Refined mucus

Abstract

The present invention relates to purified biocompatible mucus capable of forming a gel. The mucus of the present invention may be provided in a tissue model along with a cell population. The mucin or tissue model of the present invention may be useful in in vitro models of digestion, mucus penetration, and / or epithelial transport.

Description

[Technical field]

[0001] The present invention relates to a purified biocompatible mucus, and uses thereof. The present invention also relates to a tissue model comprising the purified biocompatible mucus and cells, and uses thereof. The present invention also relates to a method for purifying mucus. [Background technology]

[0002] For successful oral ingestion of active pharmaceuticals, the compound must pass through a series of barriers and retain sufficient yield to have a therapeutic effect. First, the compound must overcome the enzymatic, chemical, and physical challenges in the mouth, stomach, and small intestine during the digestive phase, including digestive enzymes, pH changes, and components such as bile that may interact with and interfere with the pharmaceutical. Once in the small intestine, the drug delivery system must then penetrate the mucus barrier so that the drug can successfully reach the epithelium and be absorbed into the bloodstream. The mucus layer acts as a barrier, excluding or trapping certain compounds based on size and charge. Any such particles are removed as the mucus layer is turned over. As a result, oral delivery of drugs, including peptide and protein therapeutics, can be hindered by the presence of mucus that covers the surface of the intestinal epithelium. Mucus is composed of 95% water and mucin, a gel-forming glycoprotein, and some mucus may also contain lipids, DNA, and actin.

[0003] It is important to test oral delivery of drugs in vitro before testing in vivo, which may provide an alternative to reduce, refine or replace the need for animal models. For this reason, separate in vitro models exist for the various stages of the digestive system.

[0004] WO2015075467 and Houghton et al. (Food Chem. 2014 May 15;151:352-7) describe a simulated model of the digestive tract that is an accumulation of the mouth, stomach, and small intestine, using physiologically relevant secretions and whole porcine bile collected from an abattoir 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 that is used to simulate nanoparticle diffusion through the mucosa. Additionally, several cell culture systems are available to model the intestinal epithelium. So far, it is not possible to put the whole process together and see how these steps interact with each other.

[0005] WO2018 / 175861 describes a macro tissue explant for use in high throughput screening, comprising a mammalian tissue explant in a multi-well matrix. The tissue explant may further comprise a mucus layer.

[0006] Gleeson and McCartney (Trends in pharmacological sciences 40.10 (2019): 720-724) describe the co-culture of Caco-2 monolayers with HT29-MTX-E12. HT29 cells are another intestinal cell line that are differentiated into mature goblet cells using methotrexate. The H29 cell line was developed to mimic a mucus-covered epithelium. However, this is not an optimal solution, since the mucus layer is 4 μm, significantly thinner than the mucus layer in the thinnest regions of the human intestine (typically around 15 μm). This means that even the co-culture system cannot integrate with the entire digestive fluid due to the possibility of cell death.

[0007] It is known to use porcine mucus in such in vitro systems. However, although porcine mucus reflects human intestinal mucus, Caco-2 cells do not survive prolonged co-incubation. To overcome this problem, biosimilar mucus has been developed. Biosimilar mucus that match Caco-2 monolayers mimic the rheological properties of porcine intestinal mucus and have been used to evaluate drug absorption in vitro. Boegh, Marie et al. (European Journal of Pharmaceutics and Biopharmaceutics 87.2 (2014): pp. 227-235) describe the design and characterization of biosimilar mucus that match Caco-2 cell monolayers cultured in vitro to establish a more representative in vitro model of the intestinal mucosa. The biosimilar mucus mixture consists of disintegrated commercial gastric mucin, BSA, cholesterol, phosphatidylcholine, linoleic acid, and polyacrylic acid (Carbopol 974P NF at 0.3-0.9% w / v). Polyacrylic acid is a synthetic polymer added to the composition to provide the desired viscoelastic properties and a microstructure equivalent to freshly isolated porcine intestinal mucus (PIM). The biosimilar mucus was optimized for lipid content to obtain cytocompatibility with well-differentiated Caco-2 cell monolayers. However, significant differences in peptide permeability were found between isolated PIM and biosimilar mucus. Furthermore, carbopol is known to interact with mucin, thus altering mucus properties and / or function. Commercially available porcine gastric mucin (Sigma Aldrich™) is denatured during isolation, resulting in loss of the ability to form mucin:mucin interactions and the inability to form gels. Furthermore, gastric mucin consists of the MUC5AC and MUC6 gene products, whereas the small intestinal mucin is MUC2.

[0008] Other approaches to model intestinal tissues, including mucus layers, have several problems. Human mucus is difficult to obtain, and the isolation process can alter the mucin. Furthermore, synthetic gastrointestinal secretions are used to mimic the natural intestinal environment, but such synthetic secretions have been found to cause cell death when applied to cell cultures in the absence of a protective mucus layer as found in vivo. Natural porcine mucus collected from animals has also been found to kill cells and is therefore not suitable for use in tissue models. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2015075467 [Patent Document 2] WO2018 / 175861 [Non-patent literature]

[0010] [Non-Patent Document 1] Houghton et al. (Food Chem. May 15, 2014; 151:352~7) [Non-Patent Document 2] Friedl et al. (J Pharm Sci. December 2013; 102(12):4406-13) [Non-Patent Document 3] Gleeson and McCartney (Trends in pharmacological sciences 40.10 (2019): 720-724) [Non-Patent Document 4] Boegh, Marie et al. (European Journal of Pharmaceutics and Biopharmaceutics 87.2 (2014): pp. 227-235) Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention seeks to overcome or ameliorate some or all of these problems associated with the prior art. [Means for solving the problem]

[0012] In a first aspect, the present invention relates to a mucus comprising: A purified biocompatible mucus is provided, comprising: i) a mucin that is substantially devoid of nucleic acids, proteins and / or lipids, and / or molecules below 100 kDa, preferably below 70 kDa; and ii) a mucin that has the ability to form a gel. Suitably, the purified biocompatible mucus contains a mucin having 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 the mucin is not substantially denatured, hydrolyzed or degraded. Suitably, the mucin present in the mucus comprises sufficient intermolecular interactions to enable the purified biocompatible mucus to form a gel. In one embodiment, the mucus is a gel.

[0013] The present invention also provides a method for producing a composition comprising the steps of: A substrate having a cell-supporting portion; A cell population provided on a substrate; A purified biocompatible mucus according to the first aspect, Optionally, body fluid provided below the mucus layer and / or matrix layer. The present invention provides an organizational model including:

[0014] In a third aspect, there is provided a method of purifying mucus comprising removing substantially all nucleic acids, proteins and / or lipids and / or molecules below 100 kDa, preferably below 70 kDa from the mucus. The removing step comprises dialysis and / or equilibrium density gradient centrifugation to provide purified biocompatible mucus. Suitably, the method substantially removes mucin from the mucus. Suitably, the method does not comprise denaturation, degradation or hydrolysis of mucin. Optionally, the method may comprise a further separation step, for example centrifugation. Suitably, the method provides a purified biocompatible mucus of the first aspect.

[0015] In one embodiment of the third aspect, the method may comprise the steps of: i) providing a sample comprising mucus; ii) solubilising any mucins in the mucus; iii) separating any nucleic acids, proteins and / or lipids from the mucus; and optionally iv) removing any contaminants, undesirable 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 isolating a fraction from the mucus having a density gradient of 1.3-1.6 g / mL. Suitably, the mucins present in the mucus have a density of 1.3-1.6 g / mL.

[0016] In one embodiment of the third aspect, the method may comprise the steps of: i) providing a sample comprising mucus; ii) dialysis of the mucus to separate any molecules below 100 kDa, preferably below 70 kDa, from the mucus; and optionally iii) separating the mucus.

[0017] In a fourth aspect, there is provided a method for determining or predicting absorption of a test compound by a tissue model, the tissue model comprising: 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, the method comprising contacting the purified biocompatible mucus with a test compound and detecting movement of the test compound in the tissue model, thereby determining the ability of the test compound to cross the purified biocompatible mucus and cell layer of the tissue model.

[0018] In one embodiment of the fourth aspect, the tissue model may comprise purified biocompatible mucus or a composition comprising purified biocompatible mucus according to the first aspect of the invention. The tissue model may be a tissue model according to the second aspect of the invention. The tissue model used in the method of detecting the effect of a 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, an optical tissue model, and / or an airway tissue model.

[0019] Detecting the movement of the test compound in the tissue model may include, but is not limited to, observing, monitoring, or measuring any one or more of the following: distance traveled by the test compound into the tissue model, absorption of the test compound into the tissue model, path of movement in the tissue model, rate of movement or perfusion into the tissue model, cellular uptake in the tissue model, and location of the test compound in the tissue model. Detecting may also include determining the concentration of the test compound at or below the substrate. Detecting the movement of the test compound may include detecting the test compound in the purified biocompatible mucus layer, in the cell population, and / or in contact with the surface of the substrate. The presence or absence of the test compound on the luminal surface and / or basolateral surface of the tissue model may be detected. The presence of the test compound on the basolateral surface indicates the ability of the compound to be absorbed by the tissue explant.

[0020] The method may include applying food to the purified biocompatible mucus or body fluid layer of the model.The method may include applying natural gut media to the purified biocompatible mucus or body fluid layer of the model.

[0021] In a fifth aspect, there is provided a kit comprising the purified biocompatible mucus or a composition comprising the purified biocompatible mucus according to the first aspect, and / or the tissue model of the second aspect, and optionally substrates, buffers, reagents, instructions for use, and other standard components known in the art.

[0022] Embodiments of the invention are further described below with reference to the accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1]Figure 1 shows Caco-2 cell viability over 4 hours in the presence of simulated digestive fluid with and without native mucus in a 24-well plate transwell. The cytotoxic effect of porcine small intestinal surface mucus on Caco-2 cell monolayers using either luminal PBS or simulated small intestinal fluid is shown by the Cell Titer Blue cell viability assay. Native mucus kills cells but partially protects remaining viable cells with GI fluid. Under agitation, the ability of native mucus to protect cells is reduced. [Diagram 2] A presentation of problems with the viscosity of commercially available mucins. Human mucus is difficult to obtain. Commercial isolation processes alter mucin glycoproteins, destroying their ability to form mucin:mucin interactions. Purification methods including boiling or exposure to proteolytic or glycolytic enzymes can alter mucins. The purification method described herein in the third aspect does not affect mucin:mucin interactions and allows mucins to retain their ability to form gels at higher concentrations. [Diagram 3] FIG. 2 is a schematic diagram of an example of how a model can be set up in accordance with the present invention. [Figure 4] Figure 1 shows the Pearson correlation of 17 active pharmaceutical ingredients (APIs) permeated through both 10 μL native mucus and 50 μL of 75 mg / mL reconstituted small intestinal mucin. A strong positive correlation (R2=0.8517) was observed. [Diagram 5] Figure 1 shows the rheological properties of purified mucus of the present invention rehydrated in phosphate buffered saline at concentrations between 5 and 200 mg / ml compared to native intestinal porcine mucus and sodium chloride washed 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 within the LVER. Panel F shows the shear strain required to break down the gel into a viscous liquid (yield point) and allow it to flow. The experiment was repeated a minimum of three times with separate batches of mucus and mucin. [Figure 6]Figure 4 shows the effect of natural porcine mucus on cell viability of Caco-2 cells grown in a 96-well, dual chamber model as in Figure 3. Natural porcine mucus significantly reduces cell viability in the 96-well plate model. [Figure 7] FIG. 1 shows the effect of concentration and volume of purified biocompatible mucus reconstituted with buffer (referred to herein as reconstituted mucus or RM) on compatibility with Caco-2 cells. In the same model used in FIG. 6, a range of concentrations and volumes of reconstituted mucus were tested for their compatibility with Caco-2 cells challenged with simulated small intestinal secretions for 4 hours. All concentrations at 50 and 70 μL were not significantly different from the control, indicating that they protect cells from simulated digestive secretions in vitro over a 4 hour time course. [Figure 8] 1 shows the effect of reconstituted mucus of the present invention in protection versus the effect of simulated small intestinal digestive fluids, and in a 24-well plate model, 50 μL of 25, 50 and 75 mg / ml were shown to protect cells from simulated small intestinal digestive fluids over a 4 hour time course. [Figure 9] Figure 1 shows the level of cell viability of the gastric cell line CRL-1739 integrated with a biocompatible mucus layer and gastric secretions.Gastric epithelial cells were covered with gastric mucus layers of various thicknesses and mucin concentrations and exposed to model gastric digestive secretions. [Figure 10] Figure 1 shows the effect of various volumes and concentrations of purified mucus of the present invention on airway cell viability over a 90 minute period. Panel A shows cell viability groups by volume of liquid added to the apical surface of Calu3 airway cells with concentrations of mucin indicated by the bars. Panel B shows the same data but groups by concentration of mucin applied, with the bars indicating the various volumes. All experiments were repeated a minimum of four times. [Figure 11] FIG. 1 shows that washed porcine small intestinal mucus dialyzed in 50,000 Da MWCO tubing is a cytocompatible gel and protects cells from simulated small intestinal fluid for 2 hours. After dialysis, the mucus is spun for 1.5 hours to remove excess water with the same results. [Figure 12]FIG. 1 shows the effect of various sterile solvents on 5 mg / ml gastric mucus after 24 h incubation with gastric cells. [Figure 13] FIG. 1 shows colony forming units present in mucus after sterilization with various solvents. [Figure 14] Figure 1 shows Caco-2 cell viability of cells incubated with layers of RM at different concentrations and volumes, where EPDF was applied on top of the mucus layer (n=3). Cells were incubated with RM and EPDF for 1 h (top left), 2 h (top right), 3 h (bottom left), and 4 h (bottom right). A live control exposed to PBS and a zero mucus control (i.e., a model in which no mucus is present but body fluids, e.g., digestive fluids, are present) exposed to EPDF were included in the analysis. The y-axis shows cell viability (%) as determined by Cell TiterBLue assay. The x-axis shows the respective volumes of live control and RM. Black bars show live control, dark grey bars represent 25 mg / mL RM, light grey bars represent 50 mg / mL RM, and white bars represent 75 mg / mL RM. Bars marked with an asterisk are significantly different from the live control. For the 2 hour graphs, bars with a pair of asterisks indicate groups that share significance with the mucus-free and live controls. [Figure 15] Graph showing all apparent permeability values ​​of all APIs for all mucus concentrations and volumes. The y-axis shows the apparent permeability of the API expressed in cm / s×10-6. The x-axis shows the API. There are also two legends on the x-axis, one showing the API's Log D or Log P value and the other showing the API charge. The APIs are classified based on their physiological charge and permeability value. Legends indicating which mucus condition is applicable for each bar are shown within the figure. PNM stands for processed native mucus, which is washed porcine small intestine mucus. RM X (X) stands for reconstituted mucus volume (outside brackets) (μL) and concentration (inside brackets) (mg / mL). APIs with higher Log D or Log P values ​​and positive charge typically have much lower apparent permeability values ​​than those that do not. [Figure 16]Figure 1 shows Pearson's correlation of apparent permeability of 11 APIs through 20, 50 and 70 μL reconstituted mucus at 25 mg / mL versus 10, 15 and 20 μL PNM (treated / washed native mucus). Permeability was repeated three times. The y-axis shows the apparent permeability of APIs through RM. The x-axis shows the apparent permeability of APIs through PNM. Apparent permeability is expressed as cm.s-1×10-6. (A) 20 μL RM vs 10 μL PNM. (B) 50 μL RM vs 10 μL PNM. (C) 70 μL RM vs 10 μL PNM. (D) 20 μL RM vs 15 μL PNM. (E) 50 μL RM vs 15 μL PNM. (F) 70 μL RM vs 15 μL PNM. (G) 20 μL RM vs 20 μL PNM. (H) 50 μL RM vs 20 μL PNM. (I) 70 μL RM vs 20 μL PNM. All relationships [Figure 17] Correlation of apparent permeability of 11 APIs through 20, 50 and 70 μL reconstituted mucus at 75 mg / mL versus 10, 15 and 20 μL PNM (treated / washed native mucus). The y-axis shows the Papp of API through RM. The x-axis shows the apparent permeability of API through PNM. Apparent permeability is expressed as cm.s-1×10-6. (A) 20 μL RM vs 10 μL PNM. (B) 50 μL RM vs 10 μL PNM. (C) 70 μL RM vs 10 μL PNM. (D) 20 μL RM vs 15 μL PNM. (E) 50 μL RM vs 15 μL PNM. (F) 70 μL RM vs 15 μL PNM. (G) 20 μL RM vs 20 μL PNM. (H) 50 μL RM vs 20 μL PNM. (I) 70 μL RM vs 20 μL PNM. C, F and I are Spearman rank correlations, others are Pearson correlations. All the correlations in I are [Figure 18]Figure 1 shows Pearson's correlation of apparent permeability of 11 APIs through 20, 50 and 70 μL reconstituted mucus at 50 mg / mL versus 10, 15 and 20 μL PNM (treated / washed native mucus). Permeability was repeated three times. The y-axis shows the apparent permeability of APIs through RM. The x-axis shows the apparent permeability of APIs through PNM. Apparent permeability is expressed as cm.s-1×10-6. (A) 20 μL RM vs 10 μL PNM. (B) 50 μL RM vs 10 μL PNM. (C) 70 μL RM vs 10 μL PNM. (D) 20 μL RM vs 15 μL PNM. (E) 50 μL RM vs 15 μL PNM. (F) 70 μL RM vs 15 μL PNM. (G) 20 μL RM vs 20 μL PNM. (H) 50 μL RM vs 20 μL PNM. (I) 70 μL RM vs 20 μL PNM. All relationships were significant. [Figure 19] Figure 1 shows Pearson's correlation of API apparent permeability data sets with positively charged API removed from the data set. The y-axis shows API apparent permeability through reconstituted mucus. The x-axis shows apparent permeability through PNM (treated / washed native mucus). Apparent permeability is expressed in cm / s x 10-6. Legend is provided above each column of figures (PNM volume) and within each figure (RM concentration and volume). All relationships were significant (p<0.05). The R2 values ​​indicate how well the data fit the correlation. Correlations ranged in strength, with the best relationships observed for 50 μL of 50 mg / mL versus 10 and 15 μL PNM (0.87 and 0.80, respectively) and 50 μL of 75 mg / mL versus 10 μL PNM (0.83). [Figure 20]Figure 1 shows Pearson's correlation of API apparent permeability data sets, with APIs with Log P>3 removed from the data set. The y-axis shows API apparent permeability through reconstituted mucus. The x-axis shows apparent permeability through PNM (treated / washed native mucus). Apparent permeability is expressed in cm / s x 10-6. It is shown in the legend, above each column of figures (PNM volume) and within each figure (RM concentration and volume). All relationships were significant (p<0.05). The R2 values ​​indicate how well the data fit the correlation. Correlations ranged in strength, with the best relationships observed with 10 μL of PNM (50 μL of 50 mg / mL, R2=0.92; 70 μL of 50 mg / mL, R2=0.87; 50 μL of 75 mg / mL, R2=0.88). [Figure 21]Figure 1 shows Spearman's rank correlation analysis of Caco-2 cell Papp and human jejunum Peff for 13 APIs permeated by the integrated model. The y-axis shows the measured Caco-2 cell Papp (measured in cm / s x 10-6). The x-axis shows human jejunum Peff values ​​expressed in cm / s x 10-6. Integrated model runs were performed using reconstituted mucus concentrations and volumes of either 50 μL of 25 mg / mL, 50 or 70 μL of 50 mg / mL, or 50 μL of 75 mg / mL. The interpretation of R2 is as follows: moderate strength has an R2 value between 0.4 and 0.69, and high strength correlations have an R2 value between 0.7 and 0.89. Significant relationships were observed for all correlations (p<0.05). (A) 50 μL RM vs Peff at 25 mg / mL. A moderate strength correlation was observed, as indicated by an R2 value of 0.6480. The equation of the fitted curve was y=0.0011x+0.1229. (B) 50μL RM at 50mg / mL vs Peff. A moderate correlation was observed as indicated by an R2 value of 0.6839. The equation of the fitted curve was y=0.0014x+0.3123. (C) 70μL RM at 50mg / mL vs Peff. A moderate correlation was observed as indicated by an R2 value of 0.68. The equation of the fitted curve was y=0.0011x+0.1229. (D) 50μL RM at 75mg / mL vs Peff. A strong correlation was observed as indicated by an R2 value of 0.70. The equation of the fitted curve was y=0.0012x+0.1957. [Figure 22]Figure 2 shows Spearman's rank correlation analysis of Caco-2 cell Papp and human jejunum Peff for eight passively absorbed APIs permeated by the integrated model. The y-axis shows Papp (cm / s x 10-6). The x-axis shows human jejunum Peff values ​​expressed in cm / s x 10-6. Integrated model runs were performed using RM concentrations and volumes of either 50 μL of 25 mg / mL, 50 or 70 μL of 50 mg / mL, or 50 μL of 75 mg / mL. Interpretation of R2 can be found in section 2.2.19. Interpretation of R2 is as follows: moderate strength has an R2 value between 0.4 and 0.69, and high strength correlations have an R2 value between 0.7 and 0.89. Significant relationships were observed for all correlations (p<0.05). (A) 50 μL RM vs Peff at 25 mg / mL. A high strength correlation was observed as indicated by an R2 value of 0.86. The equation of the fitted curve was y=0.0038x+0.64. (B) 50μL RM at 50mg / mL vs Peff. A moderate correlation was observed as indicated by an R2 value of 0.65. The equation of the fitted curve was y=0.0015x+0.3187. (C) 70μL RM at 50mg / mL vs Peff. A moderate correlation was observed as indicated by an R2 value of 0.6590. The equation of the fitted curve was y=0.0012x+0.1426. (D) 50μL RM at 75mg / mL vs Peff. A strong correlation was observed as indicated by an R2 value of 0.71. The equation of the fitted curve was y=0.0012x+0.1943. [Diagram 23] Caco-2 cell viability of cells incubated for 1, 2, 3 or 4 hours with DM (300) containing PBS or EDPF, either undiluted or diluted 1:1 prior to dialysis. The x-axis indicates the mucus group. The y-axis indicates the Caco-2 cell viability expressed in %. The legend within the figure indicates the time points of the analysis. The statistical bars indicate that the mean viability values ​​were statistically different from each other. Statistics were determined using a two-way ANOVA test. Asterisks indicate the significant level of the data. A single asterisk indicates when p<0.05. ANOVA tests were performed only for individual time points. Comparison of groups between time points is not valid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention is based on the surprising discovery by the inventors that using dialysis and / or equilibrium density gradient centrifugation it is possible to obtain purified biocompatible mucus that retains the ability to form gels. The method of the present invention does not degrade, hydrolyze or denature the mucins of the mucus, thus allowing the mucins to retain the ability to form mucin-mucin interactions and therefore provide viscoelastic properties to the purified mucus. The purified mucus is therefore capable of forming gels. It has also been shown by the inventors that the purified mucus is biocompatible (also referred to as biologically compatible) such that it can be applied to cells without producing cytotoxic effects. Thus, the present invention provides for the first time mucin formulations suitable for use in in vitro models of digestion, mucus penetration and / or epithelial transport.

[0025] definition It must be noted that as used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0026] As used herein, "about" will be understood by those of skill in the art and will vary to some extent depending on the context in which it is used. If there are uses of the term that are not clear to persons of skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0027] The terms "comprise", "comprises", and "comprising" as used herein specify the presence of stated features, integers, steps, operations, elements, and / or components of aspects or embodiments of the invention, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0028] The phrase "consisting essentially of" means that the scope of an aspect or embodiment should be interpreted to include the explicitly recited materials or steps in the aspect or embodiment, including any materials or steps that do not materially affect the invention defined by the aspect or embodiment. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not to be interpreted as being equivalent to "comprising."

[0029] "Contacting" refers to bringing two or more items into contact with one another. The items may suitably be two or more of a cell, a substrate, a bodily fluid, and a purified mucin or biosimilar mucus as defined herein. Contacting may include bringing or placing two or more of the above items into close proximity to one another.

[0030] "Detecting," "detect," and "detection" refer to the identification and / or quantification of a compound of interest (e.g., a drug, agent, etc.) in a sample. In some embodiments, detecting includes determining the absence or presence of a compound of interest in a sample. In some embodiments, detecting includes quantifying the compound of interest in a sample. In some embodiments, detecting includes identifying and / or quantifying the compound of interest in a sample at various time points. In some embodiments, detecting includes identifying and / or quantifying the compound of interest in a first sample and a second sample.

[0031] "Absorption" or "perfusion" refers to the movement of a compound, such as a test compound or drug, into the bloodstream and through tissues following administration, as well as the movement of the compound through the tissue models described herein. Absorption or perfusion depends on the physicochemical properties of the compound, the formulation, and the route of administration.

[0032] A "tissue" as referred to herein is a group of cells that function together as a unit. A tissue is a level of cellular organization between an individual cell and a complete organ. Thus, an organ is made up of multiple tissues. The tissue may be connective, muscular, nervous, or epithelial tissue. For purposes of the present invention, the tissue may be mucus-secreting epithelium.

[0033] An "epithelium" is a monolayer of cells covering all living surfaces that are exposed to the external environment and form a semi-permeable barrier, such as the respiratory tract, skin, or digestive tract of mammals.

[0034] "Gastrointestinal tract" refers to the complete system of organs and regions involved in the ingestion, digestion, and excretion of food and liquids. This system generally consists of, but is not limited to, the mouth, esophagus, stomach and / or rumen, intestine (small and large intestines), cecum (multiple caecum), fermentation sac, and anus.

[0035] "Enterocyte" refers to cells that make up the mammalian intestinal epithelium. These can be both the small intestine and the colon. The mammalian intestinal epithelium of the gastrointestinal tract has a well-defined tissue structure. The epithelium can be divided into two regions, the functional region that houses differentiated cells (absorptive enterocytes covered by villi) and the proliferative region (crypts of Lieberkühn), which is the epithelial stem cell niche. Multipotent epithelial stem cells reside in the crypts and give rise to the four main epithelial lineages: absorptive enterocytes, mucin-secreting goblet cells, peptide hormone-secreting enteroendocrine cells, and Paneth cells.

[0036] "Intestine" refers to the mammalian small intestine and the mammalian large intestine.

[0037] The term "in vitro" refers to a process performed or occurring outside of a living organism. In some embodiments, the process is performed or occurs in a culture dish.

[0038] The term "in vivo" refers to a process that takes place inside a living organism.

[0039] 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 a gel. Mucin-mucin interactions include covalent and non-covalent bonds. Covalent bonds include disulfide bonds between mucin glycoproteins.

[0040] The term "mucus" refers to an aqueous colloid that contains mucins and possibly inorganic salts, immunoglobulins, nucleic acids, proteins, and / or glycoproteins. Native mucus is swellable. It moistens and protects tissues, but can also be permeable to small compounds such as amino acids and small sugars, especially in the small intestine. Native mucus is a viscous secretion, usually rich in mucins, produced by the mucous membranes that it moistens and protects.

[0041] "Tissue explant" refers to an isolated piece of tissue.

[0042] A "substrate" as referred to herein is any surface suitable for supporting biological material, such as a cell, a cell population, a cell culture, a tissue, or a biological fluid or other biological material or composition, such as those described herein. The surface may be suitable for hosting a process or reaction, such as the growth and development of a cell or organism, a cell population, a cell culture, or a tissue.

[0043] A "gel" is a semi-fluid, jelly-like substance that does not flow when in the solid state. Gels contain a 3D crosslinked network that results in the gel being a semi-fluid structure. Gels may also be hydrogels, which contain a network of insoluble but hydrophilic polymer chains. Gels may be defined in terms of their viscoelastic or rheological properties.

[0044] A "model" as referred to herein is a three-dimensional representation of all or a portion of a natural tissue structure.

[0045] A "buffer" is a solution that can resist significant changes in pH when small amounts of acid or alkali are added. A buffer is a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0046] "Dialysis" is a process that separates molecules in a solution based on their differences in their ability to diffuse through a semi-permeable membrane. Dialysis can be used to remove small molecules, such as salts, peptides, and dyes, from larger molecules, such as DNA, proteins (including glycoproteins), and polysaccharides. The semi-permeable membrane can be cellulose, modified cellulose, or synthetic membranes. The process may require the use of a dialysis buffer that contains the sample to be dialyzed. Optimization of the dialysis process may require adjustment of parameters based on the sample volume, the size of the molecules being separated, the type of membrane, and the geometry of the membrane. Appropriate conditions will be known to those of skill in the art.

[0047] "Fractionation" is a separation process in which a mixture is divided into several smaller fractions based on specific properties of the individual components of the mixture. An example of fractionation is centrifugation.

[0048] As used herein, "cell viability" refers to the ability of a cell to remain metabolically active with respect to proliferation and function.

[0049] A "test compound" is any compound being investigated for use, eg, on the human or animal body.

[0050] The "LVE region" refers to the area in which testing can be performed without degrading or destroying the structure of the sample.

[0051] As used herein, "purified" refers to mucus that has been subjected to a process that removes some or all of the non-mucin components, such as nucleic acids, proteins, immunoglobulins, salts, enzymes, and contaminants. Purified mucus thus contains a higher concentration of mucin compared to the native mucus from which it is derived. Purified mucus may contain a lower concentration of one or more of nucleic acids, proteins, immunoglobulins, salts, enzymes, and contaminants compared to the native mucus from which it is derived. Purified mucus may be partially purified mucin or fully purified mucin, for example, where the product is substantially pure mucin (i.e., at least 95% mucin or more).

[0052] "Biocompatible," which may also be referred to as "biologically compatible," means herein that the material or substrate to which it refers is not substantially toxic or harmful to living cells.

[0053] The term "reconstituted mucus" as used herein refers to purified mucus of the present invention that has been reconstituted with a solution, such as a buffer. The reconstituted mucus is suitably biocompatible.

[0054] Mucin preparations The present invention is based on purifying mucus to provide a formulation comprising mucin that shares the rheological properties of native mucus and is capable of forming a gel without the need for synthetic or non-natural polymers. The method of the present invention can purify mucus such that the native mucin is not denatured, degraded or hydrolyzed, thus providing a formulation that retains mucin:mucin interactions. It is these interactions that provide the mucus formulation with its viscoelastic properties and ability to form a gel. The purified mucus of the present invention can share one or more rheological properties with native mucus, suitably the native mucus from which it is derived. Surprisingly, it has also been shown that the purified mucus of the present invention is biocompatible and therefore capable of supporting living cells. Thus, 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.

[0055] In one embodiment, the purified biocompatible mucus is obtained from a mucus sample, such as a tissue sample. Suitably, the purified biocompatible mucus is obtained from mucus obtained from a tissue sample by the method according to the third aspect of the invention described herein.

[0056] The mucin of the purified biocompatible mucus may be a polymer. The mucin polymer may have a molecular weight of 1 to 10 million daltons. The mucin may be a glycoprotein. The mucin glycoprotein may comprise a peptide core composed of multiple mucin monomer units linked by disulfide bonds attached to multiple carbohydrate side chains. The carbohydrate side chains may be O-linked or N-linked oligosaccharides. One or more of the carbohydrate side chains may be negatively charged. The mucin may be saturated with O- or N-linked oligosaccharides. The mucin may comprise cysteine-rich lightly glycosylated amino and carboxy terminal regions. The mucin may be a transmembrane protein comprising a cytoplasmic region, a transmembrane region, and an extracellular glycoprotein.

[0057] The purified biocompatible mucus of the present invention can form a gel when placed under appropriate conditions such as pH, concentration, temperature, etc. required for gelation. Such conditions can be understood or derived by one of skill in the art based on their knowledge in the art. Suitably, the purified biocompatible mucus of the present invention will have substantially the same viscoelastic properties as the mucus from which it is derived when both are placed under the same conditions.

[0058] In particular, the purified biocompatible mucus of the present invention may exhibit both liquid-like and solid-like properties. The purified biocompatible mucus of the present invention may exhibit linear viscoelasticity. The purified biocompatible mucus of the present invention may have a shear strain and stress strain in the linear viscoelastic region that is substantially the same as or greater than the native mucus from which it is derived, or a washed version of the native mucus from which it is derived. The purified biocompatible mucus of the present invention may have a shear strain in the linear viscoelastic region that is greater than 5%. The compositions of the present invention may have a storage modulus (G') of 1 or greater. The compositions of the present invention may have a loss modulus (G'') of 1 or greater. The compositions of the present invention may have a phase angle within the linear viscoelastic region of at least 3 to 20, or any range or integer therebetween. Suitably, the mucus formulations of the present invention may share one or more, two or more, or three or more rheological properties selected from the group of shear strain, stress strain, storage modulus, loss modulus, and phase angle with native or washed mucus.

[0059] Suitably, the purified biocompatible mucus has, in addition to the viscoelastic properties as defined above, one or more physical and / or chemical properties similar to native mucus, such as permeability, charge, and containing region-specific mucins.

[0060] 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 such components may be native or non-native to the mucus from which the purified biocompatible mucus is derived.

[0061] In one embodiment, the purified biocompatible mucus of the present invention is substantially free of lipids, nucleic acids, or proteins of native mucus. Thus, the mucus formulations of the present invention are prepared by a method that removes substantially all of the lipids, proteins, and nucleic acids from native mucus. The purified mucus of the present invention may have a density of 1.3-1.6 g / mL, suitably 1.4-1.5 g / mL, most suitably 1.42 g / mL. Suitably, the biocompatible mucus is free of components (e.g. lipids, nucleic acids, or proteins) having a density above or below 1.3-1.6 g / mL. Optionally, the purified mucus of the present invention is free of components having a molecular weight below 30 kDa, preferably 20 kDa, or preferably 15 kDa. Density and / or molecular weight may be provided in relation to the purified mucus after dialysis and / or equilibrium density gradient centrifugation, but before the addition of any other components such as buffers.

[0062] In one embodiment, the purified biocompatible mucus of the present invention is free of 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.

[0063] By biocompatible, it is meant that the purified mucus is not toxic or harmful to cells. Thus, the purified mucus is compatible with cells so as not to induce or cause cell death within a reasonable period of time. The composition may be capable of maintaining cell viability. Thus, the composition does not result in a substantial decrease in cell viability. By substantial, we mean a decrease in viability of 30% or more of the cells in the population over a period of time, e.g., minutes, an hour or more, or up to several days, depending on the nature of the composition and the cell type and the conditions of the model.

[0064] 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 domestic animal such as a pig, sheep, goat, donkey, horse, cow, dairy cow; or any other large mammal, for example a deer, antelope, elephant, camel, llama, etc. Any other suitable mammal from which mucin may be extracted will be known to the skilled person. 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 an animal, suitably a human. Suitably, the purified biocompatible mucus is derived from a pig, suitably a pig gastrointestinal system, suitably a pig intestine (large intestine or small intestine) or stomach.

[0065] The purified biocompatible mucus may be extracted from any suitable tissue of a mammal or non-mammal. When the purified biocompatible mucus is extracted from a mammal, the mucus 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, buccal, lingual, or colonic tissue; airway tissue, including, but not limited to, lung, trachea, bronchi, and bronchioles, and mouth; 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, the mucus may be extracted from the outer surface of the non-mammal.

[0066] The purified biocompatible mucus referred to herein may be 100% pure mucin or may include other non-mucin components, such as contaminants including biomolecules remaining from the extraction process, components of native mucus. The purified mucus referred to herein may include 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 includes at least 35%, suitably at least 40% mucin. The remainder of the formulation may include water, salts, lipids, proteins, sugars, glycoproteins, immunoglobulins, bacteria, reagents, and / or contaminants. When the purified biocompatible mucus of the invention is produced by the method of the third aspect, the purified biocompatible mucus may comprise 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% mucin prior to reconstitution of the purified mucus with a buffer. Suitably, the purified biocompatible mucus comprises at least 35%, suitably at least 40% mucin. Mucin is rich in serine, proline, and threonine. The purified biocompatible mucus of the 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 devoid of serine, proline, or threonine, or may contain small amounts (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.

[0067] 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).

[0068] The purified biocompatible mucus referred to herein may be a viscous, gel-like formulation. Alternatively, the mucus may be a solid, e.g., dried or lyophilized to provide a formulation for storage. Alternatively, the mucus may be a liquid (e.g., after reconstitution with an appropriate buffer or diluent).

[0069] The purified biocompatible mucus may be provided in a buffer to form a composition. The buffer may be suitable for use with cells or living tissue. The buffer may be suitable for use with acidic gastrointestinal secretions. The buffer may not alter the viscoelastic properties of the purified biocompatible mucus. The buffer may be cytocompatible. Examples of suitable buffers include, but are not limited to, deionized water, phosphate buffer (e.g., phosphate buffered saline), HEPES buffer, MOPS buffer, MES buffer, Tricene, Bicene, TAPS, ACES, MOPSO, and BES. Other suitable buffers may be used and would be known to one of skill in the art. In one embodiment, the buffer is phosphate buffered saline that corresponds to a body fluid of the correct pH and osmolality. The buffer may be supplemented with, for example, calcium and / or magnesium. The buffer may be PBS supplemented with calcium and magnesium.

[0070] The purified biocompatible mucus may be provided in a buffer of any suitable concentration. In one embodiment, the concentration of the purified biocompatible mucus in the composition is similar to that of native mucus. The purified biocompatible mucus may be present in the composition at 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 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 compositions described herein.

[0071] The purified biocompatible mucus may be provided in combination with cells or as a composition that includes cells.

[0072] The mucins present in the purified biocompatible mucus may be human MUC2, MUC5AC, MUC5B, MUC6, MUC19, MUC7, or non-human equivalents thereof, including, for example, ovamucin (egg white), spiggin (MUC19-derived mucin from stickleback), 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.

[0073] 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 buffer may be as described herein. The biocompatible mucus may be purified according to the third aspect of the invention described herein. The concentration of the mucin may be 20-120mg / ml, suitably 30-100mg / ml, suitably 40-80mg / ml, suitably 50mg / ml.

[0074] Organizational Model In a second aspect of the invention, a tissue model is provided comprising i) a cell population and ii) 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 may be useful in screening assays for, for example, drug absorption, drug dissolution, and drug toxicity. The second aspect of the invention is based on the surprising discovery that the purified biocompatible mucus of the first aspect can be used to construct a reconstituted mucus layer that can be combined with a cell population in a manner that does not kill the cells. Such a model provides a robust and physiologically relevant in vitro method by which pharmaceutical formulations can be screened and used to improve and inform human research.

[0075] The tissue model according to the second aspect of the invention may comprise a substrate comprising a cell support portion. The cell population may be in contact with the cell support portion of the substrate. The cell population cells may form a layer on the support portion.

[0076] The tissue model may be described as having a basolateral surface, which is the bottom surface of the matrix, and an apical surface, which is the top surface of the tissue model. In the tissue models of the present invention, the purified biocompatible mucin is applied to the top surface of the cell population in contact with the cell population. The purified mucin does not have to be in direct contact with the cell-supporting portion of the matrix.

[0077] In one embodiment, i. a substrate comprising a cell-supporting moiety; ii. a cell population in contact with the cell-supporting portion of the substrate; iii. Purified biocompatible mucus in contact with cells. In accordance with a first aspect of the present invention, a tissue model is provided comprising in separate layers:

[0078] As used herein, therefore, any suitable substrate having moieties suitable for hosting a cell population may be used. Suitably, the substrate is suitable for hosting or culturing mammalian cells. The substrate may be one that is easily sterilized and biocompatible. Suitable substrates are known to those skilled in the art and may include, but are not limited to, glass, carbon, organic materials (e.g., cotton), nitrocellulose, dextran, gelatin, polymeric materials (e.g., plastics, poly(methyl methacrylate), polyamides, polyesters; polystyrene; polypropylene, polyurethanes, polylacrylates, polyvinyl compounds (e.g., polyvinyl chloride); polycarbonates; polytetrafluoroethylene (PTFE, Teflon), acrylic copolymers, polyglyolic acid (PGA), fluoropolymers; fluorinated ethylene propylene; polyvinylidene fluoride; polydimethylsiloxane), metals (e.g., gold, silver, aluminum, titanium, stainless steel), silicon substrates (fused silica, polysilicon, or single crystal silicon).

[0079] The substrate may be rigid or elastic. The substrate may be porous or non-permeable. Materials that are not naturally porous can be made porous by methods available to those skilled in the art, such as sintering, etching, leaching, lithography, or laser micromachining.

[0080] The substrate may be a slide, chip, plate, flask, vial, film, microstructure (including grooves, wells, or posts), multi-well plate, dual chamber plate, or any other suitable form. The substrate may be of any suitable shape or structure, including flat, tubular, curved, spherical, elliptical, etc., including composite materials (e.g., to mimic macroanatomical structures). The substrate may include a flat surface on which the cell population may be provided. The substrate may be provided or mounted on a porous carrier (e.g., a porous membrane, mesh, inorganic grid, hydrogel, or combinations thereof) to give it structural support. The substrate may be two or three dimensional, or any combination thereof.

[0081] Suitably, the substrate may be a dual chamber plate, with each well comprising a lower chamber and an upper chamber. The upper chamber may be located above the lower chamber or may be placed within the lower chamber. Suitably, the upper and lower chambers are substantially aligned. The lower chamber may be referred to as the basolateral chamber and the upper chamber may be referred to as the apical chamber. The bottom surface of the upper chamber may be permeable to allow fluid to pass from the upper chamber to the lower chamber. Suitably, the upper chamber is located completely or partially within the lower chamber to allow fluid communication between the two chambers. The upper chamber may comprise a cell population and a purified mucin layer. The lower chamber may comprise a buffer or suitable cell culture medium to maintain the cell population. Thus, for example, in a small intestine model, the lower chamber corresponds to the basolateral side of the epithelial cells to the circulating blood and lymphatic vessels associated with the villi. The upper chamber corresponds to the lumen of the small intestine.

[0082] Suitably, the substrate may be provided with an anaerobic envelope providing an anaerobic environment. If 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, whereas the upper chamber may be an anaerobic environment.

[0083] The selection of an appropriate substrate may depend on the type of cells used in the model. Factors may include the ability of the cells to adhere to the substrate and the effect of the substrate on the cells.

[0084] The substrate can be modified to allow or improve cell adhesion to the substrate. All or part of the substrate can be modified. Suitable modifications are known to those skilled in the art and can include, for example, the application of proteins (e.g., collagen or fibronectin), extracellular matrix or components thereof, sugars, proteoglycans to the surface of the substrate.

[0085] The substrate may be a single well or may be a multi-well plate. The multi-well plate may contain any suitable number of wells, for example, 6, 12, 24, 48, 96, 384 or 1536 wells. The wells may be microwells.

[0086] In the model of the second embodiment, the cell population is placed in contact with the substrate, suitably with a flat surface of the substrate, such as the bottom surface of a well that forms the cell support portion. The cell population may comprise one or more cells, suitably two or more, up to 1000, 10,000, 1×10 6 , 2×10 6 , 3×10 6 Or it may include more.

[0087] The cell population may form a layer of cells. Suitably, the cell population forms a layer of at least 50, 60, 7, 80, 90 or 100% confluency. Hence, the cell layer may be referred to as confluent. The layer of cells in contact with the cell support portion may be a monolayer of cells (meaning the layer is one cell deep) or more than one cell layer (two or more cells deep). The cells may comprise one type of cell, or two or more different cell types. In a multi-layered cell population, each layer may substantially comprise a different cell type. The cell population provided on the substrate may be a tissue explant. The tissue explant may be a monolayer of tissue, or may be two or more layers of tissue. The tissue explant may be placed on the cell support portion of the substrate and compressed. The tissue explant may be obtained from an organism, such as a mammal, or may be cultured in vitro.

[0088] The tissue model may have the structure of the corresponding natural in vivo tissue.

[0089] When the cells form a layer on the cell-supporting portion of the substrate, the layer may be flat or may be in a folded or formed three-dimensional shape that mimics tissue structures such as, for example, the intestinal crypt or crypt-villus structures in vivo, or the vaginal stratified squamous epithelium layer.

[0090] The cell population, optionally as a tissue explant, may be derived from gastrointestinal tissue, airway tissue, nasal tissue, ocular tissue, or female reproductive tissue. The cell population, optionally as a tissue explant, may be derived from the ileum, jejunum, stomach, duodenum, esophagus, buccal, tongue, or colon of the gastrointestinal tract, or the nose, lung, bronchus, bronchioles, or mouth, or the cervix or vagina, or the eye. The cells may be obtained from a mammal as defined herein, for example a pig. The cells may be derived from a human. In one embodiment, the tissue explant is derived from the human gastrointestinal tract, suitably the ileum, jejunum, stomach, duodenum, esophagus, buccal, tongue, and / or colon of a human. In one embodiment, the cells for use in the model of the second aspect are oral cells, stomach cells, small intestinal cells, large intestinal cells, nasal cells, ocular cells, vaginal cells, cervical cells, or lung, or bronchus, or other airway cells. The cells may be epithelial cells. The cell culture may be derived from a cell line, hi one embodiment, the cell culture is a Caco-2 cell culture or a calu-3 cell culture.

[0091] The cell populations or tissues may include natural bodily fluids or tissue components, such as sputum, nasal mucus, urine, vomit, bile, blood, vaginal secretions, semen, or other biological excretions, microorganisms such as bacteria, and cellular debris.

[0092] The cell population may be placed in contact with the cell-supporting portion of the substrate in a manner that maintains native cell polarity, such that the native basal surface of the tissue may be placed in contact with the cell-supporting portion of the substrate, and the luminal surface of the tissue (which will face the lumen of the overlying tissue or organ in vivo) is separated from the cell-supporting portion of the substrate by the body of the cell population.

[0093] 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.

[0094] The substrate may contain more than one tissue explant. If the substrate is, for example, a multi-well plate, each well or cell support portion may be in contact with a different tissue explant. The explants may be the same or different tissue types. The explants may be from the same or different mammalian species. The tissue explants may all be human.

[0095] The cell population of the model of the second aspect of the invention can be maintained in culture under suitable conditions, for example for any suitable period of time, for example from a few minutes to an hour or more, 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 to 12 hours, 1 to 24 hours, 1 to 36 hours, 1 to 48 hours, 1 to 72 hours, or from 1 hour to 1 week, 2 weeks, 3 weeks, 4 weeks or more. The cell population may require exogenous growth factors to be maintained in culture, or may not require exogenous growth factors. The term "maintained in culture" means that the cell population is maintained in vitro under conditions suitable for cell growth and / or survival. This may require providing essential nutrients (e.g., amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, gases (e.g., O2, CO2), and maintenance of a suitable physicochemical environment (e.g., pH, osmolality, temperature). Conditions may be aerobic or anaerobic. Examples of substances that may be provided in cell culture to maintain cell growth and / or survival include, but are not limited to, fibronectin; fetal bovine / 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., EphrinB, EphB); bone morphogenetic proteins (BMPs, BMP-2, BMP-7); Wnt (wingless-related integration site) (e.g., Wnt3, Wnt3A, and other Wnt); notch signaling factors (notch receptors); Dlll / 4; noggin; Greml; Grem2; acetate; butyrate; propionate, desaminotyrosine, catecholamines (e.g., dopamine, noradrenaline); cytokines, and / or short chain fatty acids.

[0096] The purified biocompatible mucus may be as defined in the first aspect. Thus, in one embodiment the purified biocompatible mucus may comprise a composition comprising or consisting essentially of the purified biocompatible mucus according to the first aspect and a buffer.

[0097] The purified biocompatible mucus may be provided as a layer on the cells. The layer may be a uniform layer or a non-uniform layer. The layer may cover substantially all of the cells. The layer may be of any suitable thickness. Suitably, the purified biocompatible mucus layer mimics the thickness of mucin found in native tissue. The thickness of the mucin layer may be any suitable thickness depending on the purpose of the model, the conditions, and the drug being tested. Suitable thicknesses may 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 using the upper or lower limit of any of the aforementioned ranges, or any integer that is the upper or lower limit within the aforementioned ranges, or is within the aforementioned ranges.

[0098] The tissue models described herein may further comprise a body fluid. The body fluid may be provided to the apical surface of the mucin layer. The body fluid may therefore not be in direct contact with the cells. The body fluid may be provided to mimic a natural tissue environment and may be, for example, digestive fluid, saliva, vaginal secretions, tears, or airway secretions, or a microbial cell population. The body fluid may be a natural body fluid extracted from a mammal as described herein, or may be a synthetic body fluid. The body fluid may be an end-point digestive fluid (EPDF), for example, saliva, gastric fluid, and small intestinal fluid, optionally including bile and / or enzymes, such as pepsin and / or pancreatin. Suitable end-point digestive fluids are shown in Table 1. The composition of the dilution of the EPDF is shown in Table 2. Any suitable volume of body fluid may be applied.

[0099] [Table 1]

[0100] [Table 2]

[0101] Three or more layers of the tissue model may be stacked to fully or partially align with one or more layers of the model, fully or partially overlap, suitably two or more layers are fully aligned, suitably all of the layers are fully aligned.

[0102] The present invention may provide a model for any mucus-coated epithelial tissue.

[0103] 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 separate layers: i) a basement substrate comprising a cell support portion, ii) a cell population in contact with the cell support portion of the substrate, preferably the cell population is a Caco-2 cell population, iii) purified biocompatible mucus in contact with the cells, and optionally iv) gastric juice.

[0104] 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 separate layers: i) a base matrix comprising a cell support portion, ii) a cell population in contact with the cell support portion of the matrix, preferably the cell population is a Caco-2 cell population, iii) purified biocompatible mucus in contact with the cells, and optionally iv) gastrointestinal fluid.

[0105] 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 separate layers: i) a basement substrate comprising a cell support portion, ii) a cell population in contact with the cell support portion of the substrate, preferably the cell population is a Caco-2 cell population, iii) purified biocompatible mucus in contact with the cells, and optionally iv) gastrointestinal fluid, preferably an endpoint digestive fluid as described herein.

[0106] 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 separate layers, i) a base matrix comprising a cell support portion, ii) a cell population in contact with the cell support portion of the matrix, iii) purified biocompatible mucus in contact with the cells, and optionally iv) saliva, alveolar fluid, sputum. The cell population may comprise any suitable airway cell.

[0107] 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 separate layers: i) a basement 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.

[0108] In one embodiment, the tissue model of the second aspect of the invention is an ocular or nasal tissue model. In one embodiment, the ocular or nasal model comprises, in separate layers, i) a basement 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 mucus or tears. The cells may be any suitable cells of the ocular or nasal system.

[0109] In one embodiment, the present invention provides two or more models placed 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, and a large intestine model in series.

[0110] In one embodiment, the models of the invention may include one or more test compounds, such as pharmaceutical agents. Test compounds, such as pharmaceutical agents, may be applied to the models to assess the permeability of the agent.

[0111] In any of the tissue models provided herein, the purified biocompatible mucus may be reconstituted with a solution, such as a buffer, prior to use in the tissue model. The buffer may be any suitable buffer, for example PBS. The suitable buffer may be biocompatible. Any suitable method of reconstitution may be used that does not substantially disrupt mucin:mucin interactions. The reconstituted mucus of the invention will suitably be biocompatible.

[0112] How to purify mucus The present invention provides a method of purifying mucus, the method comprising the removal of substantially all lipids, nucleic acids and / or proteins and / or any molecules below a predetermined size, suitably below 100 kDa, more suitably below 70 kDa, the removal step comprising 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, for example centrifugation, to remove components of native mucus, contaminants, excess water and / or reagents. Suitably the method of the present invention provides a purified biocompatible mucus of the first aspect.

[0113] In one embodiment, the invention provides a method of purifying mucus, the method comprising the steps of: i) providing a sample comprising mucus; ii) solubilizing any mucins present in the sample; iii) separating any nucleic acids, proteins, lipids from the mucus; and iv) removing any contaminants, undesirable components or reagents from the mucus to provide purified biocompatible mucus. Step iii) may comprise equilibrium density gradient centrifugation. Step iv) may comprise removal of any cytotoxic components, reagents and / or enzymes and may comprise dialysis of the mucus. Suitably, the method comprises isolating a fraction from the mucus having a density gradient of 1.3-1.6 g / mL.

[0114] Solubilization can be performed using any suitable method to increase the solubility of any mucins in the sample. Suitable methods can include, but are not limited to, incubation of the tissue sample with chaotropic agents (e.g., urea, thiourea, particularly 8M urea, or a combination of 2M urea and 5-8M thiourea), ionic, non-ionic, or zwitterionic detergents (e.g., SDS, NP-40, Triton X, CHAPS, and sulfobetaine), carrier ampholytes, or reducing agents (e.g., DTT or TBP). After solubilization, any insoluble material can be removed, for example, by centrifugation.

[0115] 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 isolate a fraction 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 a fraction identified as having the highest proportion of mucin glycoproteins and the lowest proportion of nucleic acids, lipids or proteins, which may be selected using methods available to the skilled artisan, for example periodic schiffs acid analysis and OD260 / OD280 analysis. Alternatively, dialysis may be used to remove any molecules having a molecular weight below 100 kDa, suitably below 70 kDa, most suitably below 50 kDa. A combination of equilibrium density gradient centrifugation and dialysis may be used to provide purified mucus substantially devoid of nucleic acids, lipids and proteins, suitably also substantially devoid of any chemical reagents or enzymes. A suitable combination of centrifugation and dialysis to provide purified mucus with low nucleic acid content and high glycoprotein content, or a density of 1.3-1.6 g / mL, or lacking molecules below 100 kDa, suitably below 70 kDa, most suitably below 50 kDa, can be identified by one skilled in the art. When ultracentrifugation is used, suitably a caesium salt gradient is used, suitably caesium chloride. It is envisaged that other suitable methods may be used that have the effect of substantially purifying or separating mucin from mucus. Such methods, such as fractionation, centrifugation, etc., will be known and available to those skilled in the art.

[0116] Suitably, the step of separating any nucleic acids, proteins, lipids or molecules from the mucus may include removal of any such components having a density above or below 1.3-1.6 g / mL, or that are 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 a mucus preparation in which molecules below 100 kDa, suitably below 70 kDa, more suitably below 50 kDa have been removed.

[0117] The method may include removal of any unwanted components or chemicals, such as reagents, enzyme inhibitors, etc. Any suitable method may be used, such as dialysis. Suitably, the method may include dialysis to remove any chemicals, contaminants, or reagents. Suitably, the dialysis may have a molecular weight cut off (MWCO) of 30 kDa or less, 25 kDa or less, 20 kDa or less, or 15 kDa or less. Any suitable semi-permeable membrane may be used, including, but not limited to, semi-permeable membranes of polysulfone, polyethersulfone (PES), etched polycarbonate, cellulose, or collagen dialysis tubing. Dialysis may be performed for any suitable period of time, which will depend on the MWCO of the tubing. A suitable period of time may be, for example, from 1 hour or more, to 3-5 hours, 5-10 hours, 5-12 hours, 12-14 hours, 36 hours, 48 ​​hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks or more, or any integer or range therebetween. Such dialysis may be performed on selected fractions following ultracentrifugation of the mucus sample.

[0118] In an alternative method, purified mucus can be prepared using size exclusion chromatography. The mucin-containing fraction can be separated from other components in the mucus as described herein using size exclusion chromatography. Suitable columns will be known to those skilled in the art, but can include Sepharose 2B or 4B columns. Mucin can be recovered from the column void volume.

[0119] Suitably, the method may include homogenizing the tissue sample. Homogenization may be performed before solubilization and before exposure to enzyme inhibitors. Suitable methods for homogenization include, but are not limited to, grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultrasound. Samples may be refrigerated to reduce or prevent protein damage.

[0120] 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 prior to solubilization. The enzyme inhibitors may have the effect of reducing or preventing mucin dissolution by enzymes present in the sample. Suitable enzyme inhibitors include protease inhibitors. The method of the invention may comprise exposing the sample to one or more protease inhibitors. Suitably, the method may comprise exposing the sample to an inhibitor 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. By way of non-limiting example, suitable enzyme inhibitors may be aminoheaxanoic acid, EDTA, iodoacetamide, N-ethyl maelinide, benzamidine HCL, or pmsf. Suitably, a combination of two or more enzyme inhibitors may be used. A suitable enzyme inhibitor combination may include any two or more inhibitors selected from aminohexanoic acid, EDTA, iodoacetamide, N-ethylmaleimide, benzamidine HCL, and / or pmsf. Other suitable enzyme inhibitors or combinations thereof will be 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 more. Suitably, the enzyme inhibitor will not result in mucin degradation. The amount and concentration of the enzyme inhibitor may be selected to prevent mucin degradation.

[0121] In an alternative embodiment, the method may comprise the steps of: i) providing a sample comprising mucus; ii) dialysis of the mucus to separate any molecules below 100 kDa, suitably below 70 kDa, from the mucus; and optionally iii) separating the mucus, for example by centrifugation.

[0122] In such embodiments, the mucus sample may 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 may be used, including semipermeable membranes of polysulfone, polyethersulfone (PES), etched polycarbonate, cellulose, or collagen dialysis tubing. Dialysis may be performed for any suitable period of time, which will depend on the MWCO of the tubing. A suitable period of time may be, for example, 1 hour or more, to 3-5 hours, 5-10 hours, 5-12 hours, 12-14 hours, 36 hours, 48 ​​hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks or more, or any integer or range therebetween.

[0123] The method of this embodiment may further include separation, for example by centrifugation or any other suitable method, to remove excess liquid or other components of the mucus.

[0124] The method of the invention may include obtaining a mucus sample. Obtaining a mucus sample may include scraping or extracting mucus from a tissue sample. The mucus sample may be diluted prior to purification, any suitable dilution being, for example, 1:50.

[0125] The methods of the invention may further comprise the step of combining the purified mucus with a solution, such as a buffer. The buffer may be as described herein. This step may be referred to as reconstituting the purified mucus.

[0126] The method of the invention may include combining the purified biocompatible mucus of the 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 include subjecting the purified biocompatible mucus to conditions that allow for gelation of the mucus.

[0127] The method of the present invention may further comprise the step of 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 with an appropriate buffer for use.

[0128] The invention may also provide a method of preparing 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.

[0129] The method of purifying mucus from tissue may include one or more sterilization steps. A sterilization step may be performed during the method of purifying mucus from tissue, for example, by application of one or more antimicrobial agents during the process, for example, during dialysis. A sterilization step may be performed after purification or after freeze-drying. The method of the present invention may include one, two, three or more sterilization steps. Any suitable sterilization method may be used, for example, sterilization by temperature change and / or UV light, or solvent sterilization. Suitable solvents may include, but are not limited to, ethanol, methanol, or IPA. Solvent-based sterilization may include application of a solvent, mixing with tissue, cells or purified mucus, or composition, and evaporation of the solvent. Any one or more purified mucus or compositions containing purified mucus may be sterilized.

[0130] The invention may also provide a method of 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 comprise providing the purified mucus as a composition as described herein. The method of preparing a tissue model as described herein may further comprise applying a body fluid as described herein to the purified mucus applied to the cell population.

[0131] The method of forming purified mucus may include using one or more processes to promote, enable, or maintain gelation or viscoelastic properties of the mucus. In one embodiment, the method of forming purified biocompatible mucus may include one or more temperature changes, for example, utilizing heating and cooling cycles, to promote gelation. In one embodiment, the method may include changing the pH to promote gelation of the purified biocompatible mucus. For example, increasing the pH and then decreasing it can promote gelation. Any suitable number of temperature and / or pH increase and decrease cycles may be used. In one embodiment, the method may include adding a salt to the purified biocompatible mucus to promote gelation of the mucin.

[0132] The method of preparing the purified biocompatible mucin may include storing the purified mucin. The method of storing may include lyophilizing the purified mucin, the composition, or the biosimilar mucin. Suitable conditions for lyophilization will be known to those of skill in the art.

[0133] Using the Organization Model The purified biocompatible mucus or the compositions of the invention or the tissue models of the invention may be used in many applications including, but not limited to, drug absorption studies, formulation development, GI injury / side effect profiling, nutrient uptake analysis, drug-microbiome interaction studies, particularly in colon models, intestinal injury profiling, and pre / probiotic testing.

[0134] Accordingly, the present invention provides a method for determining or predicting absorption of a test compound by a tissue model, the tissue model comprising: i) a substrate including 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, the method comprising contacting the purified biocompatible mucus with a test compound and detecting movement of the test compound in the tissue model, thereby determining the ability of the test compound to cross the purified biocompatible mucus and cell layers of the tissue model. The presence of the test compound in the substrate indicates the ability of the compound to be absorbed by the tissue model.

[0135] The tissue model may comprise the purified biocompatible mucus or a composition comprising purified biocompatible mucus according to the first aspect of the invention. The tissue model may be as described in the second aspect of the invention.

[0136] Detecting the movement of the test compound through the tissue model may include determining the concentration of the test compound at or below the substrate. Detecting the movement of the test compound through the tissue model may include detecting the test compound in the purified biocompatible mucus layer, the cell population, and / or in contact with the surface of the substrate.

[0137] The test compound may be detected on the luminal and / or basolateral surface of the tissue model, with the presence of the test compound on the basolateral surface indicating the ability of the compound to be absorbed by the tissue explant.

[0138] Detecting the test compound can be performed by any suitable means, such as physical, chemical, or visual means. Examples include, but are not limited to, antibody assays, mass spectrometry, colorimetric assays, liquid chromatography, nucleic acid sequencing, SDS PAGE, Western blotting, and in situ hybridization. The appropriate method can be selected by those skilled in the art based on the properties of the test compound.

[0139] The tissue model 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, an optical tissue model, and / or an airway tissue model described herein.

[0140] In one embodiment, the method for determining or predicting the absorption of a test compound by a tissue model may be used to determine or predict the effect of food on the absorption of a compound into gastrointestinal tissue. In such a method, the tissue model may be a gastric tissue model, a small intestine tissue model, a large intestine tissue model, or a gastrointestinal tissue model. The method may include applying food to a purified biocompatible mucus or fluid layer of the model. The food may be digested or undigested food. The steps of contacting the tissue model with the food and the test compound may be separate or simultaneous. If separate, the test compound may be contacted with the tissue model before or after contacting the tissue model with the food. The method may include testing the difference in absorption of the compound of interest in the presence or absence of food.

[0141] In one embodiment, the method for determining or predicting the absorption of a test compound by a tissue model may be used to determine or predict the effect of natural intestinal media on the absorption of a compound into gastrointestinal tissue. In such a method, the tissue model may be a gastric tissue model, a small intestinal tissue model, a large intestinal tissue model, or a gastrointestinal tissue model. The method may include applying a natural intestinal media to a purified biocompatible mucus or fluid layer of the model. The steps of contacting the tissue model with the natural intestinal media and the test compound may be separate or simultaneous. If separate, the test compound may be contacted with the tissue model before or after contacting the tissue model with the natural intestinal media. The method may include testing the difference in absorption of the compound of interest in the presence or absence of natural intestinal media.

[0142] The absorption of the test compound into the tissue model can be tested at various time points. This will provide information regarding the rate of absorption of the test compound by the tissue model, including the rate of absorption through different layers of the tissue model. Such methods can be used to predict the rate of absorption through native tissue.

[0143] The methods for determining or predicting absorption of a test compound described herein may be used to determine or predict the perfusion rate of a test compound in a tissue explant. The presence of the test compound at the basolateral surface indicates the ability of the compound to be perfused through the tissue model. By detecting the presence of the test compound at various time points, the rate of perfusion can be obtained.

[0144] The present invention may also provide a method for determining the effect of a test compound on a tissue model, the tissue model comprising: 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, the method comprising contacting the purified biocompatible mucus with a test compound and detecting the effect of the test compound on the tissue model. The method may 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 may be used to predict the effect of a test compound on a native tissue.

[0145] 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 invention. The tissue model may be as described in the second aspect of the invention.

[0146] Determining the effect of the test compound may include determining the effect of the test compound on local tissue toxicity, genetic modification of tissue, temporal changes in tissue permeability, modulation of mucus or microbiome, and modulation of hormone production and / or secretion, general health of tissue cells, viability of all or part of tissue, such as the status of purified mucin layer (e.g., with respect to cell viability, cytokine production, or transepithelial electrical resistance). The status of the cell population may be evaluated with respect to factors such as the number of live or dead cells, or the presence or absence of cell markers, or the determination of cell membrane permeability. Assays include any cell viability assay available to one of skill in the art, such as flow cytometry, high content imaging, colorimetric tetrazolium reagents, resazurin reduction and protease substrates that generate fluorescent signals, luminescent ATP assays, fluorescence microscopy, real-time assays that monitor live cells over days in culture, cell titer blue assay.

[0147] The tissue model used in the method for determining the effect of a 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, an optical tissue model, and / or an airway tissue model described herein.

[0148] The method for determining the effect of a test compound may be used to determine or predict the influence of food on the effect of the compound. In such a method, the tissue model may be a stomach tissue model, a small intestine tissue model, a large intestine tissue model, or a gastrointestinal tissue model. The method may include a step of applying food to the mucin or fluid layer of the model. The food may be digested or undigested food. The steps of contacting the tissue model with the food and the test compound may be separate or simultaneous. If separate, the test compound may be contacted with the tissue model before or after contacting the tissue model with the food. The method may include a step of testing the difference in absorption of the compound of interest in the presence or absence of food.

[0149] In one embodiment, the method for determining the effect of a test compound can be used to determine or predict the influence of natural intestinal media on the effect of a compound. In such a method, the tissue model can be a stomach tissue model, a small intestinal tissue model, a large intestinal tissue model, or a gastrointestinal tissue model. The method can include applying a natural gastrointestinal media to the mucin or fluid layer of the model. The steps of contacting the tissue model with the natural intestinal media and the test compound can be separate or simultaneous. If separate, the test compound can be contacted with the tissue model before or after contacting the tissue model with the natural intestinal media. The method can include testing the difference in absorption of the compound of interest in the presence or absence of natural intestinal media.

[0150] The effect of the test compound on the tissue model can be tested at various time points, which will provide information regarding the kinetics of the effect of the test compound on the tissue model, including the kinetics of the effect in different layers of the tissue model.

[0151] The above method may be useful in identifying optimal or suitable drug delivery mechanisms, formulations, and drug effects on the body.The above method may also be suitable for identifying regulators of protein, lipid, or fat digestion for use in the food / nutraceutical industry.The above method may also be useful in testing the effects of factors such as food, meal time, etc. on drug efficacy.

[0152] Kits for use in the methods of the invention may be provided and may contain, in suitable containers, the tissue models described herein, and optionally substrates, buffers, reagents, instructions for use, and other standard components known in the art.

[0153] Test compounds for use in the present invention may 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 extracts thereof, plant and animal extracts. Test compounds may be pharmaceuticals. Test compounds may include combinations of two or more compounds.

[0154] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context otherwise requires. In particular, where the indefinite article is used, it should be understood that the specification contemplates both the plural and the singular unless the context otherwise requires.

[0155] It should be understood that any feature, integer, property, compound, chemical moiety or chemical group described in conjunction with a particular aspect, embodiment or example of the invention applies to any other aspect, embodiment or example described herein, unless inconsistent. All of the features disclosed herein (including any accompanying claims, aspects and figures) and / or all of the 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 of the foregoing embodiments. The invention extends to any novel feature, or any novel combination of features disclosed herein (including any accompanying claims, aspects and figures) or any novel step, or any novel combination of steps of any method or process so disclosed.

[0156] The reader's attention is directed to all papers and documents relating to this application, filed contemporaneously or prior to this application, and open to public inspection herewith, the contents of which are incorporated herein by reference in their entirety. EXAMPLES

[0157] Generation of reconstituted mucus Mucus scraped from porcine intestine was mixed with an enzyme inhibitor cocktail in a 3:1 ratio of inhibitor buffer to mucus (Table 5) to prevent mucin lysis due to enzymes and other biological components such as bacteria present in the intestinal scrapings. The mucus / inhibitor buffer preparation was homogenized to solubilize the mucin. Homogenization may depend on the source of mucus and may be performed for 0-30 minutes. The homogenized material was centrifuged in an 85 ml centrifuge tube at 10,000 RPM at 4°C for 1 hour (Eppendorf F-34-6-38 fixed angle rotor and Eppendorf centrifuge 5810 R). The supernatant obtained from this centrifugation was filtered through glass wool and the pellet was discarded. The supernatant was adjusted to a density of 1.42 g / l using cesium chloride and ultracentrifuged using a P50AT2-941 rotor in a Himac CP100NX ultracentrifuge. An ultracentrifuge tube assembly kit compatible with the rotor and centrifuge described above was used, which included 40PA 40mL high speed round bottom tubes, and S-40AL caps and Ti rings. The ultracentrifuge, rotor, and tube assembly were supplied by Hitachi, Ltd. (Hitachi High-Technologies, Maidenhead, UK). This process separates the solubilized mucin from nucleic acids, lipids, and proteins by this equilibrium density centrifugation. The equilibrium density centrifugation causes the solution to form a density gradient, which is separated into eight distinct fractions. The heaviest fraction is 8 at the bottom of the tube, and the lightest fraction 1 is at the top. These fractions were previously identified by mass. Fraction samples were pipetted in order from 1 to 8. Each fraction is approximately 4 ml.

[0158] Each fraction was dialyzed using a 19 mm expanded 12-14 kDa molecular weight cut-off (MWCO) dialysis membrane (Medicell Membranes Ltd, London, UK) to remove unwanted chemicals such as inhibitor buffer and cesium chloride components. Dialysis was performed in a 20 L stirred vessel filled with deionized (DI) water, which was periodically changed six times over a two day period. The process was maintained at 4°C using a cold room.

[0159] After extensive dialysis, OD was measured using an M200 Pro spectrophotometer (TECAN UK, Reading, UK). 260 / 280 The values ​​were measured to determine the DNA / protein content of the fractions. To determine the glycoprotein content, a PAS assay was performed using porcine gastric mucin as a standard.

[0160] As mucins are carbohydrate-rich glycoproteins, the fraction with the highest glycoprotein content and lowest DNA / protein was considered to be the mucin-containing fraction, which was then lyophilized and stored at -20°C until required, at which point mucin was rehydrated to the required concentration by adding 1x PBS (Merck) and left on a tube roller overnight at 4°C.

[0161] The product can be sterilized by the addition of antimicrobial agents during the dialysis process or by solvent sterilization of the purified mucus. The sterilization process may be performed after freeze-drying. In solvent sterilization, solvents such as ethanol, methanol, and IPA may be added to the mucus, mixed, and evaporated under sterile conditions.

[0162] Small intestine absorption plate model Throughout this project, permeation experiments were performed using dual chamber microplates consisting of either 96 or 24 chambers. Each chamber was termed the basolateral or apical chamber and corresponded to a chamber present either as a lower well(s) present at the bottom of the microplate or as a smaller permeable insert chamber located within the bottom well. The apical chamber inserts from each of these microplates were lined with a bottom surface with a 3 μM pore size polycarbonate membrane separating the upper apical from the basolateral chamber.

[0163] In these experiments, the basolateral chamber corresponds to what becomes available for uptake to the circulating blood and lymphatic vessels associated with the villi upon exiting the basolateral side of the epithelial cells, and the apical chamber corresponds to the lumen of the intestine.

[0164] In all plates, a simulated digestive fluid (e.g., EPDF) containing the substance of interest is applied to the apical side, and the basolateral chamber contains PBS (or any suitable alternative) that represents body fluids at the correct pH and osmolarity.

[0165] In all plates, the basolateral chamber is filled before fluid application to the apical chamber, and the time course occurs immediately after apical fluid application. 100 μL samples are taken from the basolateral chamber at 5, 60, 120, 180, and 240 min and replaced with PBS (or any suitable alternative) pre-warmed to 37° C. All plates are incubated at 37° C.

[0166] The basolateral chamber of each plate contains the following volumes of PBS: 300 μL (96-well plate), 600 μL (24-well plate). The apical chamber of each plate contains the following volumes of simulated digestion solution and components of interest: 100 μL (96-well plate), 200 μL (24-well plate).

[0167] Cell monolayer Caco-2 cells passages 22-36 are grown on permeable insert membranes (initially 5000 cells per well in 96-well plates and 20,000 cells per well in 24-well plates). After 21 days, the cells show normal epithelial resistance and transport activity.

[0168] Preparation and application of reconstituted mucus in permeability studies versus native mucus All permeation validation experiments were performed in 96-well plates. Purified mucus prepared as described above was reconstituted in 1× PBS at concentrations of 25, 50 and 75 mg / ml the day before the experiment and hydrated on a tube roller at 4° C. The reconstituted mucus was applied to the apical insert membrane in volumes of 20, 50 and 70 μL, which correspond to 1.4 mm, 3.5 mm and 4.9 mm, respectively.

[0169] Drugs were present at 100 μM in the simulated digestive fluid and applied (gently) to the mucus layer - thus initiating the time course. Drug permeation was paralleled through native porcine small intestinal mucus in volumes of 10, 15 and 20 μL, respectively.

[0170] Transmission verification data Correlation analysis of the 10 drugs revealed that the best correlations were obtained with 50 μL of 25, 50 and 75 mg / ml, respectively, and 70 μL of 50 mg / ml. These concentrations and volumes were used for further analysis, excluding the others.

[0171] Correlation analysis of the 11 drugs revealed positive correlations between the variables, with the strongest correlations being 50 μL at 50 mg / ml and 50 μL at 75 mg / ml versus the three volumes of native mucus (Spearman's ρ R-squared, Table 3; Figure 4 (50 μL in 75 mg / ml). See Figures 4, 5, and 6.

[0172] Rheological validation of reconstituted mucus The purified mucus described above was reconstituted in phosphate buffered saline (PBS) at a series of concentrations (5–200 mg / ml) overnight at 4°C. The rheological properties of the rehydrated mucin (reconstituted mucus) were assessed using a Kinexus Pro Rheometer (Malvern, UK). The properties of native (unwashed mucus) collected from the small intestine of freshly slaughtered pigs and flash frozen were also assessed once thawed in parallel with native mucus that had undergone a gentle washing process to remove any food residues. The linear viscoelastic region (LVER) was calculated using an amplitude sweep (0.01–600% shear strain) with a frequency set at 1 Hz. The resolution (yield point) shear strain (%) was also calculated along with the phase angle, storage modulus (G'), and loss modulus (G'') within the LVER. One-way ANOVA with Tukey's multiple comparisons was used to assess any statistically significant changes.

[0173] The shear strain and stress in the LVERs and the yield point were similar for native and washed mucus. An increase in the storage and loss modulus was observed for washed mucus compared to native mucus, but the phase angle remained similar. No statistical differences were observed between native or washed mucus for LVER shear stress, shear strain, and phase angle at any of the mucin concentrations. No statistical differences were observed between native mucus and concentrations of mucin below 100 mg / ml for LVER G', or resolution shear stress. The only significant difference between mucin concentrations and native mucus was seen in G'', with only concentrations above 100 mg / ml not significantly decreasing G'' from native (Figure 5 - Rheological Data). Figure 5 shows the rheological properties of rehydrated mucus compared to native and washed 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 LVERs. Panel F shows the shear strain required to break down the gel into a viscous liquid (yield point) and cause it to flow. The experiment was repeated a minimum of three times with separate batches of mucus and mucin.

[0174] To define rheologically relevant mucus, resolution shear strain, LVER, phase angle, and G' are used as the most relevant aspects and concentrations of mucin around 50 mg / ml are therefore classified as similar.

[0175] Compatibility of reconstituted mucous cells Experiments show that native porcine mucus significantly reduces cell viability in a 96-well plate model (Figure 6).

[0176] In the same model, a range of concentrations and volumes of reconstituted mucus was tested for its compatibility with Caco2 cells pulsed with small intestinal digestive secretions for 4 hours (Figure 7).

[0177] All concentrations (5-200 mg / ml) at 50 and 70 μL were not significantly different from control, demonstrating that they protected cells from simulated digestive secretions in vitro over a 4-hour time course.

[0178] In a 24-well plate model, 50 μL of 25, 50 and 75 mg / ml also protected cells from simulated small intestinal digestive fluids over a 4 hour time course (FIG. 8).

[0179] Small intestine model: an overview · Rheological characterization of the reconstituted mucus versus native mucus. Reconstituted mucus protects cells from small intestinal digestive fluids for 4 hours in 24- and 96-well plate absorption models. The reconstituted mucus will be compared to native porcine mucus for drug permeation.

[0180] Stomach model The same system as for modeling the gastric epithelium was used in gastric applications using a reconstituted gastric mucin layer with a gastric cell line (CRL-1739) and synthetic gastric secretions. This model was used to test gastric damage due to delivery of nonsteroidal anti-inflammatory drugs. Using this model, we were able to collect data on cell viability after 2 hours of exposure to test formulations in various conditions. It can be seen that the inclusion of the mucus layer protects against cell death, which further allows for accurate modeling of gastric secretions with pepsin and at pH 2.0, and that the mucus layer can be used to identify formulations that are still cytotoxic. Exemplary data from the gastric model are shown in FIG. 9.

[0181] Large Intestine Model The same method was applied to a large intestine model. This system also includes a cell monolayer and a mucus layer in a transwell system (Figure 10). However, to create an aerobic / anaerobic system, this custom biocompatible 3D printed plate will be placed in a vessel with a GasPaK anaerobic envelope. The plate has a top that allows access to the apical side of the transwell, a sealed lower chamber with an O-ring around the transwell to ensure a tight fit, a lid to prevent medium evaporation, and tubing leading to the outside of the vessel to allow air to enter the basolateral side of the transwell to create an aerobic environment. The upper chamber of the transwell is then inoculated (fecal sample inoculated) with a bacterial population grown in a three-stage fermentation model. This is the first system that can model the microbiome, mucus layer, and epithelium in a single integrated system.

[0182] When a Caco-2 monolayer and a transwell containing only medium are placed in the above plates under anaerobic conditions (normal anaer & normal anaer 2), a slight decrease in cell viability is observed compared to cells in a transwell containing medium under normal aerobic conditions (normal aerobic plate).

[0183] Inclusion of 50 μl of a 50 mg / ml reconstituted mucin layer (Reconstituted-A) or 50 μl of a 100 mg / ml reconstituted mucin layer on top of the cells along with a 50 μl native porcine mucus layer (Native-B) affected cell viability in aerobic conditions, but when the transwells were exposed to anaerobic apical conditions (generated by GasPak, in the 3D printed plate described above) along with a reconstituted mucus layer (C) or native mucus layer (D), the gel was sufficient to protect the Caco-2 cells from the anaerobic apical conditions over a 3 hour incubation period (FIG. 11).

[0184] Further addition of fecal microbial content to the reconstituted (E) or native (F) layers did not negatively affect cell viability.

[0185] The fact that no significant differences were observed between normal cells in anaerobic conditions and cells together with reconstituted mucus and bacteria demonstrates the feasibility of the model.

[0186] Airway Model Calu3 airway epithelial cells were grown in 96-well trusswell cell culture plates. Wells were seeded at 40,000 cells per well (100 μl), grown to confluence as assessed visually and by transepithelial electrical resistance measurements, and then grown as an air-liquid interface (ALI), with the surface liquid removed and cells fed basolaterally.

[0187] Mucus isolated as described above from the stomach of a freshly killed pig, containing primarily the mucin gene product of MUC5AC found in the respiratory tract, was rehydrated in sterile PBS containing calcium and magnesium.

[0188] Four different volumes of five concentrations of mucin were evaluated: 25, 50, 75, and 100 μl of either 100, 75, 50, 25, 12.5 mg / ml of mucus applied apically to cells and incubated for 90 minutes.

[0189] Cell viability was assessed by the Cell Vitality Blue assay, in which metabolically active cells reduce resazurin to the dye resorufin, which can be measured spectrophotometrically. Experiments were repeated a minimum of four times (Figure 10). Two-way ANOVA with Tukey's multiple comparisons was used to assess significant differences. Panel A shows cell viability groups by volume of liquid added to the apical surface of the cells with concentrations of mucin indicated by the bars. Panel B shows the same data but groups by concentration of mucin applied, with the bars indicating the various volumes.

[0190] No statistical differences were observed between any concentration of mucin applied at any volume compared to untreated cells, therefore the applied mucin did not adversely affect cell viability over 90 min.

[0191] Preparation of purified mucus Freshly slaughtered pig small intestines were collected from a local slaughterhouse and transported to the laboratory on ice. The tissue was opened by incision with a scalpel and the epithelial tissue surface was gently rinsed with DI water. The epithelial surface was gently scraped with a microscope slide to remove mucus. Mucus, either undiluted or diluted 1 in 50 in DI water, was added to 50mDa MWCO dialysis tubing.

[0192] The dialysis tubing was placed in a 10 L bucket of 4° C. DI water and incubated at 4° C. The dialysis water was changed six times over a two day period.

[0193] Mucus was collected from the dialysis tubing and centrifuged at 9000 RPM for 90 minutes.

[0194] Purified mucus was collected and frozen at -20.

[0195] Mucus penetration experiment For drug permeation through porcine native mucus (PNM), 0, 10, 15 and 20 μl of PNM were applied in triplicate to the apical insert membrane of the wells using a Handy Step repeater pipette (BRANDTECH Scientific, Inc., CT, USA) with a compatible 500 μL Eppendorf Combitip® (Eppendorf, Hamburg, Germany). Nineteen drugs (Table 3) were prepared at 100 μM in simulated small intestinal fluid. Drug solutions were added to the apical chamber of the test wells at 0 min and 100 μL samples were taken from the basolateral chamber at 5, 60, 120, 180 and 240 min at 37°C. Samples were replaced with pre-warmed PBS. For API permeation through reconstituted mucus, mucus was prepared and prepared as described above at 12.5, 25, 50, 75 or 100 mg / mL the day before the experiment. Each mucus concentration was added to the apical insert membrane in volumes of 0, 20, 50 and 70 μL. Permeation data were analyzed by HPLC. All conditions were screened in triplicate.

[0196] [Table 3]

[0197] Caco-2 cell culture All tissue culture was performed under sterile conditions using a S@FEFLOW 1.2 Biosafety cabinet (Bio Air Instruments, Pavia, Italy). Caco-2 cells were obtained from ATCC (ATCC® HTB-37™) (American Type Culture Collection, VA, USA). Cells arrived frozen. They were thawed according to the manufacturer's instructions and seeded onto 25 cm2 Nunc™ EasYFlask™ (Thermo Scientific, NY, USA). Upon reaching approximately 80% confluence, cells were then plated onto 75 cm2 plates. 2 The cells were further subcultured in Nunc™ EasYFlask™ flasks. The medium was replaced every other day, and the cells were cultured in 25 cm 2 and 75cm 2 The flasks received 7 mL and 20 mL, respectively. The 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). Cultures were grown in MCO-20AIC CO. 2 The animals were incubated in an incubator (SANYO Electric Co., Ltd., Japan) at 37°C and 5% CO 2 The passage numbers ranged from 22 to 38.

[0198] For 96-well plate experiments, Caco-2 cells were grown for a minimum of 21 days on the apical surface of a 3 μm pore size polycarbonate membrane in 96 dual chamber microplates (HTS Transwell® - 96-well permeable support with barcode, lid, and receiver plate; Corning Incorporated, ME, USA). Caco-2 cells were plated on a 75 cm 2 Subculture from flask to 1cm 2 Cells were seeded at 80,000 cells per well. The volume of medium in each apical well was 100 μL. 300 μL of medium was added to the basolateral chamber.

[0199] Integrated model experiment Prior to the day of the experiment, RM was prepared at 25, 50 and 75 mg / mL as before. On the day of the experiment, Caco-2 cells grown for a minimum of 21 days were transferred to a clean, sterile receiver plate by transferring onto the apical scaffold. Growth medium was then removed from the apical chamber with a multichannel pipette. 300 μL 1× PBS was added to the basolateral chamber of all test wells. All outer wells were excluded from the study due to edge effects. For these wells, 300 μL 1× PBS was added to the basolateral chamber and 100 μL to the apical chamber to reduce edge effects within the experiment. For the test wells, four different RM conditions were used for the analysis. These were 25 (50), 50 (50), 50 (70) and 75 (50), where X (X) corresponds to X mg / mL (X μL). The individual concentrations and volumes were gently added to the Caco-2 monolayer with a repeater pipette and allowed to settle for 5 min. Sixteen APIs (Table 4) were prepared at 100 μM in EPDF and 100 μL was gently applied to the mucus layer with a 200 μL multichannel pipette at T0. Plates were then incubated at 37° C. for 4 h and 100 μL samples were taken from the basolateral chamber at 60, 120, 180, and 240 min and replaced with pre-warmed 37° C. 1× PBS. Samples were incubated overnight at 37° C., resuspended in 50% methanol, and analyzed by HPLC using the ammonium acetate method.

[0200] At the end of the experiment, EPDFs were carefully removed from the apical chamber without disturbing the mucus layer. With EPDFs removed, the apical chamber was gently washed with 1x PBS by pipetting to remove the mucus layer. Cells were washed a minimum of two times. The apical scaffolds were then transferred to another sterile receiver plate and subjected to the CellTitre-Blue assay. All conditions were screened in triplicate.

[0201] [Table 4]

[0202] [Table 5]

Claims

1. i. A substrate having a cell support portion, ii. A population of cells provided on a substrate, iii. Purified biocompatible mucus, iv. Physiological fluids provided on and / or beneath the mucus layer, as applicable. An organizational model that includes these in separate layers, A tissue model comprising purified biocompatible mucus which is substantially devoid of nucleic acids, proteins, or lipids, or molecules with a magnitude less than 100 kDa, preferably less than 70 kDa, and ii) mucin having the ability to form a gel.

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

3. The tissue model according to claim 1, wherein the purified biocompatible mucus does not contain synthetic polymers and / or is substantially not denatured, hydrolyzed, or degraded.

4. The tissue model according to claim 1, wherein 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 claim 1, wherein the cells of the cell population are the ileum, jejunum, stomach, duodenum, esophagus, cheek, tongue, or colon of the gastrointestinal tract, or the nose, lung, bronchi, bronchioles, or mouth, or the cervix or vagina, or the eye, or any combination thereof.

6. The tissue model according to claim 1, wherein the body fluid is digestive fluid, saliva, vaginal secretions, tears, or respiratory secretions, synthetic body fluid, or a population of microbial cells, or any combination thereof.

7. The tissue model according to claim 1, which is a gastric 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 two or more tissue models are arranged in sequence, and preferably two or more tissue models are selected from the group consisting of an airway model, a gastric tissue model, a small intestine tissue model, and a large intestine tissue model.

9. i) substantially lacking nucleic acids, proteins or lipids, or molecules below 100 kDa, preferably below 70 kDa, and ii) containing mucin having the ability to form a gel.

10. A purified biocompatible mucus according to claim 9, which is a gel.

11. A purified biocompatible mucus according to claim 9, which does not contain synthetic polymers.

12. The purified biocompatible mucus according to claim 9, wherein the mucin is substantially not denatured, hydrolyzed, or degraded.

13. The purified biocompatible mucus according to claim 9, 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 by a tissue model according to any one of claims 1 to 8, comprising the step of contacting a purified biocompatible mucus with the test compound and detecting the migration of the test compound in the tissue model.

15. The method according to claim 14, comprising the steps of detecting and optionally determining the concentration of a test compound in a purified biocompatible mucus layer, in a cell population, in contact with the surface of the substrate, beneath the substrate, on the luminal surface and / or lateral base.

16. 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, and if applicable The method according to claim 14, wherein the test compound is a small molecule, organic molecule, antibody, peptide, protein, hormone, antagonist, nucleic acid (e.g., antisense, siRNA, shRNA, RNAi, expressible coding sequence), sugar, fatty acid, steroid, purine, pyrimidine; derivatives or structural analogs thereof; bacteria, fungi or extracts thereof; plant or animal extracts; or any combination of two or more thereof.

17. The method according to claim 14, wherein the test compound is a pharmaceutical product.

18. Use of the method of claim 14 in any one or more of the following: drug absorption studies, formulation development, GI damage / adverse event profiling, nutritional intake analysis, drug-microbiome interaction studies, intestinal damage profiling, and pre / probiotic studies, as applicable. The process includes applying food and / or intestinal media to a tissue model, and / or The test compound is measured at one or more different time intervals.

19. A kit comprising, in a suitable container, the purified mucus according to claim 9, or the tissue model according to claim 1, and optionally a substrate, buffer, reagents, and instructions for use.

20. A method for purifying mucus, the method comprising the removal of 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.

21. The method according to claim 20, comprising the steps of dialysis of mucus to remove nucleic acids, proteins, or lipids, or molecules with a magnitude less than 100 kDa, preferably less than 70 kDa, and optionally separating the mucus.

22. The method according to claim 20, comprising the steps of i) providing a sample containing mucus, ii) solubilizing any mucin in the mucus, iii) separating any nucleic acids, proteins, or lipids from the mucus, and iv) dialyzing the mucus, wherein A method comprising the step of separation of equilibrium density gradient centrifugation and, optionally, selecting fractions having a density of 1.3 to 1.6 g / mL.

23. The method according to claim 20, wherein a cesium salt density gradient is used.

24. i) a step of providing a tissue sample containing mucus, and optionally a step of extracting mucus from the tissue sample, and / or ii) A step of sterilizing one or more components of the purified mucus, and / or iii) A step of combining purified mucin with a buffer, lipids, salts, proteins, nucleic acids (DNA or RNA), glycoproteins, cells, and / or water, and / or iv) A step of placing the purified mucus under conditions that enable mucin gelation, and / or v) A freeze-drying step, a storage step, and / or a step of combining the purified mucus with a buffer, and / or vi) A step of forming a tissue model by applying purified mucus to a cell population provided on a substrate, and optionally a step of applying body fluid to purified mucin. The method according to claim 20, further comprising:

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