Purified mucus

Abstract

The present invention relates to a purified biocompatible mucus, said mucus having the ability to form a gel. The mucus according to the invention can be provided in a tissue model together with a population of cells. The mucoprotein or tissue model disclosed by the invention can be used for an in-vitro model for digestion, mucus permeation and / or epithelium transport.

Description

Technical Field

[0001] The present invention relates to purified biocompatible mucus and its use. The present invention also relates to a tissue model comprising purified biocompatible mucus and cells and its use. The present invention also relates to a method for purifying mucus. Background Art

[0002] In order to successfully take an active agent orally, the compound must pass through a series of barriers and maintain sufficient intake (yield) to produce a therapeutic effect. First, the compound must overcome the enzymatic challenges, chemical challenges and physical challenges during the digestive stages of the mouth, stomach and small intestine, including, for example, digestive enzymes, pH changes, and components (such as bile) that may interact with and hinder the agent. Even if it reaches the small intestine, the drug delivery system must penetrate the mucus barrier so that the agent can successfully reach the epithelial cells and be absorbed into the bloodstream. The mucus layer acts as a barrier, excluding or capturing certain compounds based on size and charge. When the mucus layer is turned over, any such particles will be cleared. Therefore, oral delivery of drugs (including peptides and protein therapeutics) may be hindered by the presence of mucus on the surface of the intestinal epithelium. The mucus is composed of 95% water and mucin, a glycoprotein that forms a gel, and lipids, DNA and actin may be present in some mucus.

[0003] It is important to test oral delivery of drugs in vitro prior to in vivo testing. This may provide an alternative to reduce, improve or replace the need for animal models. Therefore, separate in vitro models exist for different stages of the digestive system.

[0004] WO 2015075467 and Houghton et al. (Food Chem. 2014 May 15; 151: 352-7) describe a digestive tract simulation model consisting of the mouth, stomach and small intestine, which uses physiologically relevant secretions and whole pig bile collected from slaughterhouses to provide a model closely related to the human digestive tract. Friedl et al. (J Pharm Sci. 2013 Dec; 102 (12): 4406-13) describe an in vitro transwell mucus permeation model for simulating the diffusion of nanoparticles through the mucosa. In addition, many cell culture systems can be used to simulate the intestinal epithelium. So far, it has not been possible to observe the entire process and how these stages interact at the same time.

[0005] WO2018 / 175861 describes a macroscopic tissue explant for high throughput screening, comprising a mammalian tissue explant in a porous substrate. The tissue explant may further comprise a mucus layer.

[0006] Gleeson & McCartney (Trends in pharmacological sciences 40.10 (2019): 720-724) described the co-culture of Caco-2 monolayers with HT29-MTX-E12, where HT29 cells are another intestinal cell line that was differentiated into mature goblet cells using methotrexate. The H29 cell line was developed to mimic the mucus-lined epithelium. However, this is not an optimal solution because the mucus layer is 4 μm, which is much thinner than the mucus layer in the thinnest areas of the human intestine, which is usually around 15 μm. This means that even a co-culture system cannot be combined with the entire digestive fluid because the cells may die.

[0007] The use of porcine mucus in such in vitro systems is well known. However, although porcine mucus reflects human intestinal mucus, Caco-2 cells cannot survive after prolonged co-incubation. To overcome this problem, biosimilar mucus has been developed. The biosimilar mucus is compatible with Caco-2 monolayers, mimics the rheological properties of porcine intestinal mucus, and has been used to evaluate in vitro drug absorption. Boegh, Marie et al. (European Journal of Pharmaceutics and Biopharmaceutics 87.2 (2014): 227-235) describe the design and characterization of biosimilar mucus compatible with in vitro cultured Caco-2 cell monolayers to establish a more representative in vitro model of intestinal mucosa. The biosimilar mucus mixture consists of degraded commercial gastric mucin, BSA, cholesterol, phosphatidylcholine, linoleic acid, and polyacrylic acid (Carbopol 974PNF, 0.3 to 0.9% w / v). Polyacrylic acid is a synthetic polymer added to the composition to provide the desired viscoelastic properties and a microstructure comparable to freshly isolated porcine intestinal mucus (PIM). The biosimilar mucus was optimized with respect to lipid content for cytocompatibility with well-differentiated Caco-2 cell monolayers. However, there were significant differences in peptide permeability between isolated PIM and the biosimilar mucus. In addition, Carbopol is known to interact with mucins, thereby altering the properties and / or function of the mucus. Commercially available porcine gastric mucin (Sigma Aldrich TM ) has been denatured during isolation and therefore has lost the ability to form mucin:mucin interactions, resulting in its inability to form a gel. In addition, gastric mucins are composed of the MUC5AC and MUC6 gene products, while small intestinal mucins are MUC2.

[0008] Other approaches to modeling intestinal tissue that include a mucus layer have a number of problems. Human mucus is difficult to obtain, and the isolation process can alter the mucin. Additionally, synthetic gastrointestinal secretions have been used to mimic the native intestinal environment, but it has been found that in the absence of a protective mucus layer like that found in vivo, such synthetic secretions can cause cell death when applied to cell cultures. Native porcine mucus harvested from animals has also been found to kill cells, making it unsuitable for use in tissue models.

[0009] The present invention is directed to overcoming or ameliorating some or all of these problems associated with the prior art. Summary of the invention

[0010] In a first aspect, the present invention provides purified biocompatible mucus, wherein the mucus: i) substantially free of nucleic acids, proteins and / or lipids and / or molecules below 100 kDa, preferably below 70 kDa; and ii) comprising mucin, wherein the mucin has the ability to form a gel. Suitably, the purified biocompatible mucus contains mucin with a density of 1.3 to 1.6 g / mL. Suitably, the purified biocompatible mucus does not contain synthetic polymers. Suitably, the purified biocompatible mucus contains mucin, and wherein the mucin is substantially not denatured, hydrolyzed or degraded. Suitably, the intermolecular interactions contained in the mucin present in the mucus are sufficient to enable the purified biocompatible mucus to form a gel. In one embodiment, the mucus is a gel.

[0011] In a second aspect, the present invention also provides a tissue model comprising: a substrate having a cell-supporting portion; a population of cells provided on said substrate; The purified biocompatible mucus according to the first aspect; Optionally, a fluid is provided on the mucus layer and / or beneath the basal layer.

[0012] In a third aspect, there is provided a method for purifying mucus, wherein the method comprises removing substantially all nucleic acids, proteins and / or lipids and / or molecules below 100 kDa, preferably below 70 kDa, from the mucus, wherein the removal step comprises dialysis and / or equilibrium density gradient centrifugation; to provide purified biocompatible mucus. Suitably, the method does not substantially remove mucin from the mucus. Suitably, the method does not comprise denaturation, degradation or hydrolysis of the mucin. Optionally, the method may comprise a further separation step, such as centrifugation. Suitably, the method provides the purified biocompatible mucus of the first aspect.

[0013] In one embodiment of the third aspect, the method may comprise i) providing a sample comprising mucus; ii) solubilizing any mucin in the mucus; iii) separating any nucleic acids, proteins and / or lipids from the mucus; and optionally iv) removing any contaminants, unwanted components or reagents from the mucus; to provide purified biocompatible mucus. Step iii) may comprise equilibrium density gradient centrifugation. Step iv) may comprise removing any cytotoxic components, reagents and / or enzymes, and may comprise dialysis of the mucus. Suitably, the method comprises separating a fraction having a density gradient of 1.3 to 1.6 g / mL from the mucus. Suitably, the density of the mucin present in the mucus is 1.3 to 1.6 g / mL.

[0014] In one embodiment of the third aspect, the method may comprise i) providing a sample comprising mucus; ii) dialyzing the mucus to separate any molecules below 100 kDa, preferably below 70 kDa from the mucus; and optionally iii) separating the mucus.

[0015] In a fourth aspect, a method for determining or predicting the absorption of a test compound through a tissue model is provided, wherein the tissue model comprises i) a substrate comprising a cell-supporting portion; ii) a cell population in contact with the cell-supporting portion of the substrate; and iii) a purified biocompatible mucus in contact with the cells; and wherein the method comprises contacting the purified biocompatible mucus with the test compound and detecting the movement of the test compound in the tissue model; thereby determining the ability of the test compound to pass through the purified biocompatible mucus and cell layer of the tissue model.

[0016] In one embodiment of the fourth aspect, the tissue model may comprise the purified biocompatible mucus as described in the first aspect of the present invention or a composition comprising the purified biocompatible mucus. The tissue model may be as described in the second aspect of the present invention. The tissue model used in the method for detecting the effect of the test compound may be a stomach tissue model, a small intestine tissue model, a large intestine tissue model, a gastrointestinal tissue model, a female reproductive tissue model, a nasal tissue model, a visual tissue model and / or an airway tissue model.

[0017] The step of detecting the movement of the test compound in the tissue model may include observing, monitoring or measuring any one or more of the following (but not limited to): the distance traveled by the test compound into the tissue model, the absorption of the test compound into the tissue model, the path traveled in the tissue model, the rate of travel or perfusion into the tissue model, the cellular uptake in the tissue model, and the position of the test compound in the tissue model. The detection may also include determining the concentration of the test compound at or below the substrate. The step of detecting the movement of the test compound may include detecting the test compound in a purified biocompatible mucus layer, in a cell population, and / or in contact with the substrate surface. The presence of the test compound at the luminal surface and / or the basolateral surface of the tissue model may be detected. The presence of the test compound at the basolateral surface indicates the ability of the compound to be absorbed by the tissue explant.

[0018] The method may comprise the step of applying the food to the purified biocompatible mucus or fluid layer of the model. The method may comprise the step of applying native intestinal media to the purified biocompatible mucus or fluid layer of the model.

[0019] In a fifth aspect, a kit is provided, comprising the purified biocompatible mucus as described in the first aspect or a composition comprising the purified biocompatible mucus, and / or the tissue model of the second aspect in a suitable container, and optionally substrates, buffers, reagents, instructions for use and other standard components well known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Embodiments of the present invention will be further described below with reference to the accompanying drawings, in which:

[0021] Figure 1 Figure 2 shows the viability of Caco-2 cells in simulated digestive fluid (with and without native mucus) in a 24-well plate transport chamber over a 4-hour period. The cytotoxic effect of porcine small intestinal surface mucus on Caco-2 cell monolayers (containing luminal PBS or simulated small intestinal fluid) was shown using a Cell titre Blue cell viability assay. Native mucus kills cells, but provides some protection to the remaining live cells by GI fluid. Under agitation, the ability of native mucus to protect cells is reduced.

[0022] Figure 2 The viscosity of commercial mucins is problematic. Human mucus is difficult to obtain. Commercial separation processes alter the mucin glycoproteins and destroy the ability to form mucin:mucin interactions. Purification methods that include boiling or exposure to proteolytic or glycolytic enzymes alter mucins. The purification method described in the third aspect of the present invention does not affect mucin:mucin interactions and allows mucins to retain the ability to form gels at higher concentrations.

[0023] Figure 3 is a diagram of how to build a model instance according to the present invention.

[0024] Figure 4 Pearson correlations are shown for 17 active pharmaceutical ingredients (APIs) permeating through 10 μL native mucus and 50 μL of 75 mg / mL reconstituted intestinal mucin. A strong positive correlation (R 2 =0.8517).

[0025] Figure 5 The rheological properties of the purified mucus of the present invention (at a concentration of 5-200 mg / ml) rehydrated in phosphate buffered saline are shown in comparison to native small intestinal porcine mucus and sodium chloride cleaned porcine small intestinal mucus. Panels A and B show the maximum shear strain and stress in the linear viscoelastic region, respectively. Panels C, D and E show the storage modulus, loss modulus and phase angle in the LVER. Panel F shows the shear strain required for the gel to break into a viscous liquid (yield point) and allow it to flow. The experiments were repeated at least three times with different batches of mucus and mucin.

[0026] Figure 6 It shows that native pig mucus has an effect on Figure 3 Effects on viability of Caco-2 cells grown in a 96-well dual chamber model are shown. In the 96-well plate model, native porcine mucus significantly reduced cell viability.

[0027] Figure 7 The effect of the concentration and volume of the purified biocompatible mucus reconstituted in buffer (referred to herein as reconstituted mucus or RM) on compatibility with Caco-2 cells is shown. Figure 6 In the same model used in , a range of concentrations and volumes of reconstituted mucus were tested for their compatibility with caco2 cells with simulated intestinal secretions applied for 4 hours. All concentrations of mucus at 50 μL and 70 μL were not significantly different from the control, indicating that these mucuses protected cells from simulated digestive secretions in vitro over a 4-hour time course.

[0028] Figure 8 The effect of the reconstructed mucus of the present invention in protecting cells from the effects of simulated intestinal digestive fluid was shown. In a 24-well plate model, 50 μL of 25 mg / ml, 50 mg / ml and 75 mg / ml mucus was shown to protect cells from the effects of simulated intestinal digestive fluid over a 4-hour time course.

[0029] Fig. 9 Cell viability levels of the gastric cell line CRL-1739 integrated with a biocompatible mucus layer and gastric secretions are shown. Gastric epithelial cells were covered with gastric mucus layers of varying thickness and mucin concentration and exposed to model gastric digestive secretions.

[0030] Fig.10 The effects of different volumes and concentrations of purified mucus of the present invention on airway cell viability at 90 minutes are shown. Panel A shows the cell viability group, where the volume of liquid was added to the apical surface of Calu3 airway cells, and the mucin concentration is shown as a bar. Panel B shows the same data, but different mucin concentrations were applied to each group, and the bars show different volumes. All experiments were repeated at least four times.

[0031] Fig.11 Clean porcine intestinal mucus dialyzed in 50,000Da MWCO tubing was shown to be a cytocompatible gel and protected cells from simulated intestinal fluid for 2 hours. The same results were obtained when the mucus was spun for 1.5 hours after dialysis to remove excess water.

[0032] Fig.12 Shown are the effects of various sterile solvents on 5 mg / ml gastric mucus after 24 hours of incubation with gastric cells.

[0033] Fig.13 Shown are the colony forming units present in mucus after sterilization with various solvents.

[0034] Fig.14 The Caco-2 cell viability of cells incubated with RM layers of different concentrations and volumes is shown, and EPDF is applied to the mucus layer surface (n=3). Cells were incubated with RM and EPDF for 1 hour (upper left), 2 hours (upper right), 3 hours (lower left), and 4 hours (lower right). The analysis included a live control exposed to PBS and a 0 mucus control exposed to EPDF (i.e., a model without mucus but with a fluid such as digestive fluid). The y-axis shows the cell viability (%) determined by the cell titer blue assay. The y-axis shows the corresponding volume of the live control and RM. The black bar shows the live control, the dark gray bar represents 25 mg / mL RM, the light gray bar represents 50 mg / mL RM, and the white bar represents 75 mg / mL RM. The bars marked with asterisks are significantly different from the live control. In the 2-hour figure, a pair of asterisks are shown to represent groups that share significance with the mucus-free control group and the live control group.

[0035] Fig.15 The graph depicts all apparent permeability values ​​for all APIs at all mucus concentrations and volumes. The y-axis shows the apparent permeability of the API in cm / s x 10 -6Indicates. The x-axis shows the API. The x-axis also has two legends, one indicating the Log D value or Log P value of the API, and the other indicating the API charge. The APIs are grouped based on their physiological charge and permeability values. The legends shown in the graph indicate the mucus conditions to which each bar applies. PNM stands for processed native mucus, i.e., cleaned porcine small intestinal mucus. RM X(X) represents the reconstituted mucus volume (outside brackets) in μL; and the concentration (inside brackets) in mg / mL. The apparent permeability values ​​of APIs with higher Log D values ​​or Log P values ​​and positive charges are generally much lower than the apparent permeability values ​​of APIs that do not have higher Log D values ​​or Log P values ​​and positive charges.

[0036] Fig.16 Pearson correlation of apparent permeability of 11 APIs through 20 μL, 50 μL and 70 μL of 25 mg / mL reconstituted mucus and 10 μL, 15 μL and 20 μL of PNM (processed / cleaned native mucus) is shown. The permeation process was repeated three times. The y-axis shows the apparent permeability of the API through the RM. The x-axis shows the apparent permeability of the API through the PNM. The apparent permeability is expressed as cm.s -1 x10 -6 (A) 20μL RM and 10μL PNM. (B) 50μL RM and 10μL PNM. (C) 70μL RM and 10μL PNM. (D) 20μL RM and 15μL PNM. (E) 50μL RM and 15μL. (F) 70μL RM and 15μL PNM. (G) 20μL RM and 20μL PNM. (H) 50μL RM and 20μL PNM. (I) 70μL RM and 20μL PNM. All relationships

[0037] Fig.17 Figure 2 shows the correlation of the apparent permeability of 11 APIs through 20 μL, 50 μL, and 70 μL of 75 mg / mL reconstituted mucus versus 10 μL, 15 μL, and 20 μL of PNM (processed / cleaned native mucus). The y-axis shows the P value of the API through the RM. app The x-axis shows the apparent permeability of the API through the PNM. The apparent permeability is expressed in cm.s -1 x10 -6Indicates. (A) 20μL RM and 10μL PNM. (B) 50μL RM and 10μL PNM. (C) 70μL RM and 10μL PNM. (D) 20μL RM and 15μL PNM. (E) 50μL RM and 15μL PNM. (F) 70μL RM and 15μL PNM. (G) 20μL RM and 20μL PNM. (H) 50μL RM and 20μL PNM. (I) 70μL RM and 20μL PNM. Figures C, F, and I are Spearman rank correlations, and the others are Pearson correlations. All relations except those in Figure I are

[0038] Fig.18 Pearson correlations of the apparent permeability of 11 APIs through 20 μL, 50 μL, and 70 μL of 50 mg / mL reconstituted mucus versus 10 μL, 15 μL, and 20 μL PNM (processed / cleaned native mucus) are shown. The permeation process was repeated three times. The y-axis shows the apparent permeability of the API through the RM. The x-axis shows the apparent permeability of the API through the PNM. The apparent permeability is expressed in cm.s - 1 x10 -6 Indicated. (A) 20μL RM and 10μL PNM. (B) 50μL RM and 10μL PNM. (C) 70μL RM and 10μL PNM. (D) 20μL RM and 15μL PNM. (E) 50μL RM and 15μL. (F) 70μL RM and 15μL. (G) 20μL RM and 20μL PNM. (H) 50μL RM and 20μL PNM. (I) 70μL RM and 20μL PNM. All relationships are significant.

[0039] Fig.19 Pearson correlations for the API apparent permeability dataset (positively charged API removed from the dataset) are shown. The y-axis shows the apparent permeability of the API through the reconstituted mucus. The x-axis shows the apparent permeability through the PNM (processed / cleaned native mucus). The apparent permeability is expressed in cm / s x10 -6 Legends are shown above each column of graphs (PNM volume) and within each graph (RM concentration and volume). All relationships are significant (p < 0.05). 2 The values ​​indicate how well the data fit the correlation. The correlations varied in strength, with the best relationships observed at 50 μL of 50 mg / mL with 10 μL and 15 μL of PNM (0.87 and 0.80, respectively) and 50 μL of 75 mg / mL with 10 μL of PNM (0.83).

[0040] Fig. 20 Pearson correlations for the API apparent permeability dataset (API with Log P>3 was removed from the dataset) are shown. The y-axis shows the apparent permeability of the API through the reconstituted mucus. The x-axis shows the apparent permeability through the PNM (processed / cleaned native mucus). The apparent permeability is expressed in cm / s x10 -6 Legends are shown above each column of graphs (PNM volume) and within each graph (RM concentration and volume). All relationships are significant (p < 0.05). 2 The value indicates the degree of fit between the data and the correlation. The correlation strength varies. At 10 μL PNM (50 μL of 50 mg / mL, R 2 =0.92; 70 μL of 50 mg / L, R 2 =0.87; 50 μL of 75 mg / mL, R 2 =0.88) observed the best relationship.

[0041] Fig.21 Figure 2 shows the permeation of 13 APIs through Caco-2 cells of the integrated model. app P eff The y-axis shows the measured Caco-2 cell P app , in cm / s x10 -6 The y-axis shows the x10 -6 Human jejunum P eff The integrated model runs were performed using 50 μL of 25 mg / mL, 50 μL or 70 μL of 50 mg / mL, or 50 μL of 75 mg / mL reconstituted mucus concentrations and volumes. 2 The explanation is as follows: moderate intensity R 2 The values ​​ranged from 0.4 to 0.69, with a high correlation R 2 The values ​​ranged from 0.7 to 0.89. In all correlations, significant relationships were observed (p < 0.05). (A) 50 μL of 25 mg / mL RM and P eff A moderately strong correlation was observed, as evidenced by an R of 0.6480. 2 The trend line equation is y = 0.0011x + 0.1229. (B) 50 μL of 50 mg / mL RM and P eff A moderately strong correlation was observed, as evidenced by an R of 0.6839. 2 The trend line equation is y = 0.0014x + 0.3123. (C) 70 μL of 50 mg / mL RM and P eff A moderately strong correlation was observed, as evidenced by an R of 0.68.2 The trend line equation is y = 0.0011x + 0.1229. (D) 50 μL of 75 mg / mL RM and P eff A strong correlation was observed, as indicated by an R of 0.70. 2 The value shows that the trend line equation is y = 0.0012x + 0.1957.

[0042] Fig. 22 Caco-2 cell P showing passive absorption and permeation of 8 APIs through the integrated model app P eff Spearman rank correlation analysis. The y axis shows the P app , unit is cm / sx10 -6 y shows the value in cm / sx10 -6 Human jejunum P eff The integrated model runs were performed using RM concentrations and volumes of 50 μL of 25 mg / mL, 50 μL or 70 μL of 50 mg / mL, or 50 μL of 75 mg / mL. 2 For interpretation of values, see Section 2.2.19. 2 The explanation is as follows: moderate intensity R 2 The values ​​ranged from 0.4 to 0.69, while the R 2 The values ​​ranged from 0.7 to 0.89. In all correlations, significant relationships were observed (p < 0.05). (A) 50 μL of 25 mg / mL RM and P eff A strong correlation was observed, with an R of 0.86. 2 The trend line equation is y = 0.0038x + 0.64. (B) 50 μL of 50 mg / mL RM and P eff A moderate correlation was observed, with an R of 0.65. 2 The trend line equation is y = 0.0015x + 0.3187. (C) 70 μL of 50 mg / mL RM and P eff A moderate correlation was observed, with an R of 0.6590. 2 The trend line equation is y = 0.0012x + 0.1426. (D) 50 μL of 75 mg / mL RM and P eff A strong correlation was observed, with an R of 0.71. 2 The value shows that the trend line equation is y = 0.0012x + 0.1943.

[0043] Fig.23Caco-2 cell viability of cells incubated with DM (300) (neat or diluted 1:1 before dialysis) with PBS or EDPF for 1 hour, 2 hours, 3 hours, or 4 hours is shown. The x-axis shows the mucus group. The y-axis shows the Caco-2 cell viability in %. The legend of the graph indicates the time point analyzed. The statistical bars indicate where there is a statistical difference in the mean viability values. Statistics were determined by a two-way ANOVA test. Asterisks indicate the significance level of the data. Single asterisks indicate p<0.05. ANOVA was performed only at a single time point. Intergroup comparisons between time points are invalid. DETAILED DESCRIPTION

[0044] The present invention is based on the unexpected discovery of the inventors that purified biocompatible mucus can be obtained using dialysis and / or equilibrium density gradient centrifugation, which mucus retains the ability to form a gel. The method described in the present invention does not degrade, hydrolyze or denature the mucins of the mucus, thereby enabling the mucins to retain the ability to form mucin-mucin interactions, thereby providing viscoelastic properties to the purified mucus. Therefore, the purified mucus is able to form a gel. The inventors have also shown that the purified mucus is biocompatible (also called biocompatible) and can therefore be applied to cells without producing cytotoxic effects. Therefore, the present invention provides for the first time a mucin preparation suitable for an in vitro model of digestion, mucus penetration and / or epithelial transport. definition

[0045] It must be noted that, as used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0046] As used herein, "about" will be understood by a person of ordinary skill and will vary to some extent depending on the context in which it is used. If the use of the term is unclear to a person of ordinary skill in view of the context in which it is used, "about" will mean within plus or minus 10% of the specified value.

[0047] As used herein, the terms "comprise", "comprises" and "comprising" specify the presence of features, integers, steps, operations, elements and / or components described in one aspect or embodiment of the present invention, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0048] The expression "consisting essentially of..." means that the scope of an aspect or embodiment should be interpreted as covering the specific materials or steps listed in the aspect or embodiment, including any materials or steps that have no substantial impact on the invention defined in the aspect or embodiment. Therefore, the term "consisting essentially of..." when used in the claims of the present invention is not intended to be interpreted as equivalent to "comprising".

[0049] "Contacting" means bringing two or more items into contact with each other. The items may be two or more of cells, substrates, fluids, and purified mucin or biosimilar mucus as appropriately defined herein. Contacting may include bringing two or more of the above items into close physical relationship with each other.

[0050] "Detecting" ("detect" and "detection") refers to identifying and / or quantifying a compound of interest (e.g., a drug, an agent, etc.) in a sample. In some embodiments, detecting comprises determining whether a compound of interest is present in a sample. In some embodiments, detecting comprises quantifying the compound of interest in a sample. In some embodiments, detecting comprises identifying and / or quantifying the compound of interest in a sample at different time points. In some embodiments, detecting comprises identifying and / or quantifying the compound of interest in a first sample and a second sample.

[0051] "Absorption" or "perfusion" refers to the movement of a compound (e.g., a test compound or drug) into the bloodstream and through tissues after administration, as well as the movement of a compound through tissue models described herein. Absorption or perfusion is determined by the physicochemical properties of the compound, the formulation, and the route of administration.

[0052] As used herein, "tissue" is a group of cells that function together as a unit. Tissue is a level of cellular organization between a single cell and a complete organ. Thus, an organ is composed of multiple tissues. Tissue can be connective tissue, muscle tissue, neural tissue, or epithelial tissue. For purposes of the present invention, tissue can be epithelial tissue that secretes mucus.

[0053] "Epithelium" is a single layer of cells that covers all biological surfaces exposed to the external environment, forming a semipermeable barrier. These surfaces include the airways, skin, or digestive tract of mammals.

[0054] The "alimentary canal" refers to the complete system of organs and regions involved in the intake, digestion, and excretion of food and fluids. This system is generally composed of, but not limited to, the mouth, esophagus, stomach and / or rumen, intestines (large and small intestines), cecum (multiple blind sacs), fermentation sacs, and anus.

[0055] "Enterocytes" refer to cells that make up the intestinal epithelium of mammals. These cells can be found in both the small intestine and the colon. The intestinal epithelium of the mammalian digestive tract has a well-defined organizational structure. The epithelium can be divided into two regions, a functional region that accommodates differentiated cells (absorptive enterocytes wrapped in villi) and a proliferative region (Lieberkuhn crypts) that represents the microenvironment of epithelial stem cells. Multipotent epithelial stem cells are present in the crypts and produce four major epithelial lineages: absorptive enterocytes, goblet cells that secrete mucin, enteroendocrine cells that secrete peptide hormones, and Paneth cells.

[0056] "Intestine" refers to the mammalian small intestine and the mammalian large intestine.

[0057] The term "in vitro" refers to a process that is performed or occurs outside a living organism. In some embodiments, the process is performed or occurs in a culture dish.

[0058] The term "in vivo" refers to processes that occur within a living organism.

[0059] The term "mucin" refers to a high molecular weight glycosylated protein that is a component of mucus and has the ability to form mucin-mucin interactions to form gels. Mucin-mucin interactions include covalent and non-covalent bonding. Covalent bonding includes disulfide bonds between mucin glycoproteins.

[0060] The term "mucus" refers to an aqueous colloid comprising mucin and optionally inorganic salts, immunoglobulins, nucleic acids, proteins and / or glycoproteins. Native mucus is swellable. Native mucus moisturizes and protects tissues, but may also permeate small compounds (such as amino acids and small molecule sugars), especially in the small intestine. Native mucus is a viscous secretion, usually rich in mucin, produced by the mucosa, moisturizing and protecting the mucosa.

[0061] "Tissue explant" refers to an isolated piece or pieces of tissue.

[0062] As used herein, "substrate" refers to any surface suitable for supporting biological materials (e.g., cells, cell groups, cell cultures, tissues or fluids or other biological materials or compositions as described herein). The surface can be suitable for supporting processes or reactions, such as the growth and development of cells or organisms, cell groups, cell cultures or tissues.

[0063] A "gel" is a semisolid, jelly-like substance that does not flow when in a solid state. A gel contains a 3D cross-linked network, making it a semisolid structure. A gel may be a hydrogel, containing a network of insoluble but hydrophilic polymer chains. A gel may be defined by its viscoelastic or rheological properties.

[0064] A "model" as referred to herein is a three-dimensional representation of all or part of a native tissue structure.

[0065] A "buffer" is a solution that resists a significant change in pH after a small amount of acid or base is added. A buffer is a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid.

[0066] "Dialysis" is a process of separating molecules in solution based on the difference in the diffusion capacity of molecules through a semipermeable membrane. Dialysis can be used to remove small molecules, such as salts, peptides and dyes, from larger molecules (e.g., DNA, proteins (including glycoproteins) and polysaccharides). The semipermeable membrane can be cellulose, modified cellulose or synthetic parameters. The process may require the use of a dialysis buffer into which the sample to be dialyzed is placed. Optimization of the dialysis process may require adjustment parameters based on sample size, the size of the separated molecules, the type of membrane and the geometry of the membrane. Suitable conditions are known to those skilled in the art.

[0067] "Fractionation" is the separation process of dividing a mixture into several smaller fractions based on the specific properties of each component of the mixture. An example of fractionation is centrifugation.

[0068] As used herein, "cell viability" refers to the ability of a cell to remain metabolically active in order to grow and function.

[0069] A "test compound" is any chemical compound to be studied, such as a compound for use in humans or animals.

[0070] "LVE region" refers to the range in which testing can be performed without damaging or disturbing the sample structure.

[0071] "Purified" herein refers to mucus that has been subjected to a process of removing some or all components other than mucin (e.g., nucleic acids, proteins, immunoglobulins, salts, enzymes, and contaminants). Thus, the purified mucus contains a higher concentration of mucin than the native mucus from which it is derived. The purified mucus may contain a lower concentration of one or more of nucleic acids, proteins, immunoglobulins, salts, enzymes, and contaminants than the native mucus from which it is derived. The purified mucus may be partially purified mucin, or completely purified mucin, for example, the product is substantially pure mucin (i.e., at least 95% or more mucin).

[0072] "Biocompatibility" may also be referred to as "biological compatibility" and in this context means that the substance or substrate to which it refers is substantially non-toxic or non-detrimental to living cells.

[0073] As used herein, the term "reconstituted mucus" refers to the purified mucus of the present invention that has been reconstituted in a solution (eg, a buffer). The reconstituted mucus has suitable biocompatibility. Mucus preparation

[0074] The present invention is based on purifying mucus to provide a preparation containing mucin, wherein the preparation has the rheological properties of native mucus and can form a gel without the need for synthetic polymers or non-natural polymers. The method described in the present invention can purify mucus to provide a preparation in which the native mucin is not denatured, degraded or hydrolyzed, thereby maintaining the interaction of mucin: mucin. It is these interactions that provide viscoelasticity and the ability to form a gel for the mucus preparation. The purified mucus described in the present invention may have one or more rheological properties of native mucus (suitably the native mucus of its source). The purified mucus described in the present invention is also unexpectedly shown to be biocompatible and can therefore support living cells. Therefore, the present invention provides a purified biocompatible mucus according to the first aspect. The purified biocompatible mucus of the present invention can be prepared using the method of the third aspect.

[0075] In one embodiment, the purified biocompatible mucus is obtained from a mucus sample, such as from a tissue sample. Suitably, the purified biocompatible mucus is obtained from mucus obtained from a tissue sample by a method according to the third aspect of the invention as described herein.

[0076] The mucin of the purified biocompatible mucus can be a polymer. The molecular weight of the mucin polymer can be 1 million to 10 million Daltons. Mucin can be a glycoprotein. Mucin glycoprotein can include a peptide core composed of a plurality of mucin monomer units connected by disulfide bonds, and the peptide core is attached to a plurality of carbohydrate side chains. The carbohydrate side chains can be O-connected or N-connected oligosaccharides. One or more carbohydrate side chains can be negatively charged. Mucin can be saturated with O- or N-connected oligosaccharides. Mucin can include cysteine-rich, lightly glycosylated amino and carboxyl terminal regions. Mucin can be a transmembrane protein, including a cytoplasmic region, a transmembrane region, and an extracellular glycoprotein.

[0077] The purified biocompatible mucus obtained by the present invention can form a gel when placed under appropriate conditions such as pH, concentration, temperature, etc. required for gelation. These conditions are known or can be derived by a technician based on his or her own knowledge of the field. Suitably, when both are placed under the same conditions, the purified biocompatible mucus of the present invention will have substantially the same viscoelasticity as the mucus from which it is derived.

[0078] Specifically, the purified biocompatible mucus of the present invention may exhibit liquid-like and solid-like properties. The purified biocompatible mucus of the present invention may exhibit linear viscoelasticity. The shear strain and stress strain of the purified biocompatible mucus of the present invention in the linear viscoelastic region may be substantially equal to or greater than the shear strain and stress strain of the native mucus from which it is derived or the cleaned version of the native mucus from which it is derived. The shear strain of the purified biocompatible mucus of the present invention in the linear viscoelastic region may be greater than 5%. The storage modulus (G') of the composition of the present invention may be 1 or greater. The loss modulus (G") of the composition of the present invention may be 1 or greater. The phase angle of the composition of the present invention in the linear viscoelastic region may be at least 3 to 20, or any range or integer therebetween. Suitably, the mucus preparation of the present invention may have one or more, two or more, or three or more rheological properties of native mucus or cleaned mucus, wherein the rheological properties are selected from the group consisting of shear strain, stress strain, storage modulus, loss modulus and phase angle.

[0079] The purified biocompatible mucus, in addition to the above-defined viscoelastic properties, also has one or more physical and / or chemical properties similar to native mucus, such as osmotic pressure, charge, and containing region-specific mucins.

[0080] The purified biocompatible mucus of the present invention may contain one or more additional components, including but not limited to lipids, salts, immunoglobulins, enzymes, proteins, nucleic acids (DNA or RNA), glycoproteins, cells and water. One or more of these components may be native components or non-native components of the mucus from which the purified biocompatible mucus is derived.

[0081] In one embodiment, the purified biocompatible mucus of the present invention does not substantially contain lipids, nucleic acids or proteins of native mucus. Therefore, the mucus preparation of the present invention is prepared by a method of removing substantially all lipids, proteins and nucleic acids from native mucus. The density of the purified mucus of the present invention may be 1.3 to 1.6 g / mL, appropriately 1.4-1.5 g / mL, and most appropriately 1.42 g / mL. Suitably, the biocompatible mucus does not contain components (e.g., lipids, nucleic acids or proteins) having a density higher or lower than 1.3-1.6 g / mL. Optionally, the purified mucus of the present invention does not contain components having a molecular weight lower than 30 kDa, preferably 20 kDa or preferably 15 kDa. After dialysis and / or equilibrium density gradient centrifugation, but before adding any other components (e.g., buffers), the density and / or molecular weight associated with the purified mucus can be provided.

[0082] In one embodiment, the purified biocompatible mucus of the present invention does not contain components having a molecular weight of 100 kDa or less, preferably 70 kDa or less, preferably 60 kDa or less or preferably 50 kDa or less.

[0083] Biocompatibility means that the purified mucus is not toxic or harmful to cells. Therefore, the purified mucus is compatible with cells and does not induce or cause cell death within an appropriate period of time. The composition is able to maintain the viability of cells. Therefore, the composition does not cause a significant loss of cell viability. Significantly means that 30% or more of the cells in the cell population lose viability within a specified time period (e.g., a few minutes, one or more hours, or up to several days), depending on the nature of the composition, the cell type, and the conditions of the model.

[0084] In one embodiment, the mucus is purified from mammalian or non-mammalian mucus. The mammal may be a human; a domestic animal such as a dog, cat, guinea pig, rabbit, rat, mouse; a farm animal such as a pig, sheep, goat, donkey, horse, cattle, cow; or any other large mammal such as a deer, antelope, elephant, camel, llama, etc. Those skilled in the art will be aware of any other suitable mammal from which mucin may be extracted. The mammal may be a pig. The mammal may be a human. The non-mammal may be a fish, an amphibian, a snail, a slug or any other suitable animal. Suitably, the purified biocompatible mucus is derived from a mammal, suitably from an animal, suitably from a human. Suitably, the purified biocompatible mucus is derived from a pig, suitably from the gastrointestinal system of a pig, suitably from the intestine (large intestine or small intestine) or stomach of a pig.

[0085] The purified biocompatible mucus may be extracted from any suitable tissue of the mammal or non-mammal. When the purified biocompatible mucus is extracted from a mammal, it may be extracted from any tissue that naturally contains or secretes mucus. For example, the tissue from which mucin is extracted according to the present invention may be gastrointestinal tissue, including but not limited to stomach, duodenum, esophagus, cheek, tongue or colon tissue; airway tissue, including but not limited to lung, trachea, bronchi and bronchioles and oral cavity; female reproductive tissue, including but not limited to cervical tissue and vaginal tissue; and nasal tissue. In one embodiment, the tissue from which the biocompatible mucus is extracted may include epithelial cells. When the biocompatible mucus is extracted from a non-mammal, it may be extracted from the outer surface of the non-mammal.

[0086] The purified biocompatible mucus described herein can be 100% pure mucin, or can contain other non-mucin components (e.g., contaminants), native mucus components as described herein, including biomolecules remaining during extraction. The purified mucus described herein can contain 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% mucin. Suitably, the purified biocompatible mucus contains at least 35%, suitably at least 40% mucin. The remainder of the preparation can include water, salts, lipids, proteins, sugars, glycoproteins, immunoglobulins, bacteria, reagents and / or contaminants. If the purified biocompatible mucus of the present invention is produced by the method of the third aspect, before the purified mucus is reconstituted in a buffer, the purified biocompatible mucus may contain 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% mucin. Suitably, the purified biocompatible mucus contains at least 35%, suitably at least 40% mucin. Mucin is rich in serine, proline and threonine, and the purified biocompatible mucus of the present invention may be rich in serine, proline and threonine. For example, the purified biocompatible mucus of the present invention may contain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% serine, proline and threonine, and most suitably at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% serine, proline and threonine. The purified biocompatible mucus of the present invention may be substantially free of or contain a low content (e.g., less than 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) of amino acids other than serine, threonine or proline, such as valine,

[0087] Suitably, the purified biocompatible mucus of the present invention may contain less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% impurities (i.e. non-mucin).

[0088] The purified biocompatible mucus described herein can be a viscous gel-like preparation. Alternatively, it can be a solid, for example, it can be dried or lyophilized to provide a preparation for storage. Alternatively, it can be a liquid, for example, a liquid after reconstitution in a suitable buffer or diluent.

[0089] Purified biocompatible mucus can be provided in a buffer to form a composition. The buffer may be suitable for use with cells or living tissues. The buffer may be suitable for use with acidic gastrointestinal secretions. The buffer may not change the viscoelasticity of the purified biocompatible mucus. The buffer may be cell compatible. Examples of suitable buffers include, but are not limited to, deionized water, phosphate buffers (e.g., phosphate buffered saline), HEPES buffers, MOPS buffers, MES buffers, Tricene, Bicene, TAPS, ACES, MOPSO, and BES. Other suitable buffers may be used, which are known to those skilled in the art. In one embodiment, the buffer is phosphate buffered saline, representing a body fluid with the correct pH and osmotic pressure. The buffer may be supplemented with, for example, calcium and / or magnesium. The buffer may be PBS supplemented with calcium and magnesium.

[0090] The purified biocompatible mucus may be provided in a buffer at any suitable concentration. In one embodiment, the concentration of the purified biocompatible mucus in the composition is similar to that of the native mucus. The purified biocompatible mucus may be present in the composition at a concentration of 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml or more. In one embodiment, the biocompatible mucus is present in the composition at a concentration of 5-120 mg / ml, suitably 30-100 mg / ml, suitably 40-80 mg / ml. The concentration will depend on the presence and amount of other components in the composition described herein.

[0091] The purified biocompatible mucus can be provided together with cells, or as a composition comprising cells.

[0092] The mucin present in the purified biocompatible mucus can be human MUC2, MUC5AC, MUC5B, MUC6, MUC19, MUC7 or its non-human equivalent, including, for example, ovomucin (egg white), spiggin (MUC19-derived mucin from stickleback fish), SCO-spondin or otogelin (see also Lang et al Mol Biol Evol. 2016 Aug; 33(8): 1921-1936). In one embodiment, the mucin is MUC5AC or MUC5B. In one embodiment, the mucin is MUC2.

[0093] In one embodiment, there is provided a composition consisting essentially of the purified biocompatible mucus of the first aspect and a buffer. The biocompatible mucus and the buffer may be as described herein. The biocompatible mucus may be purified according to the third aspect of the invention as described herein. The concentration of mucin may be 20-120 mg / ml, suitably 30-100 mg / ml, suitably 40-80 mg / ml, suitably 50 mg / ml. Organizational Model

[0094] In a second aspect of the invention, a tissue model is provided, comprising: i) a cell population and ii) a purified biocompatible mucus according to the first aspect. Suitably, the purified mucus is in contact with the cell population. The tissue model according to the second aspect of the invention can be used for screening assays, such as screening assays for drug absorption, drug dissolution and drug toxicity. The second aspect of the invention is based on the unexpected discovery that the purified biocompatible mucus of the first aspect can be used to construct a reconstructed mucus layer that can bind to a cell population without killing the cells. This model provides a robust and physiologically relevant in vitro method in which drug preparations can be screened and used to improve and guide human studies.

[0095] The tissue model according to the second aspect of the present invention may comprise a substrate, the substrate comprising a cell support portion, the cell population may be in contact with the cell support portion of the substrate, and the cell population may form a layer on the cell support portion.

[0096] The tissue model can be described as having a basolateral surface (i.e., the bottom of the substrate) and an apical surface (i.e., the top surface of the tissue model). In the tissue model of the present invention, the purified biocompatible mucin is applied to the top surface of the cell population and contacts the cell population. The purified mucin may not be in direct contact with the cell-supporting portion of the substrate.

[0097] In one embodiment, there is provided a tissue model of the first aspect, comprising in discrete layers: i. a substrate comprising a cell support portion; ii. a cell population in contact with a cell-supporting portion of a substrate; iii. Purified biocompatible mucus contacted with cells.

[0098] In this article, any suitable substrate with a suitable carrying cell group part can be used. Suitably, the substrate is suitable for carrying or culturing mammalian cells. The substrate can be a substrate that is easy to sterilize and has biocompatibility. Suitable substrates will be known to those skilled in the art, and may include but are not limited to glass, carbon, organic materials (such as cotton), nitrocellulose, dextran, gelatin, polymer materials (such as plastics, polymethyl methacrylate, polyamide, polyester; polystyrene; polypropylene, polyurethane polyacrylate, polyethylene compounds (such as polyvinyl chloride); polycarbonate; polytetrafluoroethylene (PTFE, Teflon), acrylic copolymer polyglycolic acid (PGA)); fluoropolymers; fluorinated ethylene propylene; polyvinylidene; polydimethylsiloxane), metals (such as gold, silver, aluminum, titanium, stainless steel), silicon substrates (fused quartz, polycrystalline silicon or monocrystalline silicon).

[0099] The substrate can be rigid, or can be elastic. The substrate can be porous, or can be non-permeable. Non-natural porous materials can be made into porous materials by methods available to the skilled person (e.g., sintering, etching, leaching, photolithography, or laser micromachining).

[0100] The substrate can be a slide, a chip, a plate, a bottle (flask), a vial, a film, a microstructure (including grooves, holes or columns), a porous plate, a dual chamber plate or any other suitable form. The substrate can have any suitable shape or structure, including flat, tubular, curved, spherical, ellipsoidal, etc., including composite shapes or structures (for example, for simulating macroscopic anatomical structures). The substrate can include a plane, on which a cell population can be provided. The substrate can provide or be installed on a porous carrier (such as a porous membrane, a net, an inorganic grid, a hydrogel or a combination thereof) to provide structural support thereto. The substrate can be two-dimensional or three-dimensional, or any combination thereof.

[0101] Suitably, the substrate can be a dual chamber plate, wherein each well comprises a lower chamber and an upper chamber. The upper chamber can be located on top of the lower chamber or placed within the lower chamber. Suitably, the upper chamber and the lower chamber are substantially aligned. The lower chamber can be referred to as a basolateral chamber and the upper chamber can be referred to as an apical chamber. The bottom plane of the upper chamber can be permeable, thereby enabling fluid to flow from the upper chamber to the lower chamber. Suitably, the upper chamber is located entirely or partially within the lower chamber to achieve fluid communication between the two chambers. The upper chamber can contain a cell population and a purified mucin layer. The lower chamber can contain a buffer or a suitable cell culture medium to maintain the cell population. Thus, for example, in a small intestine model, the lower chamber represents the basolateral side of the epithelial cells of the villus-associated circulating blood vessels and lymphatic vessels. The upper chamber represents the lumen of the small intestine.

[0102] Suitably, the substrate may be provided in an anaerobic envelope to provide an anaerobic environment. Where the substrate is a dual chamber plate as described above, the lower chamber may be provided with an air supply to create an aerobic environment, while the upper chamber may be an anaerobic environment.

[0103] Choosing an appropriate substrate may depend on the cell type used in the model. Factors may include the ability of the cells to adhere to the substrate, as well as the effects of the substrate on the cells.

[0104] The substrate can be modified to achieve or improve cell adhesion to it. All or part of the substrate can be modified. Suitable modifications are known to those skilled in the art and can include, for example, applying proteins (e.g., collagen or fibronectin), extracellular matrix or its components, sugars, proteoglycans to the substrate surface.

[0105] The substrate can be a single-well plate, or can be a multi-well plate. The multi-well plate can contain any suitable number of holes, such as 6, 12, 24, 48, 96, 384 or 1536 holes. The hole can be a micropore.

[0106] In the model of the second aspect, the cell population is provided in contact with a substrate, suitably in contact with a substrate plane, such as the bottom of a well forming a cell support portion. The cell population may comprise one or more cells, suitably two or more, up to 1000, 10000, 1 x 10 6 pcs, 2x 10 6 pcs, 3x 10 6 or more cells.

[0107] The cell group can form a cell layer. Suitably, the cell group forms a layer of at least 50%, 60%, 7%, 80%, 90% or 100% confluence. Therefore, the cell layer can be referred to as confluence. The cell layer in contact with the cell support portion can be a monolayer of cells (meaning that the depth of the layer is one cell), or can be more than one layer of cells (the depth is two or more cells). The cell can include one type of cell, or two or more different cell types. In a multilayer cell group, each layer can basically include different cell types. The cell group provided on the substrate can be a tissue explant. The tissue explant can be a monolayer of tissue, or can be two or more layers of tissue. The tissue explant can be provided on the cell support portion of the substrate and compressed. The tissue explant can be derived from an organism (e.g., a mammal), or can be cultured in vitro.

[0108] Tissue models can have the architecture of corresponding native in vivo tissues.

[0109] When cells form layers on the cell-supporting portion of the substrate, they layers may be flat, or may be folded or formed into three-dimensional shapes, for example to mimic tissue structures such as the crypt structure or crypt-villus structure of the intestine in vivo, or the stratified squamous epithelial layer of the vagina.

[0110] The cell group, optionally as a tissue explant, can be derived from gastrointestinal tissue, airway tissue, nasal tissue, visual tissue or female reproductive tissue. The cell group, optionally as a tissue explant, can be derived from the ileum, jejunum, stomach, duodenum, esophagus, cheek, tongue or colon of the digestive tract, or from the nose, lung, bronchi, bronchioles or oral cavity; or from the cervix or vagina or from the eye. The cell can be obtained from a mammal (e.g., pig) as defined herein. The cell may be derived from the human body. In one embodiment, the tissue explant is derived from the digestive tract of the human body, appropriately from the ileum, jejunum, stomach, duodenum, esophagus, cheek, tongue and / or colon of the human body. In one embodiment, the cell used for the second aspect model is an oral cell, a gastric cell, a small intestinal cell, a large intestinal cell, a nasal cell, a visual cell, a vaginal cell, a cervical cell or a lung cell, a bronchial cell or other airway cell. The cell can be an epithelial cell. The cell culture can be derived from a cell line. In one embodiment, the cell culture is a caco-2 cell culture or a calu-3 cell culture.

[0111] The cell population or tissue may include native fluid or tissue components, such as sputum, nasal discharge, urine, vomit, bile, blood, vaginal secretions, semen or other biological secretions, microorganisms (eg, bacteria), and cellular debris.

[0112] The cell population can be in contact with the cell supporting portion of the substrate in a manner that maintains native cell polarity. Thus, the native basal surface of the tissue can be in contact with the cell supporting portion of the substrate, with the luminal surface of the tissue (which would face the lumen of the tissue or organ in vivo) being uppermost and separated from the cell supporting portion of the substrate by the bulk of the cell population.

[0113] The top surface of the tissue model may be referred to herein as the apical surface and the bottom surface may be referred to as the basolateral surface.

[0114] The substrate can contain two or more tissue explants. For example, when the substrate is a multi-well plate, each hole or cell support portion can be in contact with a different tissue explant. The explants can be the same or different tissue types. The explants can be from the same type of mammal or different types of mammals. The tissue explants can all be from the human body.

[0115] The cell mass of the second aspect model of the present invention can maintain culture for example any suitable time period under appropriate conditions, for example a few minutes to one hour or longer, 30 minutes to 2 hours, 30 minutes to 3 hours, 30 minutes to 4 hours, 30 minutes to 5 hours, 30 minutes to 6 hours, 1 hour to 12 hours, 1 hour to 24 hours, 1 hour to 36 hours, 1 hour to 48 hours, 1 hour to 72 hours or 1 hour to 1 week, 2 weeks, 3 weeks, 4 weeks or longer. Cell mass may need exogenous growth factor to maintain culture, or may not need exogenous growth factor. Term " maintain culture " refers to that cell mass is suitable for cell growth and / or survival under the condition of maintaining in vitro. This may need to provide essential nutrients (such as amino acid, carbohydrate, vitamin, mineral substance), growth factor, hormone, gas (such as O , CO ), and maintain suitable physicochemical environment (such as pH, osmotic pressure, temperature). Condition can be aerobic or anaerobic. Examples of substances that can be provided in cell culture to maintain cell growth and / or survival include, but are not limited to: fibronectin; fetal bovine serum / bovine serum; laminin; epidermal growth factor (EGF); R-spondin; noggin; cytokines (e.g., interleukins (e.g., IL-6, IL-17, IL-22), tumor necrosis factor (TNF)); ephrin receptors (e.g., ephrin B, EphB); bone morphogenetic proteins (BMPs, BMP-2, BMP-7); Wnts (wingless-related integration sites) (e.g., Wnt3, Wnt3A and other Wnts); notch signaling factors (notch receptors); Dlll / 4; Noggin; Greml; Grem2; acetate; butyrate; propionate, p-hydroxyphenylpropionic acid, catecholamines (e.g., dopamine, norepinephrine) cytokines and / or short chain fatty acids.

[0116] The purified biocompatible mucus may be as defined in the first aspect. Therefore, in one embodiment, the purified biocompatible mucus may be a composition comprising or mainly consisting of the purified biocompatible mucus as described in the first aspect and a buffer.

[0117] Purified biocompatible mucus can be provided on the cell as a layer. The layer can be a uniform layer or a non-uniform layer. The layer can substantially cover all cells. The layer can have any suitable thickness. Suitably, the purified biocompatible mucus layer simulates the thickness of mucin in native tissue. The thickness of the mucin layer can be any suitable thickness, depending on the purpose, conditions and drug to be tested of the model. Suitable thickness can be 500 μm to 15 mm, 400 μm to 15 mm, 300 μm to 15 mm, 200 μm to 15 mm, 100 μm to 15 mm, 10 μm to 15 mm, 1 μm to 15 mm, 0.1 mm to 15 mm, 0.5 mm to 15 mm, 1 mm to 15 mm or 3 mm to 15 mm, or any range of the upper or lower limit of any of the above ranges, or as the upper or lower limit in the above range or any integer falling therein.

[0118] The tissue model described herein may also include a fluid. The fluid may be provided on the top surface of the mucin layer. Therefore, the fluid may not be in direct contact with the cells. The fluid may be provided to simulate the native tissue environment, and may be, for example, digestive fluid, saliva, vaginal secretions, tears or airway secretions, or a microbial cell population. The fluid may be a native fluid extracted from a mammal as described herein, or may be a synthetic fluid. The fluid may be an end-point digestive fluid (EPDF), such as a fluid comprising saliva, gastric juice and small intestinal fluid, optionally bile and / or enzymes (e.g., pepsin and / or pancreatin). Suitable end-point digestive fluids are shown in Table 1. The composition of the EPDF diluent is shown in Table 2. Any suitable volume of fluid may be applied. Table 1. Component ratios of components involved in the preparation of EPDF Table 2. Compositions of the three diluents involved in the preparation of EPDF.

[0119] Three or more layers of the organizational model may be stacked so that they are fully or partially aligned with one or more layers of the model. Fully or partially overlapped. Suitably, two or more layers are fully aligned. Suitably, all layers are fully aligned.

[0120] The present invention can provide a model of any mucus-covered epithelial tissue.

[0121] In one embodiment, the tissue model of the second aspect of the invention is a stomach model. In one embodiment, the stomach tissue model comprises in discrete layers: i) a base substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; preferably, wherein the cell population is a Caco-2 cell population; iii) purified biocompatible mucus in contact with the cells; and optionally iv) gastric juice.

[0122] In one embodiment, the tissue model of the second aspect of the invention is a small intestine model. In one embodiment, the small intestine tissue model comprises in discrete layers: i) a base substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; preferably, wherein the cell population is a Caco-2 cell population; iii) purified biocompatible mucus in contact with the cells; and optionally iv) gastrointestinal fluid.

[0123] In one embodiment, the tissue model of the second aspect of the invention is a large intestine model. In one embodiment, the large intestine tissue model comprises in discrete layers: i) a base substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; preferably, wherein the cell population is a Caco-2 cell population; iii) purified biocompatible mucus in contact with the cells; and optionally iv) a gastrointestinal fluid, preferably an end-point digestion fluid as described herein.

[0124] In one embodiment, the tissue model of the second aspect of the invention is an airway tissue model. In one embodiment, the airway tissue model comprises in discrete layers: i) a base substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; iii) purified biocompatible mucus in contact with the cells; and optionally iv) saliva, alveolar fluid, sputum. The cell population may comprise any suitable airway cells.

[0125] In one embodiment, the tissue model of the second aspect of the invention is a female reproductive tissue model. In one embodiment, the female reproductive tissue model comprises in discrete layers: i) a base substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; iii) purified biocompatible mucus in contact with the cells; and optionally iv) vaginal fluid. The cell population may comprise any suitable cell of the female reproductive system, including, for example, vaginal cells, cervical cells, uterine cells, fallopian tube cells, or ovarian cells.

[0126] In one embodiment, the tissue model of the second aspect of the invention is a visual or nasal tissue model. In one embodiment, the visual or nasal model comprises in discrete layers: i) a base substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; iii) purified biocompatible mucus in contact with the cells; and optionally iv) nasal secretions or tears. The cells may be any suitable cells of the visual or nasal system.

[0127] In one embodiment, the present invention provides two or more models arranged in series to provide a gastrointestinal model. The gastrointestinal model may include two or more of an airway model, a stomach model, a small intestine model and a large intestine model in series.

[0128] In one embodiment, the models of the invention can include one or more test compounds, such as pharmaceutical agents. The test compound (eg, pharmaceutical agent) can be applied to the model to assess the permeability of the pharmaceutical agent.

[0129] In any tissue model provided herein, the purified biocompatible mucus can be reconstituted in a solution (e.g., a buffer) prior to use in the tissue model. The buffer can be any suitable buffer, such as PBS. The suitable buffer can be biocompatible. Any suitable reconstitution method can be used that does not significantly disrupt the mucin:mucin interaction. The reconstituted mucus of the present invention will be appropriately biocompatible. Method for purifying mucus

[0130] The invention provides a method for purifying mucus, wherein the method comprises removing substantially all lipids, nucleic acids and / or proteins, and / or any molecules below a predetermined size (suitably 100 kDa, more suitably 70 kDa), wherein the removal step comprises dialysis and / or equilibrium density gradient centrifugation. Suitably, the method does not comprise denaturation, degradation or hydrolysis of mucin. Optionally, the method may comprise a further separation step, such as centrifugation, to remove native mucus components, contaminants, excess water and / or reagents. Suitably, the method of the invention provides the purified biocompatible mucus of the first aspect.

[0131] In one embodiment, the present invention provides a method for purifying mucus, wherein the method comprises i) providing a sample comprising mucus; ii) solubilizing any mucin present in the sample; iii) separating any nucleic acids, proteins, lipids from the mucus; and iv) removing any contaminants, unwanted components or reagents from the mucus to provide purified biocompatible mucus. Step iii) may comprise equilibrium density gradient centrifugation. Step iv) may comprise removing any cytotoxic components, reagents and / or enzymes, and may comprise dialysis of the mucus. Suitably, the method comprises separating a fraction having a density gradient of 1.3 to 1.6 g / mL from the mucus.

[0132] Any suitable method may be used for solubilization to increase the solubility of any mucin in the sample. Suitable methods may include, but are not limited to, incubating the tissue sample with a chaotropic agent (e.g., urea, thiourea, particularly 8M urea, or a combination of 2M urea and 5-8M thiourea), an ionic, nonionic or zwitterionic detergent (e.g., SDS, NP-40, Triton X, CHAPS and sulfobetaine), a carrier ampholyte or a reducing agent (e.g., DTT or TBP). After solubilization, any insoluble material may be removed, for example, by centrifugation.

[0133] The step of separating any nucleic acids, proteins, lipids from the mucus may suitably comprise equilibrium density gradient centrifugation and / or dialysis. The method may comprise equilibrium density gradient centrifugation to separate fractions having a density of 1.3-1.6 g / mL, suitably 1.4-1.5 g / mL or most suitably 1.42 g / mL, or fractions identified as having the highest proportion of mucin glycoproteins and the lowest proportion of nucleic acids, lipids or proteins, the fractions being selected using methods available to those skilled in the art, such as periodic Schiff acid analysis and OD260 / OD280 analysis. Alternatively, dialysis may be used to remove any molecule having a molecular weight below 100 kDa, suitably 70 kDa, and most suitably 50 kDa. A combination of equilibrium density gradient centrifugation and dialysis may be used to provide a purified mucus that is substantially free of nucleic acids, lipids and proteins, and suitably free of any chemical reagents or enzymes. The skilled person can determine a suitable combination of centrifugation and dialysis to provide a purified mucus having a low nucleic acid content and a high glycoprotein content, or a density between 1.3-1.6 g / mL, or being free of molecules below 100 kDa, suitably 70 kDa, most suitably 50 kDa. When ultracentrifugation is used, a caesium salt gradient is suitably used, suitably caesium chloride. It is envisaged that other suitable methods having the effect of substantially purifying or isolating mucin from mucus may be used. Such methods (e.g. fractionation, centrifugation, etc.) are known and available to the skilled person.

[0134] Suitably, the step of separating any nucleic acids, proteins, lipids or molecules from the mucus may comprise removing any such components having a density above or below 1.3-1.6 g / mL or below 100 kDa, more suitably below 70 kDa. Any suitable combination of ultracentrifugation and / or dialysis may be used to provide a mucus preparation having a density of 1.3-1.6 g / mL or having removed molecules below 100 kDa, suitably 70 kDa, more suitably 50 kDa.

[0135] A method can include removing any unwanted components or chemicals, such as reagents, enzyme inhibitors, etc. Any suitable method can be used, such as dialysis. Suitably, the method can include dialysis to remove any chemical, pollutant or reagent. Suitably, the molecular weight cutoff (MWCO) of dialysis can be 30kDa or lower, 25kDa or lower, 20kDa or lower, or 15kDa or lower. Any suitable semipermeable membrane can be used, including but not limited to polysulfone, polyethersulfone (PES), etched polycarbonate, cellulose or collagen dialysis tubing. Dialysis can be performed for any suitable time period, depending on the MWCO of the dialysis tubing. An example of a suitable time period can be 1 hour or longer, 3 hours to 5 hours, 5 hours to 10 hours, 5 hours to 12 hours, 12 hours to 14 hours, 36 hours, 48 ​​hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks or longer, or any integer or range therebetween. This dialysis can be performed on the selected fraction after ultracentrifugation of the mucus sample.

[0136] In an alternative method, size exclusion chromatography can be used to prepare purified mucus. The fraction containing mucin can be separated from other components of mucus as described herein using size exclusion chromatography. Suitable chromatographic columns are known to those skilled in the art, but may include Sepharose 2B or 4B columns. Mucin can be collected from the void volume of the chromatographic column.

[0137] Suitably, the method may comprise homogenization of the tissue sample. Homogenization may be performed prior to solubilization and prior to exposure to enzyme inhibitors. Suitable homogenization methods include, but are not limited to, grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thaw, and ultrasound. The sample may be refrigerated to reduce or prevent protein damage.

[0138] Suitably, the method further comprises exposing the sample to one or more enzyme inhibitors. Suitably, the sample is exposed to one or more enzyme inhibitors before solubilization. The enzyme inhibitor may have the effect of reducing or preventing the enzyme present in the sample from cleaving mucin. Suitable enzyme inhibitors include protease inhibitors. The method described in the present invention may comprise exposing the sample to one or more protease inhibitors. Suitably, the method may comprise exposing the sample to inhibitors of aspartic proteases, cysteine ​​proteases, serine proteases, metalloproteases, thiol proteases and / or threonine proteases. Suitable inhibitors or combinations of enzyme inhibitors will be known and available in the art. As non-limiting examples, suitable enzyme inhibitors may be aminocaproic acid, EDTA, iodoacetamide, N-ethylmaleimide, benzamidine hydrochloride or pmsf. Suitably, a combination of two or more enzyme inhibitors may be used. Suitable enzyme inhibitor combinations may comprise any two or more inhibitors selected from aminocaproic acid, EDTA, iodoacetamide, N-ethylmaleimide, benzamidine hydrochloride and / or pmsf. Other suitable enzyme inhibitors or combinations thereof are known to those skilled in the art. The enzyme inhibitor may be applied to the tissue sample in any suitable amount, for example in a v / v ratio (enzyme inhibitor by volume:tissue sample) of 1:1, 2:1, 3:1 or higher. Suitably, the enzyme inhibitor does not cause mucin degradation. The amount and concentration of the enzyme inhibitor may be selected to prevent mucin degradation.

[0139] In an alternative embodiment, the method may comprise: i) providing a sample comprising mucus; ii) dialyzing the mucus to separate any molecules below 100 kDa, suitably below 70 kDa from the mucus; and optionally iii) isolating the mucus, for example by centrifugation.

[0140] In this embodiment, the mucus sample can be dialyzed using dialysis tubing having a MWCO of 100 kDa or less, 70 kDa or less, 60 kDa or less, or 50 kDa or less. Any suitable semipermeable membrane can be used, including but not limited to Polysulfone , polyethersulfone (PES), etched polycarbonate, cellulose or collagen dialysis tubing. Dialysis can be performed for any suitable time period, depending on the MWCO of the dialysis tubing. Examples of suitable time periods can be 1 hour or longer, 3 hours to 5 hours, 5 hours to 10 hours, 5 hours to 12 hours, 12 hours to 14 hours, 36 hours, 48 ​​hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks or longer, or any integer or range therebetween.

[0141] The method of this embodiment may further comprise separation, such as by centrifugation or any other suitable method, to remove excess liquid or other components of the mucus.

[0142] The method of the present invention may include obtaining a mucus sample. The step of obtaining a mucus sample may include scraping or extracting mucus from a tissue sample. The mucus sample may be diluted at any suitable dilution (e.g., 1:50) before purification.

[0143] The method of the present invention may further comprise combining the purified mucus with a solution (e.g., a buffer). The buffer may be as described herein. This step may be referred to as reconstitution of the purified mucus.

[0144] The method of the present invention may comprise combining the purified biocompatible mucus of the present invention with one or more components selected from lipids, salts, proteins, nucleic acids (DNA or RNA), glycoproteins, cells, and water. The buffer and / or one or more of the lipids, salts, proteins, nucleic acids (DNA or RNA), glycoproteins, cells, and water may be as described herein. The method may further comprise placing the purified biocompatible mucus under conditions that cause the mucus to gel.

[0145] The method of the present invention may also comprise drying, freezing, reconstituting or diluting the purified biocompatible mucus of the present invention. In a preferred embodiment, the purified mucus of the present invention is lyophilized. In one embodiment, the lyophilized purified mucus of the present invention is reconstituted in a suitable buffer for use.

[0146] The present invention may also provide a method for preparing the purified biocompatible mucus according to the first aspect, comprising purifying the mucus as described herein, and combining the purified mucus with a buffer and one or more of lipids, salts, proteins, nucleic acids (DNA or RNA), glycoproteins, cells and water to prepare the biocompatible mucus as described herein. The buffer and / or one or more of lipids, salts, proteins, nucleic acids (DNA or RNA), glycoproteins, cells and water may be as described herein.

[0147] The method of purifying mucus from tissue may comprise one or more sterilization steps. A sterilization step may be performed during the method of purifying mucus from tissue, for example by applying one or more antimicrobial agents during a process such as during dialysis. The sterilization step may be performed after purification or after lyophilization. The method of the present invention may comprise one, two, three or more sterilization steps. Any suitable sterilization method may be used, such as temperature change and / or UV-mediated sterilization, or solvent sterilization. Suitable solvents may include, but are not limited to, ethanol, methanol, or IPA. Solvent-based sterilization may include applying a solvent, mixing with tissue, cells, or purified mucus or composition, and evaporating the solvent. Any one or more of the purified mucus or composition comprising the purified mucus may be sterilized.

[0148] The present invention may also provide a method for preparing a tissue model according to the second aspect, comprising purifying mucus as described herein, and applying the purified mucus to a cell population provided on a substrate. The method may include providing the purified mucus as a composition as described herein. The method for preparing a tissue model as described herein may also include applying a fluid as described herein to the purified mucus (which is applied to the cell population).

[0149] The method of forming the purified mucus may comprise employing one or more processes to promote, achieve or maintain gelation or viscoelasticity of the mucus. In one embodiment, the method of forming the purified biocompatible mucus may comprise one or more temperature changes, such as utilizing heating and cooling cycles to promote gelation. In one embodiment, the method may comprise changing the pH to promote gelation of the purified biocompatible mucus. For example, raising and subsequently lowering the pH may promote gelation. Any suitable number of temperature and / or pH raising and lowering cycles may be employed. In one embodiment, the method may comprise adding salt to the purified biocompatible mucus to promote mucin gelation.

[0150] The method of preparing the purified biocompatible mucus may comprise storing the purified mucin. The storage method may comprise freeze-drying the purified mucin, composition or biosimilar mucus. Suitable conditions for freeze-drying are known to those skilled in the art. Purpose of the organizational model

[0151] The purified biocompatible mucus or composition of the present invention or the tissue model of the present invention can be used for a variety of applications, including but not limited to drug absorption testing, drug formulation development, analysis of GI damage / side effects, nutrient uptake analysis, drug-microbiome interaction studies, especially in the large intestine model, analysis of intestinal damage and prebiotic / probiotic testing.

[0152] Thus, the present invention provides a method for determining or predicting the absorption of a test compound through a tissue model, wherein the tissue model comprises: i) a substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; and iii) a purified biocompatible mucus in contact with the cells; and wherein the method comprises contacting the purified biocompatible mucus with a test compound and detecting the movement of the test compound in the tissue model, thereby determining the ability of the test compound to pass through the purified biocompatible mucus and cell layer of the tissue model. The presence of the test compound at the substrate indicates the ability of the compound to be absorbed through the tissue model.

[0153] The tissue model may comprise the purified biocompatible mucus or a composition comprising the purified biocompatible mucus as described in the first aspect of the present invention. The tissue model may be as described in the second aspect of the present invention.

[0154] The step of detecting the movement of the test compound in the tissue model can include determining the concentration of the test compound at or below the substrate. The step of detecting the movement of the test compound in the tissue model can include detecting the test compound in the purified biocompatible mucus layer, in the cell population and / or in contact with the substrate surface.

[0155] The test compound can be detected at the luminal surface and / or the basolateral surface of the tissue model. The presence of the test compound at the basolateral surface indicates the ability of the compound to be absorbed through the tissue explant.

[0156] Detection of the test compound can be carried out by any suitable means, such as physical means, chemical means or visual means. Examples include, but are not limited to, antibody assays, mass spectrometry, colorimetric assays, liquid chromatography, nucleic acid sequencing, SDS PAGE, protein blotting, in situ hybridization. Those skilled in the art can select a suitable method based on the properties of the test compound.

[0157] The tissue model can be a stomach tissue model, a small intestine tissue model, a large intestine tissue model, a gastrointestinal tissue model, a female reproductive tissue model, a nasal tissue model, a visual tissue model and / or an airway tissue model as described herein.

[0158] In one embodiment, a method for determining or predicting the absorption of a test compound by a tissue model can be used to determine or predict the effect of food on the absorption of a compound into gastrointestinal tissue. In this method, the tissue model can be a stomach tissue model, a small intestine tissue model, a large intestine tissue model, or a gastrointestinal tissue model. The method can include the step of applying food to a purified biocompatible mucus or fluid layer of the model. The food can be digested or undigested food. The steps of contacting the tissue model with the food and the test compound can be separate or simultaneous. In the case of separation, the test compound can be contacted with the tissue model before or after the tissue model is contacted with the food. The method can include testing the absorption difference of the compound of interest in the presence or absence of food.

[0159] In one embodiment, the method that determines or predicts that test compounds absorbs by tissue model can be used for determining or predicting that native intestinal medium absorbs the impact of compound in gastrointestinal tissue.In this method, tissue model can be stomach tissue model, small intestine tissue model, large intestine tissue model or gastrointestinal tissue model.The method can comprise the step of biocompatible mucus or fluid layer of the purification of native intestinal medium applied to model.The step of contacting tissue model with native intestinal medium and test compound can be separated, or can be simultaneously.In the case of separation, test compound can contact with tissue model before or after tissue model contacts with native intestinal medium.The method can be included in the absorption difference of testing concerned compound in the case of existence or absence of native intestinal medium.

[0160] The absorption of the test compound in the tissue model can be tested at different time points. This will provide information about the absorption rate of the test compound by the tissue model, including the absorption rate through the different layers of the tissue model. This method can be used to predict the absorption rate through the native tissue.

[0161] The methods for determining or predicting the uptake of a test compound as described herein can be used to determine or predict the perfusion rate of a test compound in a tissue explant. The presence of a test compound at the basolateral surface indicates the ability of the compound to be perfused in a tissue model. By detecting the presence of a test compound at different time points, the perfusion rate can be obtained.

[0162] The present invention can also provide a method for determining the effect of a test compound on a tissue model, wherein the tissue model comprises: i) a substrate comprising a cell support portion; ii) a cell population in contact with the cell support portion of the substrate; and iii) purified biocompatible mucus in contact with the cells; wherein the method comprises contacting the purified biocompatible mucus with the test compound and detecting the effect of the test compound on the tissue model. The method can comprise contacting the tissue model with the test compound and contacting the tissue model with a control substance to compare the effect of the test compound on the tissue model. The method can be used to predict the effect of the test compound on the native tissue.

[0163] The tissue model used in the method for determining the effect of a test compound may comprise the purified biocompatible mucus or a composition comprising the purified biocompatible mucus as described in the first aspect of the present invention. The tissue model may be as described in the second aspect of the present invention.

[0164] Determining the effect of the test compound may comprise determining the effect of the test compound on local tissue toxicity, genetic modification of the tissue, temporal changes in tissue permeability, modulation of mucus or microbiome, modulation of hormone production and / or secretion, overall tissue cell health, whole or partial tissue viability, such as the status of a purified mucin layer (e.g., in terms of cell viability, cytokine production, or transepithelial electrical resistance). The status of a cell population may be assessed by determining the number of live or dead cells, or factors such as the presence or absence of a cell marker or cell membrane permeability. Assays include any cell viability assay available to a skilled artisan, such as flow cytometry, high content imaging, colorimetric tetrazolium reagents, protease substrates that produce a fluorescent signal and resazurin reduction, luminescent ATP assays, fluorescence microscopy, real-time assays that monitor live cells in culture for days, cell titer blue assays.

[0165] The tissue model used in the method of determining the effect of a test compound can be a stomach tissue model, a small intestine tissue model, a large intestine tissue model, a gastrointestinal tissue model, a female reproductive tissue model, a nasal tissue model, a visual tissue model and / or an airway tissue model as described herein.

[0166] The method for determining the effect of a test compound can be used to determine or predict the effect of food on the effect of a compound. In this method, the tissue model can be a stomach tissue model, a small intestine tissue model, a large intestine tissue model, or a gastrointestinal tissue model. The method can include the step of applying food to the mucin or fluid layer of the model. The food can be digested or undigested food. The steps of contacting the tissue model with the food and the test compound can be separate, or can be simultaneous. In the case of separation, the test compound can be contacted with the tissue model before or after the tissue model is contacted with the food. The method can include testing the difference in absorption of the compound of interest in the presence or absence of food.

[0167] In one embodiment, the method for determining the impact of the test compound can be used to determine or predict the impact of native intestinal medium on the impact of the compound. In this method, the tissue model can be a stomach tissue model, a small intestine tissue model, a large intestine tissue model or a gastrointestinal tissue model. The method can include the step of applying native gastrointestinal medium to the mucin or fluid layer of the model. The step of contacting the tissue model with native intestinal medium and the test compound can be separated, or can be simultaneous. In the case of separation, the test compound can contact the tissue model before or after the tissue model contacts the native intestinal medium. The method can be included in the absorption difference of the test concerned compound in the presence or absence of native intestinal medium.

[0168] The effect of the test compound introduced into the tissue model can be tested at different time points. This will provide information about the rate of effect of the test compound on the tissue model, including the rate of effect on different layers of the tissue model.

[0169] The above methods can be used to determine the best or appropriate drug delivery mechanism, drug formulation and the effect of the drug on the body. The above methods may also be suitable for identifying protein, lipid or fat digestion regulators used in the food / nutraceutical industry. The above methods can also be used to test the effect of factors such as food and meal time on the efficacy of drugs.

[0170] Kits for use in the methods of the invention may be provided. The kits may include the tissue models described herein in a suitable container, and optionally substrates, buffers, reagents, instructions for use, and other standard components well known in the art.

[0171] The test compound used for the present invention can be any compound that has or may have biological activity, including, for example but not limited to, small molecules, organic molecules, antibodies, peptides, proteins, hormones, antagonists and nucleic acids (e.g., antisense, siRNA, shRNA, RNAi, expressible coding sequences), sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, bacteria and fungi or their extracts, plants and animal extracts. The test compound can be a medicament. The test compound can include a combination of two or more compounds.

[0172] Throughout the specification and claims of this specification, the words "comprise" and "contain" and their variations mean "including but not limited to", so they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the specification and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where indefinite articles are used, the specification should be understood to contemplate both the plural and the singular unless the context requires otherwise.

[0173] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel one or any novel combination of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or any novel one or any novel combination of any method or process steps so disclosed.

[0174] The reader is invited to note all papers and documents related to the present application filed concurrently or previously with the present specification, which are open to public inspection with the present specification, and the contents of all these papers and documents are incorporated herein by reference. Example Reconstructing mucus production

[0175] The mucus scraping material of the pig small intestine is mixed with an enzyme inhibitor mixture (Table 5) at a ratio of 3:1 inhibitor buffer to mucus to prevent mucin from being lysed by enzymes and other biological components (e.g., bacteria) present in the intestinal scraping material. The mucus / inhibitor buffer mixture is homogenized to solubilize mucin. Homogenization may depend on the mucus source and can be performed for 0 to 30 minutes. The homogenized material is centrifuged at 10000RPM for 1 hour at 4°C in an 85ml centrifuge tube (Eppendorf F-34-6-38 fixed angle rotor and Eppendorf Centrifuge 5810 R). The supernatant obtained by the centrifugation is filtered through glass wool and the precipitate is discarded. The density of the supernatant is adjusted to 1.42 g / l using cesium chloride and ultracentrifuged using a P50AT2-941 rotor in a Himac CP100NX ultracentrifuge. The kit was assembled using ultracentrifuge tubes compatible with the above rotor and centrifuge, which included: 40PA 40mL high-speed round bottom tubes and S-40AL caps and Ti rings. The ultracentrifuge, rotor and tube assemblies were provided by Hitachi, Ltd. (Hitachi High-Technologies, Maidenhead, UK). This step separates the solubilized mucin from nucleic acids, lipids and proteins by equilibrium density centrifugation such that the solution forms a density gradient that is divided into 8 separate fractions. The heaviest fraction 8 is located at the bottom of the tube and the lightest fraction 1 is located at the top. These fractions have been previously determined by weight. The fractionated samples are removed in sequence from 1 to 8, each fraction being approximately 4 ml.

[0176] Each fraction was dialyzed using a 19 mm expanded 12-14 kDa molecular weight cutoff (MWCO) dialysis membrane (Medicell Membranes Ltd, London, UK) to remove unwanted chemicals such as inhibitor buffers and cesium chloride components. Dialysis was performed in a 20 L stirred vessel filled with deionized (DI) water, which was routinely replaced 6 times over 2 days. This step was maintained at 4 ° C by using a refrigerator.

[0177] After complete dialysis, OD was measured using an Infinite M200 Pro spectrophotometer (TECAN UK, Reading, UK). 260 / 280 The DNA / protein content of each fraction was determined by PAS assay using porcine gastric mucin as a standard for the determination of glycoprotein content.

[0178] The fraction with the highest glycoprotein content and the lowest amount of DNA / protein was considered to be the mucin-containing fraction, since mucin is a carbohydrate-rich glycoprotein, and the fraction was then freeze-dried and stored at -20°C until needed. When needed, mucin was rehydrated to the desired concentration by adding 1x PBS (Merck) and placed on a tube roller overnight at 4°C to hydrate.

[0179] The product can be sterilized during the dialysis process by adding an antimicrobial agent or solvent sterilizing the purified mucus. The sterilization process can be performed after lyophilization. For solvent sterilization, solvents (such as ethanol, methanol, and IPA) can be added to the mucus, mixed under sterile conditions, and evaporated. Small Intestinal Absorption Plate Model

[0180] Permeation experiments throughout the project were performed using dual-chamber microplates consisting of either 96 or 24 chambers. Each chamber is referred to as either the basolateral chamber or the apical chamber - corresponding to the lower well in the base of the microplate, or as a smaller permeable insert chamber located within the base well. The bottom of the apical chamber insert of each of these microplates is lined with a 3 μM pore size polycarbonate membrane that separates the upper apical chamber from the basolateral chamber.

[0181] In these experiments, the basolateral compartment represents materials that can be taken up by villus-associated circulatory and lymphatic vessels once they have left the basolateral side of the epithelium, and the apical compartment represents the intestinal lumen.

[0182] In all plates, a simulated digestion solution (eg, EPDF) containing the substance of interest is applied apically. In the basolateral chamber is PBS (or any suitable substitute) representing body fluid of the correct pH and osmolarity.

[0183] In all plates, the basolateral chamber was filled before applying fluid to the apical chamber, and the time course was started immediately after apical fluid application. 100 μL samples were removed from the basolateral chamber at 5 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes and replaced with 37°C pre-warmed PBS (or any suitable alternative). All plates were incubated at 37°C.

[0184] In the basolateral chamber of each plate, there was the following volume of PBS: 300 μL (96-well plate), 600 μL (24-well plate). In the apical chamber of each plate, there was the following volume of simulated digestion solution and the components of interest: 100 μL (96-well plate), 200 μL (24-well plate). Cell monolayer

[0185] Caco-2 cells at passage 22-36 were grown on permeable inserts (5000 cells per well initially for 96-well plates and 20,000 cells per well for 24-well plates). After 21 days, cells showed normal epithelial electrical resistance and transport activity. Preparation of reconstituted mucus and its application in the validation of native mucus permeability

[0186] All permeability validation experiments were done in 96-well plates. A few days before the experiment, the purified mucus prepared above was reconstituted in 1xPBS at concentrations of 25 mg / ml, 50 mg / ml, and 75 mg / ml and placed on a roller mixer at 4°C to hydrate. The reconstituted mucus was applied to the top insert membrane in volumes of 20 μL, 50 μL, and 70 μL. These corresponded to 1.4 mm, 3.5 mm, and 4.9 mm, respectively.

[0187] Drugs were formulated at 100 μM in simulated digestive fluid and (gently) applied to the mucus layer to start the time course. Drug penetration was accomplished simultaneously with permeation through native porcine small intestinal mucus (volumes of 10 μL, 15 μL, and 20 μL, respectively). Permeability Verification Data

[0188] Correlation analysis of the 10 drugs showed that the best correlations were achieved with 25 mg / ml, 50 mg / ml and 75 mg / ml in 50 μL, and 50 mg / ml in 70 μL, respectively. These concentrations and volumes were used in further analysis, while others were excluded.

[0189] Correlation analysis of the 11 drugs showed positive correlations among the variables, with the strongest being 50 μL of 50 mg / ml and 50 μL of 75 mg / ml with the three volumes of native mucus (Spearmans Rho R squared, Table 3; Figure 4 (50 μL of 75 mg / ml). Figure 4 , Figure 5 and Figure 6 . Rheological validation of reconstituted mucus

[0190] Purified mucus was reconstituted in phosphate-buffered saline (PBS) at a range of concentrations (5–200 mg / ml) overnight at 4°C as described previously. The rheological properties of the rehydrated mucin (reconstituted mucus) were evaluated using a Kinexus Pro rheometer (Malvern, UK). The properties of native mucus (uncleaned mucus) collected from the small intestine of freshly slaughtered pigs and rapidly frozen were evaluated after thawing, as well as native mucus that had undergone a gentle cleaning process to remove any food debris. The linear viscoelastic region (LVER) was calculated using an amplitude sweep (0.01% to 600% shear strain) and the frequency was set to 1 Hz. The shear strain at rupture (yield point) within the LVER was also calculated (%) as well as the phase angle, storage modulus (G'), and loss modulus (G''). One-way ANOVA and Tukey's multiple comparisons were used to assess any statistically significant changes.

[0191] The LVER shear strain and stress and yield point of native and clean mucus were similar. The storage modulus and loss modulus of clean mucus increased compared to native mucus, but the phase angle remained similar. There were no statistical differences in LVER shear stress, shear strain, and phase angle between native or clean mucus at any mucin concentration. There were no statistical differences in G' of LVER or rupture shear stress between native mucus and mucin concentrations below 100 mg / ml. The only significant difference between mucin concentrations and native mucus was G", where G" was not significantly lower than G" of native mucus only at concentrations of 100 mg / ml or above. Figure 5 - Rheological data). Figure 5 Rheological properties of rehydrated mucus compared to native and cleaned porcine intestinal mucus are shown. Panels A and B show the maximum shear strain and stress in the linear viscoelastic region, respectively. Panels C, D and E show the storage modulus, loss modulus and phase angle in the LVER. Panel F shows the shear strain required to break the gel into a viscous liquid (yield point) and allow it to flow. Experiments were repeated at least three times with different batches of mucus and mucin.

[0192] Using rupture shear strain, LVER, phase angle and G' as the most relevant aspects to define rheologically relevant mucins, mucins at concentrations of 50 mg / ml and around 50 mg / ml were therefore classified as similar. Reconstructing mucus cytocompatibility

[0193] The experiment showed that native porcine mucus significantly reduced cell viability in a 96-well plate model. Figure 6 )

[0194] In the same model, a range of concentrations and volumes of reconstituted mucus were tested for compatibility with Caco2 cells using simulated intestinal secretions applied for 4 hours ( Figure 7 ).

[0195] All concentrations (5-200 mg / ml) at 50 μL and 70 μL were not significantly different from the control, indicating that these mucus protected cells from simulated digestive secretions in vitro over a 4-hour time course.

[0196] In the 24-well plate model, 50 μL of 25 mg / ml, 50 mg / ml, and 75 mg / ml also protected cells from simulated intestinal digestive fluid over a 4-hour time course ( Figure 8 ).

[0197] Small Intestine Model: Summary The reconstituted mucus was rheologically validated compared to native mucus. In both 24-well and 96-well plate absorption models, reconstituted mucus protected cells from small intestinal digestive juices for 4 hours. The reconstituted mucus has been validated for drug penetration compared to native porcine mucus. Stomach Model

[0198] The same system has been used for gastric applications, such as a system that models the gastric epithelium using a reconstituted gastric mucin layer with a gastric cell line (CRL-1739) and synthetic gastric secretions. This model has been used to test gastric injury from the delivery of non-steroidal anti-inflammatory drugs. Using this model, we were able to collect cell viability data after two hours of exposure to test formulations under different conditions. It was seen that including the mucus layer prevented cell death, and furthermore, this allowed for appropriate modeling of gastric secretions (including pepsin), at pH 2.0, allowing for the identification of formulations that were still cytotoxic to the mucus layer. Example data for the gastric model is available at Fig. 9 Shown in. Large Intestine Model

[0199] The same approach has been applied to the large intestine model. This system also includes a cell monolayer and a mucus layer in a transport chamber system ( Fig.10 ). However, to set up the aerobic / anaerobic system, this custom biocompatible 3D printed plate will be placed in a container with a GasPaK anaerobic envelope and have an upper portion with access to the apical side of the transport chamber, a sealed bottom chamber (with an O-ring around the transport chamber to ensure an airtight fit), a lid to prevent evaporation of the medium, and tubing (to the outside of the container to allow air to enter the basolateral side of the transport chamber, thereby creating an aerobic environment). The upper chamber of the transport chamber will then be inoculated with a bacterial population grown in a 3-stage fermentation model (inoculated with fecal samples). This is the first system that can model the microbiome, mucus layer, and epithelium in a single integrated system.

[0200] When transport chambers containing only Caco-2 monolayer and medium were placed in the above plates under anaerobic conditions (Normal Anaerobic Plates and Normal Anaerobic Plate 2), cell viability was slightly reduced compared to cells and medium in the transport chambers under normal aerobic conditions (Normal Aerobic Plates).

[0201] Adding 50 μl of a 50 mg / ml reconstituted mucin layer (reconstituted-A) or 50 μl of a 100 mg / ml reconstituted mucin layer and 50 μl of a native porcine mucus layer (native-B) above the cells affected cell viability under aerobic conditions, but when the transport chamber was exposed to anaerobic apical conditions with a reconstituted mucus layer (C) or a native mucus layer (D) (in the 3D-printed plates generated by the GasPak described above), the gel was sufficient to protect Caco-2 cells from anaerobic apical conditions over a 3-hour incubation period ( Fig.11 ).

[0202] Further addition of fecal microbial content above the reconstructed layer (E) or native layer (F) did not negatively affect cell viability.

[0203] The fact that there were no significant differences between normal cells under anaerobic conditions and cells with reconstituted mucus and bacteria indicates the feasibility of this model. Airway Model

[0204] Calu3 airway epithelial cells were grown in 96-well transport chamber cell culture plates. 40,000 cells (100 μl) were seeded per well and grown to confluence (assessed by visual inspection and transepithelial electrical resistance measurements) and then grown as an air-liquid interface (ALI), with surface liquid removed to feed the cells from the basolateral side.

[0205] As described above, mucus isolated from freshly slaughtered pig stomachs, containing primarily the MUC5AC mucin gene product found in the airways, was rehydrated in sterile PBS containing calcium and magnesium.

[0206] Five concentrations of mucin were evaluated in four different volumes; 25 μl, 50 μl, 75 μl and 100 μl of 100 mg / ml, 75 mg / ml, 50 mg / ml, 25 mg / ml and 12.5 mg / ml, respectively, were applied to the top of the cells and incubated for 90 min.

[0207] Cell viability was assessed by the cell titer blue assay, in which metabolically active cells reduce resazurin to the dye resorufin, which can be measured spectrophotometrically. Experiments were repeated at least four times ( Fig.10). Significant differences were assessed using two-way ANOVA with Tukey's multiple comparisons. Panel A shows cell viability grouped by volume of fluid added to the apical surface of the cells, with mucin concentrations shown as bars. Panel B shows the same data grouped by mucin concentration applied, with the bars showing different volumes.

[0208] No statistical differences were seen between any concentration of mucin applied at any volume compared to untreated cells. Thus, over the course of 90 minutes, the applied mucin did not adversely affect cell viability. Preparation of purified mucus

[0209] Small intestines of freshly slaughtered pigs were collected from a local slaughterhouse and transported to the laboratory on ice. The tissue was cut open with a scalpel and the epithelial tissue surface was gently rinsed with deionized water. The epithelial surface was gently scraped with a microscope slide to remove mucus. Mucus, either neat or diluted with deionized water at a 1 / 50 dilution, was added to a 50 mDa mwco dialysis tube.

[0210] Place the dialysis tubing in a 10L bucket of 4°C deionized water and incubate at 4°C. Change the dialysis water 6 times within 2 days.

[0211] The mucus was collected from the dialysis tube and centrifuged at 9000 RPM for 90 minutes.

[0212] The purified mucus was collected and frozen at -20°C. Mucus penetration test

[0213] For drug permeation through porcine native mucus (PNM), a Handy Step repeating pipette (BrandTech Scientific Inc., CT, USA) and a compatible 500 μl Eppendorf- (Eppendorf, Hamburg, Germany) 0μl, 10μl, 15μl and 20μl PNM were applied to the top insert membrane of the well in triplicate. 19 drugs were prepared at 100μM in simulated intestinal fluid (Table 3). The drug solution was added to the top chamber of the test well at 0 minutes, and 100μL samples were collected from the basolateral chamber at 37°C at 5 minutes, 60 minutes, 120 minutes, 180 minutes and 240 minutes. The sample was replaced with pre-heated PBS. Regarding the penetration of API through reconstructed mucus, as described above, mucus was prepared and formulated at 12.5mg / mL, 25mg / mL, 50mg / mL, 75mg / mL or 100mg / mL the day before the experiment. Each mucus concentration was added to the top insert membrane at a volume of 0μL, 20μL, 50μL and 70μL. The permeation data was analyzed by HPLC. All conditions were screened in triplicate. Table 3. List of APIs used in the permeation studies. Caco-2 cell culture

[0214] All tissue cultures were performed under sterile conditions using a S@FEFLOW 1.2 biosafety cabinet (Bio Air Instruments, Pavia, Italy). Caco-2 cells were purchased from ATCC ( HTB-37 TM )(American Type Culture Selection, VA, USA). The cells were delivered frozen and thawed and plated into 25 cm 2 Nunc TM EasYFlask TM (Thermo Scientific, NY, USA). After reaching about 80% confluence, the cells were further subcultured to 75 cm 2 Nunc TM EasYFlask TM Culture flask. The culture medium is changed every other day. 25cm 2 and 75cm 2 The culture flask was replaced with 7 mL and 20 mL, respectively. The culture medium used for Caco-2 cell growth was Dulbecco's modified Eagle's medium-high glucose (Sigma-Aldrich; D5797-500ML) supplemented with 50 mL fetal bovine serum, 5 mL 200 mM L-glutamine, 5 mL penicillin / streptomycin (penicillin: 5 mg / mL; streptomycin: 10 mg / mL), and 5 mL modified Eagle's medium non-essential amino acid solution (100x) (Merck; M7145). The cultures were cultured in MCO-20AIC CO 2 The cells were incubated in an incubator (SANYO Electric Co., Ltd, Japan) at 37°C and 5% CO2. The number of passages ranged from 22 to 38.

[0215] For 96-well plate experiments, Caco-2 cells were grown in 96 dual-chamber microplates (HTS -96-well permeable support with barcode, lid, receiver plate; Corning Inc., ME, USA) on the top surface of a 3 μm pore size polycarbonate membrane for at least 21 days. Caco-2 cells were grown from 75 cm 2 Subculture in culture flasks, each cm 2Seed 80,000 cells in a volume of 100 μL of medium in each apical well. Add 300 μL of medium to the basolateral chamber. Integrated model experiments

[0216] Prior to the day of the experiment, RM was prepared as previously described at 25 mg / mL, 50 mg / mL, and 75 mg / mL. On the day of the experiment, Caco-2 cells grown for at least 21 days were transferred to a clean, sterile receiver plate by transferring the apical holder. The growth medium was then removed from the apical chamber using a multichannel pipette. 300 μL of 1x PBS was added to the basolateral chamber of all test wells. All lateral wells were excluded from the test due to edge effects. To these wells, 300 μL of 1x PBS was added to the basolateral chamber and 100 μL was added to the apical chamber to reduce edge effects in the experiment. For the test wells, four different RM conditions were used in the analysis. These conditions were 25 (50), 50 (50), 50 (70), and 75 (50), where X (X) is equivalent to X mg / mL (X μL). Each concentration and volume of RM was gently added to the Caco-2 monolayer using a repeating pipette and allowed to settle for 5 minutes. 16 API solutions (Table 4) were prepared at 100 μM in EPDF, and 100 μL was gently applied to the mucus layer using a 200 μL multichannel pipette at T0. The plate was then incubated at 37°C for 4 hours, and 100 μL samples were removed from the basolateral chamber at 60 minutes, 120 minutes, 180 minutes, and 240 minutes and replaced with pre-warmed 37°C 1xPBS. The samples were incubated at 37°C overnight, resuspended in 50% methanol, and analyzed by HPLC using the ammonium acetate method.

[0217] At the end of the experiment, the EPDF was removed from the apical chamber, taking care not to disturb the mucus layer. After removing the EPDF, the apical chamber was gently washed with 1xPBS by pipetting to remove the mucus layer. The cells were washed at least twice. The apical scaffolds were then transferred to another sterile receiving plate and the cell titer blue assay was performed. All conditions were screened in triplicate. Table 4: Drugs used in the ensemble model penetration Table 5. Protease inhibitor cocktails for reconstituted mucin production. Inhibition mechanisms are detailed in the middle column.

Claims

1. An organizational model consisting of: i. a substrate having a cell-supporting portion; ii. a cell population provided on said substrate; iii. purified biocompatible mucus; iv. Optionally, a physiological fluid provided on the mucus layer and / or below the basal layer, The purified biocompatible mucus: i) is substantially free of nucleic acids, proteins or lipids or molecules below 100 kDa, preferably 70 kDa; and ii) contains mucin, wherein the mucin has the ability to form a gel.

2. The tissue model of claim 1, wherein the purified biocompatible mucus is a gel.

3. A tissue model according to claim 1 or claim 2, wherein the purified biocompatible mucus is free of synthetic polymers and / or is substantially free of denaturation, hydrolysis or degradation.

4. The tissue model according to any one of claims 1 to 3, wherein the purified biocompatible mucus is provided in combination with one or more of lipids, salts, proteins, nucleic acids (DNA or RNA), carbohydrates, glycoproteins, cells and water.

5. The tissue model according to any one of claims 1 to 4, wherein the cells of the cell population are or originate from the ileum, jejunum, stomach, duodenum, esophagus, cheek, tongue or colon of the digestive tract, or from the nose, lung, bronchi, bronchioles or oral cavity; or from the cervix or vagina; or from the eyes; or any combination thereof.

6. The tissue model according to any one of claims 1 to 5, wherein the fluid is digestive fluid, saliva, vaginal secretions, tears or airway secretions, synthetic fluids or microbial cell populations; or any combination thereof. 7 . The tissue model according to any one of claims 1 to 6 , wherein the tissue model is a stomach tissue model, a small intestine tissue model, a large intestine tissue model or a gastrointestinal tissue model.

8. A combination of two or more tissue models according to any one of claims 1 to 7, wherein the two or more tissue models are arranged in series, and preferably wherein the two or more tissue models are each selected from the group consisting of: an airway model, a stomach tissue model, a small intestine tissue model, and a large intestine tissue model.

9. Purified biocompatible mucus, wherein the mucus: i) is substantially free of nucleic acids, proteins or lipids or molecules below 100 kDa, preferably 70 kDa; and ii) comprises mucin, wherein the mucin has the ability to form a gel.

10. The purified biocompatible mucus according to claim 9, wherein the purified biocompatible mucus is a gel.

11. The purified biocompatible mucus according to claim 9 or claim 10, wherein the purified biocompatible mucus does not contain synthetic polymers.

12. The purified biocompatible mucus according to any one of claims 9 to 11, wherein the mucin is not substantially denatured, hydrolyzed or degraded.

13. The purified biocompatible mucus according to any one of claims 9 to 12, and one or more of lipids, salts, proteins, nucleic acids (DNA or RNA), carbohydrates, glycoproteins, cells and water.

14. A method for determining or predicting the absorption of a test compound through a tissue model as defined in any one of claims 1 to 8; wherein the method comprises contacting the purified biocompatible mucus with the test compound and detecting the movement of the test compound in the tissue model.

15. The method of claim 14, wherein the method comprises detecting and optionally determining the concentration of the test compound in the purified biocompatible mucus layer, in the cell population, in contact with the substrate surface, beneath the substrate, at the luminal surface and / or at the basolateral surface.

16. The method of claim 14 or 15, wherein the test compound is detected by binding assay, mass spectrometry, colorimetric assay, liquid chromatography, nucleic acid sequencing, SDS PAGE, Western blotting, or in situ hybridization.

17. The method of claim 16, wherein the test compound is a small molecule, an organic molecule, an antibody, a peptide, a protein, a hormone, an antagonist, a nucleic acid (e.g., antisense, siRNA, shRNA, RNAi, an expressible coding sequence), a sugar, a fatty acid, a steroid, a purine, a pyrimidine; a derivative or structural analog thereof; a bacterium, a fungus or an extract thereof; a plant or animal extract; or any combination of two or more thereof.

18. The method according to any one of claims 14 to 17, wherein the test compound is a pharmaceutical agent.

19. The method according to any one of claims 14 to 18, for use in any one or more of drug absorption testing, drug formulation development, analyzing GI damage / side effects, nutrient uptake analysis, drug-microbiome interaction studies, analyzing intestinal damage, and prebiotic / probiotic testing.

20. The method according to any one of claims 14 to 19, wherein the method comprises the step of applying food and / or enteric medium to the tissue model.

21. The method of any one of claims 14 to 20, wherein the test compound is measured at one or more different time intervals.

22. A kit comprising, in a suitable container, a purified mucus as defined in any one of claims 9 to 13 or a tissue model as defined in any one of claims 1 to 8, and optionally substrates, buffers, reagents and instructions for use.

23. A method for purifying mucus, wherein the method comprises removing substantially all nucleic acids, proteins and / or lipids and / or molecules below 100 kDa, preferably below 70 kDa, from the mucus, wherein the removal step comprises dialysis and / or equilibrium density gradient centrifugation.

24. The method according to claim 23, comprising dialyzing the mucus to remove nucleic acids, proteins or lipids or molecules below 100 kDa, preferably below 70 kDa; and optionally separating the mucus.

25. The method of claim 23, comprising i) providing a sample comprising mucus; ii) solubilizing any mucin in the mucus; iii) separating any nucleic acids, proteins, lipids from the mucus; and iv) dialyzing the mucus.

26. The method of claim 25, wherein the separation step comprises equilibrium density gradient centrifugation and optionally selecting a fraction with a density of 1.3-1.6 g / mL.

27. The method of any one of claims 23 to 26, wherein the method uses a cesium salt density gradient.

28. The method according to any one of claims 23 to 27, further comprising providing a tissue sample comprising mucus; and optionally extracting the mucus from the tissue sample.

29. The method of any one of claims 20 to 25, further comprising sterilizing one or more components of the purified mucus.

30. The method of any one of claims 20 to 26, further comprising combining the purified mucin with a buffer, lipids, salts, proteins, nucleic acids (DNA or RNA), glycoproteins, cells and / or water.

31. The method of any one of claims 20 to 27, further comprising subjecting the purified mucus to conditions capable of gelling the mucin.

32. The method of any one of claims 20 to 28, further comprising lyophilizing, storing and / or combining the purified mucus with a buffer.

33. The method of any one of claims 20 to 29, further comprising applying the purified mucin to a cell population provided on a substrate to form a tissue model, and optionally applying a fluid to the purified mucin.

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