-5 days, endoderm-origin cells (e.g. intestinal epithelial cells, “lECs” or “ECs”) as dissociated from an organoid culture generated using an IntestiCult™ kit (STEMCELL Technologies), were layered on the mesoderm monolayer at a density of ~1 x 106cells / cm2. The layered cells were cocultured in IntestiCult™ ODMh (STEMCELL Technologies) for about 1 to 2 weeks until the apical chamber co-culture (e.g. IF-IEC gut mucosa model) was established.

[0103] After establishing the apical chamber co-culture, immune cells (i.e. second mesodermorigin cell type) were seeded in the basal compartment of the culture receptacle, and cultured 1-6 days in IntestiCult™ ODMh (STEMCELL Technologies) in the presence or absence of a selection of maturation cytokines (“±MAT”), such as one or more of TNFa, IFNy, IL-ip, IL-4, and anti-CD40 at a concentration ranging between 20-50 ng / mL. A first exemplary immune cell type corresponds to dendritic cells (“DC”), obtained by pre-culturing whole blood- or PBMC-derived monocytes for 5-6 days in a suitable medium supplemented with critical factors such as GM-CSF (~50 ng / mL) and IL-4 (~20 ng / mL), thereby yielding either matured or immature DCs seeded into the basal compartment at a density of ~1 x 10'4cells / well. A second exemplary immune cell type corresponds to macrophages (M<t>), obtained by differentiating whole blood- or PBMC-derived monocytes using ImmunoCult™ branded medium(lmmunoCultTM-SF Macrophage Medium, STEMCELL Technologies) to yield naTve / unpolarized macrophages (MO M<t>), or by exposing MO naTve / unpolarized macrophages to 10 ng / mL LPS + 50 ng / mL IFN-y to generate classically activated M1-like M<t>, or to 10 ng / mL IL-4 to generate alternatively activated M2-like M<t>. Thereby, MO, M1 , or M2 M<t> may be produced that were seeded into the basal compartment at a density of ~1 x 105cells / well.Example 2: Assessing structural and functional characteristics of the co-culture model

[0104] Establishment of an epithelial barrier, viability of cells of the barrier, and synthesis of ECM by cells underlying / supporting the barrier, essentially as generated in Example 1 , were characterized by i) Trans-epithelial electrical resistance (TEER) measurements, ii) visualization of a uniform network of tight junctions by Zonula Occludents-1 (ZO-1) immunostaining, and iii) formation of a sealed intestinal barrier by paracellular permeability assay to FITC-dextran 4 kDa (FD4).

[0105] The TEER for each of four independent co-culture models (Gut mucosa 1-4), established by all pair-wise combinations of mesoderm- and endoderm-origin cells from two donors, was measured for a period of 6 weeks using the epithelial volt-ohm meter EVOM2 or EVOM3 Epithelial Voltmeter (World Precision instruments). A blank coated insert without cells served as normalization control. At day-14, the cell culture plates were taken out of the incubator and TEER measurements were performed at room temperature in a biosafety cabinet, by gently positioning electrodes in either compartment (apical and basal) of a receptacle / well. First, the resistance of an empty cell culture insert submerged with the same type and volume of cell culture medium used in the experiments, was measured to determine the baseline TEER value and subtracted from all subsequent measurements. Then, all TEER measurements on sample cultures were performed in the same way as the blank, and the mean TEER value and standard deviation were calculated.

[0106] Within the first 7 days of generating the apical co-culture, TEER measurements ranged between 200 and 3000 Q*cm2for all models analyzed (Figure 1A). Data also indicate that the established epithelial cells can be maintained for up to 6 weeks, as there was little or no appreciable change in TEER value over such a long period. Endoderm-origin cells (as layered with mesoderm-origin cells) established acceptable barrier integrity, as determined by day-14 ZO-1 staining of methanol or paraformaldehyde (PFA) fixed cells using an anti-ZO-1 monoclonal antibody (clone ZO1-1A12) (ThermoFisher), due to a continuous and uniform network of tight junctions in all 4 models examined (Figure 1 B, as indicated by arrows). In addition, no detectable permeability of a day-14 barrier was observed 3 hours after administering 1 mg / mL of the fluorescent tracer FITC-Dextran 4 kDa (FD4) into the apicalchamber; whilst 134±11 g FD4 leaked through an empty insert. No FD4 was detected in the basal chambers of inserts containing an established cellular barrier (Figure 1C).

[0107] These data indicate that the co-culture model comprises a sealed barrier, which could be maintained throughout the duration of a prolonged culture period in a common culture medium.

[0108] The established co-culture model was also assessed for the production / deposition of extracellular matrix (ECM) and cell viability. On the one hand, immunofluorescence images showed expression of Collagen I and Collagen III (anti-Collagen I and anti-Collagen III, both Abeam) by cells within the apical compartment of the two day- 14 co-culture models examined (Figure 1 D). ECM production by endoderm-origin cells (e.g. lECs) was undetectable, suggesting the ECM was generated by mesoderm-origin cells (e.g. IFs) (data not shown). On the other hand, viability of the various cell types of a day-14 co-culture, essentially as established in Example 1 , was assessed by fixable-dye-based (AO-DAPI) flow cytometry analysis. For all cell types, a viability of at least 80% was shown (Figure 2). Among the mesoderm- and endoderm-origin cells of the apical compartment, ~85% of the IFs and lECs were viable when assessed after 6 days in the co-culture model (Figure 2A). Among cells present in the basal compartment, DCs exhibited comparable viabilities of 89.9%±6.0% and 93.9%±3.2% when exposed or not exposed to maturation conditions (±MAT) (Figure 2B), and MO-like, M1-like, or M2-like M<t> remained viable (93.7% ± 4.0%, 95.4 ± 3.2%, and 94.7% ± 4.4% respectively) when assessed after 48 hours in the co-culture model (Figure 2C).

[0109] Altogether, these data demonstrated the formation of a confluent, long lasting, intact and functional barrier comprising a first cell type of mesoderm origin and a second cell type of endodermal origin, and that cells of mesoderm origin may support cells of endoderm origin layered therewith, such as by producing physiologically relevant extracellular matrix protein(s).Example 3: Assessing phenotypes and other properties ofco-cultured antigen presenting cells

[0110] Cells of a second mesoderm type (i.e. immune cells) of the co-culture model were further assessed for expression of relevant markers by flow cytometry. In addition, DCs were also assessed for cytokine production by enzyme-linked immunosorbent assay (ELISA).

[0111] Immunostaining confirmed that DCs and M<t> expressed CD45 (Anti-Human CD45 Antibody, Clone HI30, STEMCELL Technologies), and also that such cells efficiently attached to the basal side of the insert / substrate (indicated by arrows) in every co-culture model analyzed (Figure 3A and 3B, respectively). Staining in the background corresponds to DAPI staining within mesoderm- and endoderm-origin cells of the apical compartment.

[0112] DCs in the co-culture model exhibited phenotypic changes upon exposure to a pathogen trigger condition (+MAT), as described in Example 1. Exposure of the DCs to an environment resembling inflammation revealed acceptable stability of the co-culture model and increased / high expression levels of immune activation markers (CD80, CD83, CD86), and tissue residency / migration / adhesion marker (CD108), in comparison to DCs not exposed to the inflammatory environment (Figure 3C). In addition, ELISA revealed increased secretion of the inflammatory cytokine, IL-12p40, by DCs exposed to the inflammatory environment (+MAT) in comparison to the -MAT condition (Figure 3D). With regard to M<t>, the pan- hematopoietic marker CD45 was observed among all macrophage states (MO, M1 , and M2), and typical M1 activation markers (i.e. CD80 and CD86) and typical M2 activation markers (i.e. CD206 and CD209) were upregulated in macrophages cultured in the presence of M1 and M2 activation factors, respectively (Figure 3E).

[0113] Overall, these data confirm the viability, attachment, and marker expression by immune cells in the described co-culture model, and also the functionality of DCs in the coculture model to secrete cytokines when challenged.

Claims

CLAIMS1) A method for generating an in vitro co-culture model, the method comprising: a) providing a substrate in a culture receptacle that physically separates the culture receptacle into a first compartment and a second compartment; and b) co-culturing in a common culture medium; i) at least two mesoderm lineage cell types comprising a first mesoderm lineage cell type as a monolayer and a second mesoderm lineage cell type, and ii) at least one endoderm lineage cell type.2) The method according to claim 1 , further comprising layering the first mesoderm lineage cell type of the at least two mesoderm lineage cell types and the at least one endoderm lineage cell type in the first compartment.3) The method according to claim 2, further comprising seeding the second mesoderm lineage cell type of the at least two mesoderm lineage cell types in the second compartment.4) The method according to according to claim 2 or 3, wherein the first mesoderm lineage cell type and the at least one endoderm lineage cell type are either submerged in the common culture medium, or exposed to the common culture medium at an air liquid interface.5) The method according to any one of claims 1 to 4, wherein the at least two mesoderm lineage cell types and the at least one endoderm lineage cell type are cultured in the absence of an exogenously added extracellular matrix protein.6) The method according to any one of claims 1-5, wherein a) the first mesoderm lineage cell type is a mesenchymal cell; and / or b) the second mesoderm lineage cell type is an immune cell; and / or c) the at least one endoderm lineage cell type is an epithelial cell.7) The method according to claim 6, wherein a) the immune cell is an antigen-presenting cell; and / or b) the epithelial cell is an intestinal cell.8) The method according to claim 6, wherein the epithelial cell is obtained from a small intestine or a colon, and / or the epithelial cell is obtained from a dissociated organoid or monolayer culture.9) The method according to claim 1 , wherein the substrate comprises a porous member, optionally a cell culture insert that is removable from the culture receptacle.10) The method according to claim 1 , wherein the common culture medium comprises one or more of an agonist of WNT signaling, an inhibitor of BMP signaling, and a mitogen.11) The method according to claim 2 or 3, wherein co-culturing elapses between 1 and 42 days.12) An in vitro co-culture model comprising: a) at least two mesoderm lineage cell types comprising a first mesoderm lineage cell type as a monolayer and a second mesoderm lineage cell type; and b) at least one endoderm lineage cell type, in a culture receptacle, wherein a first mesoderm lineage cell type of the at least two mesoderm cell types and the at least one endoderm lineage cell type are physically separated from a second mesoderm lineage cell type of the at least two mesoderm cell types, and wherein the cell types of a) and b) are exposed to a common culture medium.13) The co-culture model according to claim 12, wherein the first mesoderm lineage cell type and the at least one endoderm lineage cell type are physically separated from the second mesoderm lineages cell type by a substrate that separates the culture receptacle into a first compartment and a second compartment.14) The co-culture model according to claim 12 or 13, wherein the co-culture lacks an exogenously added extracellular matrix protein.15) The co-culture model according to any one of claims 12 to 14, wherein a) the first mesoderm lineage cell type is a mesenchymal cell; and / or b) the second mesoderm lineage cell type is an immune cell; and / or c) the at least one endoderm lineage cell type is an epithelial cell.16) The co-culture model according to claim 15, wherein the immune cell is an antigen- presenting cell, and / or the epithelial cell is an intestinal cell.17) The co-culture model according to claim 15, wherein the epithelial cell is obtained from a small intestine or a colon, and / or the epithelial cell is obtained from a dissociated organoid or monolayer culture.18) The co-culture model according to any one of claims 12-17, wherein the first mesoderm lineage cell type and the at least one endoderm lineage cell-type are comprised in the first compartment either submerged in the common culture medium or at an air liquid interface.19) The co-culture model according to claim 12, wherein the substrate comprises a porous member, optionally a cell culture insert that is removable from the culture receptacle.20) The co-culture model according to claim 12, wherein the common culture medium comprises one or more of an agonist of Wnt signaling, an inhibitor of BMP signaling, and a mitogen.

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