System for calculating human oral bioavailability

Abstract

The present invention relates to methods and systems for co-culture of primary liver cells and primary intestinal cells. In particular, the present invention applies to estimating bioavailability.

Description

Technical Field

[0001] This invention relates to methods and systems for co-culturing primary liver cells and primary intestinal cells. Specifically, embodiments of the invention relate to such systems for calculating bioavailability. Background Technology

[0002] Cell culture refers to the growth of cells in an artificially controlled environment. Primary cell culture is the in vitro culture of cells obtained directly from multicellular organisms (rather than immortalized cell lines). Generally speaking, primary cell cultures are more representative of in vivo tissues than cell lines; however, maintaining appropriate conditions for primary cell cultures is often more difficult to achieve than for cell lines.

[0003] Co-culture is used to study the interactions (cross-talk) between two or more cell populations. Since any process in vivo typically involves multiple cell types, co-culture systems are crucial for understanding how different cell populations interact and influence each other. Direct co-culture refers to cell cultures where cells are in direct physical contact with each other. Indirect co-culture, on the other hand, involves culturing cells under different conditions where different cell populations do not have direct physical contact with each other.

[0004] The liver and intestines both play crucial roles in the metabolism and uptake of orally ingested drugs, nutrients, and metabolites. The liver contains enzymes that metabolize these compounds, while the intestines are the primary site of absorption. Therefore, both liver cells and intestinal cells are key to determining the bioavailability of compounds such as drugs, metabolites, and nutrients.

[0005] Bioavailability refers to the amount of a compound (e.g., a drug or metabolite) that enters circulation. Oral bioavailability is defined as the amount of a compound that reaches systemic circulation after oral ingestion, absorption through the intestines, and metabolism in the liver. Currently, the industry standard for estimating oral bioavailability is through in vivo human clinical trials and animal studies, in which the treatment is administered orally and intravenously to calculate oral bioavailability. However, human clinical trials are extremely expensive, so it is best to have a preliminary assessment of the predicted bioavailability before proceeding to this stage of research. This is typically done through animal studies; however, animal models are not always representative of humans when it comes to bioavailability, which limits the usefulness of such studies. In vitro studies are much cheaper than in vivo studies in humans and animals and can be performed at much higher throughput. In addition, the FDA will accept the use of in vitro methods to determine the bioavailability of compounds. However, few methods are suitable for this purpose. Although an assay has been developed for co-culturing liver and intestinal cells to estimate bioavailability, this assay is only applicable to co-culturing liver and intestinal cell lines, and primary liver and intestinal cell lines. Currently, there is a lack of assays available for co-culturing primary liver and intestinal cells. This has been problematic because cell lines may not possess the same metabolic activity or transporter protein expression levels as primary cells. This ultimately means that these models cannot provide accurate or reliable estimates of bioavailability. Therefore, there is a need for methods and systems for co-culturing primary liver and intestinal cells that can be used to estimate bioavailability. This invention addresses this need. Summary of the Invention

[0006] The inventors have developed methods and systems suitable for the co-culture of primary liver cells and primary intestinal cells, with different culture medium requirements to maintain the functionality and metabolic activity of each cell type. Specifically, these methods and systems have applications in studies estimating bioavailability.

[0007] One aspect of the present invention is a method for co-culturing primary liver cells and primary intestinal cells, the method comprising:

[0008] a. A system is provided comprising at least one three-compartment group, the three-compartment group including a liver compartment, a lateral compartment and a basolateral compartment, wherein the system includes a first fluid circulation pathway thereby fluidly connecting the basolateral compartment to the liver compartment, the first fluid circulation pathway being selectively interruptible to fluidly isolate the liver compartment from the basolateral compartment;

[0009] b. Inoculate primary liver cells into liver compartments;

[0010] c. Add primary intestinal cells to the top side compartment, wherein the primary intestinal cells form a barrier between the top side compartment and the basal lateral compartment;

[0011] d. Circulate the culture medium that does not contain epidermal growth factor (EGF) through the first fluid circulation pathway;

[0012] e. Add EGF-containing culture medium to the top side compartment.

[0013] The culture medium in the apical compartment and the culture medium in the liver compartment and the basal lateral compartment should be considered separate, meaning that fluid in the apical compartment will not flow into the liver compartment or the basal lateral compartment. Only the culture medium in the apical compartment contains EGF. The culture medium in the liver compartment and the basal lateral compartment does not contain EGF.

[0014] In one embodiment, the first fluid circulation pathway includes, for example, but not limited to, a mechanism such as a switch, valve, pump, or closure capable of selectively interrupting the first fluid circulation pathway. When the mechanism in the first fluid circulation pathway is "activated" or "open," the basolateral compartment and the liver compartment are in fluid connection. When the mechanism is "deactivated" or "closed," the basolateral compartment and the liver compartment are not in fluid connection.

[0015] In a preferred embodiment, the mechanism capable of selectively interrupting the first fluid circulation path is an interconnecting pump.

[0016] In one embodiment, when the liver compartment and the basolateral compartment are fluidly connected, the system may further include a second and / or a third fluid circulation pathway that recirculates the culture medium in the liver compartment and / or the basolateral compartment, respectively.

[0017] In one embodiment, when the liver compartment and the basolateral compartment are fluidly isolated, the system may further include a second and / or a third fluid circulation pathway that recirculates the culture medium in the liver compartment and / or the basolateral compartment, respectively.

[0018] The second and / or third fluid circulation paths may include, for example, but not limited to, mechanisms such as switches, valves, pumps, or closures.

[0019] In one embodiment, a liver pump recirculates the culture medium in the liver compartment, and / or a basolateral pump recirculates the culture medium in the basolateral compartment.

[0020] In one embodiment, the three-compartment assembly further includes a second fluid circulation pathway, wherein fluid in the liver compartments is recirculated when the liver pump is "activated" or "on". When the liver pump is "deactivated" or "off", fluid in the second fluid circulation pathway is not recirculated. In one embodiment, the recirculation of fluid in the liver compartments helps maintain hepatocyte function.

[0021] In one embodiment, the three-compartment assembly further includes a third fluid circulation path in which fluid in the outer substrate compartment is recirculated when the outer substrate pump is "activated" or "on". When the outer substrate compartment is "deactivated" or "off", fluid in the third fluid circulation path is not recirculated. In one embodiment, the recirculation of fluid in the third circulation path facilitates the mixing of the fluid components.

[0022] In one embodiment, the three-compartment assembly includes the first fluid circulation pathway described above.

[0023] In one embodiment, the three-compartment assembly includes the first and second fluid circulation pathways described above.

[0024] In one embodiment, the three-compartment assembly includes the first and third fluid circulation pathways described above.

[0025] In a preferred embodiment, the three-compartment assembly includes the first fluid circulation passage, the second fluid circulation passage, and the third fluid circulation passage described above.

[0026] The culture medium in the apical compartment, as well as in the fluidly connected liver compartment and the basal lateral compartment, should be considered separate, meaning that fluid in the apical compartment will not flow into the first, second, or third fluid circulation pathway, and vice versa. Only the culture medium in the apical compartment contains EGF. The culture media in the first, second, and third fluid circulation pathways do not contain EGF.

[0027] In the method and system of the present invention, the system includes at least one three-compartment group, at least two three-compartment groups, at least three three-compartment groups, at least four three-compartment groups, at least five three-compartment groups, at least six three-compartment groups, or more three-compartment groups. Preferably, the method and system of the present invention includes at least six three-compartment groups. The system of the present invention can be used independently or simultaneously with additional systems.

[0028] In the methods and systems of the present invention, the culture medium does not flow from the top side compartment to the liver compartment and / or the basal side compartment.

[0029] In the context of this invention, the culture medium added to the top compartment may already contain EGF. Alternatively, EGF may be added separately to the culture medium in the top compartment. In other words, EGF may be added simultaneously with the culture medium or separately from the culture medium; however, both are considered as adding a culture medium containing EGF.

[0030] In a preferred embodiment, the culture medium circulated through the first, second, and third fluid circulation pathways is the same as the culture medium in the top compartment, except that the culture medium in the top compartment contains EGF.

[0031] In an alternative implementation, the culture medium circulated through the first, second, and third fluid circulation pathways is different from the culture medium in the top compartment, and only the culture medium in the top compartment contains EGF.

[0032] In a preferred embodiment, the culture medium in the top compartment contains 0.1-1000 ng / ml EGF. More preferably, the culture medium in the top compartment contains 2.5-50 ng / ml EGF.

[0033] In some embodiments, primary liver cells and / or primary intestinal cells are seeded on a biomimetic scaffold or a 3D scaffold. Preferably, the primary liver cells are seeded on a porous collagen-coated scaffold and / or the primary intestinal cells are seeded on a biomimetic scaffold. Seeding primary liver cells and primary intestinal cells onto such scaffolds provides a culture environment that more closely resembles that of organs in vivo.

[0034] In one embodiment, at least one of the top-side compartment, the basal lateral compartment, and the liver compartment comprises a 3D scaffold. In the context of this invention, a 3D scaffold is a three-dimensional structure that provides physical support for cell growth. 3D scaffolds with varying porosities, stiffness, and degradability can be selected. Those skilled in the art can select appropriate 3D scaffolds to meet their needs. 3D scaffolds allow cells to grow in environments more representative of those seen in vivo. A biomimetic scaffold refers to a scaffold incorporating biomimetic materials. Biomimetic materials are materials designed to replicate the structure and characteristics of biological materials found in nature.

[0035] A variety of 3D scaffolds can be used in cell culture, including but not limited to: hydrogel scaffolds, nanofiber scaffolds, collagen scaffolds, polystyrene scaffolds, and polycaprolactone scaffolds.

[0036] In one embodiment, the liver compartment comprises a porous collagen-coated scaffold and / or the top compartment comprises a biomimetic scaffold.

[0037] In one embodiment, primary liver cells and / or primary intestinal cells are seeded on a biomimetic scaffold or a 3D scaffold. Preferably, primary liver cells are seeded on a porous collagen-coated scaffold and / or primary intestinal cells are seeded on a biomimetic scaffold.

[0038] In one embodiment, the primary liver cells seeded in the liver compartment may include at least one cell type selected from hepatocytes, Kupffer cells, sinusoidal endothelial cells, stellate cells, or any other cell type found in the liver. Preferably, the primary liver cells are human.

[0039] The primary intestinal cells seeded in the top side compartment and the base side compartment may include at least one cell type selected from intestinal epithelial cells, goblet cells, Panette cells, enteroendocrine cells, or any other cell type found in the intestine. Preferably, the primary intestinal cells are human.

[0040] In one embodiment, primary intestinal cells are cultured in an expansion medium, and once the primary intestinal cells form a confluent monolayer, they are cultured in a differentiation medium and then seeded into apical compartments. Preferably, the primary intestinal cells are cultured in the expansion medium for approximately 8 days, and then subsequently cultured in the differentiation medium for approximately 5 days. The expansion medium is a nutrient-rich medium that promotes proliferation to obtain a large cell population. The differentiation medium is a specialized medium containing specific molecules (e.g., growth factors and hormones) that induce cells to develop along a specific lineage. Many types of expansion and differentiation media are commercially available. Those skilled in the art are able to select appropriate expansion and differentiation media to suit their needs and can modify the culture times in the expansion and differentiation media to meet their specific requirements. In some embodiments, the method may not include this step at all.

[0041] In a preferred embodiment, primary hepatocytes are seeded into liver compartments, and then primary enterocytes are transferred to the top compartment. Preferably, the hepatocytes are seeded into the liver compartments approximately 4 days before the primary enterocytes are transferred to the top compartment. More preferably, primary enterocytes are seeded onto a biomimetic scaffold on a cell culture insert and then cultured in expansion medium for approximately 8 days, followed by transfer to differentiation medium; and after culturing in differentiation medium for approximately 5 days, the cell culture insert is transferred to the top compartment; wherein the transfer of the cell culture insert with primary enterocytes occurs approximately 4 days after the hepatocytes are seeded into the liver compartments of the three-compartment group.

[0042] In some embodiments, the method further includes adding at least one treatment agent to at least one of the top lateral compartment, the basal lateral compartment, and / or the liver compartment. Preferably, at least one treatment agent is added to the top lateral compartment, and at least one treatment agent is added separately to the liver compartment. Those skilled in the art can select any treatment agent for their desired application. Examples of treatment agents that can be used with the present invention include, but are not limited to: pharmaceuticals, nutrients, metabolites, vitamins, cytokines, chemokines, hormones, lipids, carbohydrates, nucleic acids, and peptides.

[0043] In one embodiment, at least one culture medium sample is collected from at least one of the top side compartment, the basal lateral compartment, and / or the liver compartment. The one or more samples may be collected at the beginning, during, or end of the co-culture time process. One sample may be collected from one or more of the top side compartment, the basal lateral compartment, and / or the liver compartment. Preferably, multiple samples are collected from one or more of the top side compartment, the basal lateral compartment, and / or the liver compartment, and more preferably, multiple samples are collected from both the top side compartment and the liver compartment.

[0044] In a preferred embodiment, culture medium samples are periodically collected from one of the top side compartment, the basal side compartment, and / or the liver compartment. More preferably, culture medium samples are periodically collected from both the top side compartment and the liver compartment. Those skilled in the art can select an appropriate co-culture timeframe and an appropriate frequency for periodic sample collection to meet their specific needs.

[0045] In one embodiment, after primary hepatocytes are seeded in the liver compartment and / or primary intestinal cells are seeded in the top side compartment, at least one treatment agent is added to at least one compartment. Preferably, at least one treatment agent is added to the liver compartment, and in a separate three-compartment group, after primary hepatocytes are seeded in the liver compartment and primary intestinal cells are seeded in the top side compartment, at least one treatment agent is added to the top side compartment. For primary co-culture, once both hepatocytes and intestinal cells have been added to at least one three-compartment group of the system, and preferably at least one treatment agent has been added to at least one compartment of the three-compartment group of the system, the experiment can be run for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours or more, depending on the user's needs. At least one culture medium sample shall be collected from at least one compartment at intervals of one hour, one every two hours, one every three hours, one every four hours, one every five hours, one every six hours, one every twelve hours, one every 24 hours, or at any other interval required by the user.

[0046] In a preferred embodiment, at least one treatment agent is added to the top-side compartment, and in a separate three-compartment group, after primary hepatocytes are seeded in the liver compartment and primary intestinal cells are seeded in the top-side compartment, at least one treatment agent is added to the liver compartment, and the experiment is run for 48 hours, during which at least one culture medium sample is collected from at least one of the liver compartment, the top-side compartment, and / or the basal lateral compartment at 0, 1, 4, 6, 24, and 48 hours, more preferably, the culture medium sample is collected from the top-side compartment and the liver compartment.

[0047] In some embodiments, a sample is collected from the top side compartment of at least one three-compartment group at the end of the experiment. In alternative embodiments, multiple samples are collected periodically from the top side compartment of at least one three-compartment group.

[0048] Preferably, the at least one treatment agent added to the top side compartment is the same as the at least one treatment agent added to the liver compartment. More preferably, the treatment agent is added to the top side compartment at a different concentration than the concentration of the treatment agent added to the liver compartment. Most preferably, the treatment agent is added to the top side compartment at a higher concentration than it is added to the liver compartment. Even more preferably, the treatment agent is added to the top side compartment at a concentration approximately 10 times higher than its concentration in the liver compartment.

[0049] Preferably, the culture medium sample is analyzed from any one of the top side compartment, the base side compartment, and / or the liver compartment. The levels of the applied treatment agent, generated metabolites, release of signaling molecules, levels of waste products, levels of cytokines, or any other parameters of interest to those skilled in the art can be analyzed in the sample. Such parameters can be analyzed using techniques known in the art, a non-limiting example of which is LC-MS. LC-MS is an analytical chemistry technique involving the separation of analytes and subsequent mass-based detection. Analytes can be separated based on a variety of parameters. Partition chromatography separates analytes based on their solubility and hydrophobicity. Ion exchange chromatography separates analytes based on their ionic charge. Size exclusion chromatography utilizes the size differences of the analytes. Affinity chromatography separates analytes based on the affinity of the analyte for the immobilizer. Those skilled in the art can select an appropriate chromatogram to suit their needs and the specific analyte to be measured. This technique can be used to quantify the amount of a target compound and measure its concentration over time.

[0050] In one implementation, the bioavailability of at least one treatment agent is calculated. Bioavailability refers to the proportion of a compound or substance that enters the circulation after being introduced into the body.

[0051] Preferably, the method and system of the present invention can be used to estimate oral bioavailability. Oral bioavailability is the proportion of an orally administered compound or substance that enters the circulation.

[0052] In some embodiments, the method includes periodically collecting culture medium samples from at least one compartment and measuring the concentration of the at least one treatment agent in the samples, as described above. Preferably, the method further includes calculating the bioavailability of the at least one treatment agent using the following formula:

[0053] .

[0055] Preferably, the dosage is the concentration of at least one treatment agent in moles.

[0056] In one embodiment, at least one treatment agent is added to the top compartment of at least one three-compartment group of the system, and the top compartment simulates an oral administration regimen; and / or at least one treatment agent is added individually to the liver compartment of at least one three-compartment group of the system, and the liver compartment simulates an intravenous (IV) administration regimen. Preferably, oral and IV administration regimens are established simultaneously in separate three-compartment groups. Oral and IV administration regimens may be established in separate three-compartment groups within the same system, and / or in separate three-compartment groups within different systems.

[0057] In another aspect of the invention, at least one system having at least two three-compartment groups is provided, wherein the first three-compartment group comprises:

[0058] • Liver compartments, which contain primary hepatocytes;

[0059] • The lateral compartment, which contains primary intestinal cells; and

[0060] • The outer compartment of the base;

[0061] The primary intestinal cells form a barrier between the apical compartment and the basal lateral compartment, and the three-compartment group includes a first fluid circulation pathway containing culture medium, thereby fluidly connecting the basal lateral compartment to the liver compartment; a second fluid circulation pathway containing culture medium, thereby recirculating the fluid in the liver compartment; and / or a third fluid circulation pathway, thereby allowing the fluid in the basal lateral compartment to be recirculated, and the culture medium in only the apical compartment contains EGF.

[0062] And the second and third compartment groups include;

[0063] • Liver compartments, which contain primary hepatocytes;

[0064] • Top-side compartment;

[0065] • The outer compartment of the base;

[0066] The apical and basal lateral compartments do not contain primary intestinal cells, and the three-compartment group includes: a first fluid circulation pathway containing culture medium, thereby fluidly connecting the basal lateral compartment to the liver compartment; a second fluid circulation pathway containing culture medium, thereby recirculating the fluid in the liver compartment; and a third fluid circulation pathway, thereby allowing the fluid in the basal lateral compartment to be recirculated, and the culture medium in the liver compartment and the basal lateral compartment does not contain EGF.

[0067] In one embodiment, in an experiment simulating an oral dosing regimen, a first three-compartment group is used, wherein culture medium and primary hepatocytes are added to the liver compartment, and culture medium containing EGF and primary intestinal cells is added to the top compartment. Preferably, at least one treatment agent is added to the top compartment of the three-compartment group, and the mechanism is activated such that the liver compartment and the basal lateral compartment are fluidly connected. In this embodiment, no treatment agent is added to the liver compartment.

[0068] In one embodiment, in experiments simulating intravenous administration, a second- and third-compartment group is used, wherein culture medium and primary hepatocytes are added to the liver compartments. Preferably, at least one treatment agent is added to the liver compartments of the third-compartment group, and the mechanism is activated such that the liver compartments and the basal lateral compartments are fluidly connected. In this embodiment, primary intestinal cells are not seeded in the top lateral compartment; preferably, the top lateral compartment does not contain these cells and no treatment agent is added to the top lateral compartment.

[0069] In one embodiment, experiments simulating oral dosing and simulating IV dosing are conducted separately, i.e., not simultaneously in the same three-compartment group. Preferably, multiple three-compartment groups, some simulating oral dosing, and some simulating IV dosing are used simultaneously. In some embodiments, multiple three-compartment groups, some simulating oral dosing, and some simulating IV dosing are used simultaneously in the same system. In alternative embodiments, multiple three-compartment groups, some simulating oral dosing, and some simulating IV dosing are used simultaneously in different systems.

[0070] In one implementation, samples are collected from the liver compartment to calculate the area under the curve (AUC) for both oral and intravenous (IV) administration regimens.

[0071] In one embodiment, samples are collected from the top side compartment to analyze absorption across the intestinal barrier and metabolism by primary intestinal cells.

[0072] The present invention also provides a method for determining bioavailability, the method comprising:

[0073] a. Providing at least one system comprising at least two three-compartment groups, the three-compartment groups comprising a liver compartment, a lateral compartment, and a basolateral compartment, wherein the three-compartment groups comprise: a first fluid circulation pathway, wherein the basolateral compartment is fluidly connected to the liver compartment; a second fluid circulation pathway, wherein fluid in the liver compartment is recirculated; and a third fluid circulation pathway, wherein fluid in the basolateral compartment can be recirculated.

[0074] b. In the first three-compartment group, primary hepatocytes are seeded in the liver compartment and primary intestinal cells are added to the top compartment, wherein the primary intestinal cells form a barrier between the top compartment and the basal lateral compartment;

[0075] c. In the second and third compartment groups, primary liver cells are seeded in the liver compartments without adding primary intestinal cells to the top compartments;

[0076] d. Culture medium without EGF is circulated through the first fluid circulation pathway, the second fluid circulation pathway, and the third fluid circulation pathway of the first three-compartment group and the second three-compartment group;

[0077] e. Add EGF-free culture medium to the top compartment of the first three-compartment group;

[0078] f. Add at least one treatment agent to the top side compartment of the first three-compartment group, and add at least one treatment agent separately to the liver compartment of the second three-compartment group;

[0079] g. Periodically collect samples from the top side compartment and the liver compartment of the first and second three-compartment groups;

[0080] h. Measure the concentration of at least one treatment agent in the sample;

[0081] i. Calculate bioavailability using the following formula;

[0082]

[0083] In one implementation, samples are collected from the liver compartments in at least two tricompartment groups to calculate the area under the curve for both oral and intravenous (IV) administration regimens.

[0084] In one embodiment, samples are collected from the top side compartment to analyze absorption across the intestinal barrier and metabolism by primary intestinal cells.

[0085] Any number of first and second three-compartment groups can be used simultaneously to meet the user's experimental needs. In some implementations, multiple three-compartment groups are used simultaneously in the same system. In alternative implementations, multiple three-compartment groups are used in multiple different systems. The user will be able to select the number of three-compartment groups and the number of systems to meet their specific needs.

[0086] In another aspect of the invention, a system for co-culturing primary liver cells and primary intestinal cells is provided, wherein the system comprises at least one three-compartment assembly, the three-compartment assembly comprising:

[0087] • Liver compartments, which contain primary hepatocytes;

[0088] • Top lateral compartments, which optionally contain primary intestinal cells; and

[0089] • The outer compartment of the base;

[0090] The primary intestinal cells form a barrier between the apical and basolateral compartments, and the three-compartment group includes a first fluid circulation pathway containing culture medium, thereby fluidly connecting the basolateral compartment to the liver compartment. The first fluid circulation pathway is selectively interruptible to fluidly isolate the liver compartment from the basolateral compartment. The three-compartment group may also include a second fluid circulation pathway containing culture medium, thereby recirculating the fluid in the liver compartment, and / or a third fluid circulation pathway, thereby recirculating the fluid in the basolateral compartment. Only the culture medium in the apical compartment contains EGF.

[0091] In one embodiment of the invention, the culture medium in the first, second, and third fluid circulation pathways cannot enter the top side compartment, and the culture medium in the top side compartment does not flow into the first, second, and third fluid circulation pathways. Preferably, although the culture medium in the first, second, and third fluid circulation pathways cannot enter the top side compartment, and vice versa, components of the culture medium from the first, second, and third fluid circulation pathways can be transported to the top side compartment by active or passive transport, and vice versa.

[0092] In one embodiment, the culture medium does not flow from the top side compartment of the system to the liver compartment.

[0093] In one embodiment, the system of the present invention is suitable for experiments simulating oral dosing regimens and / or simulating IV dosing regimens, as described above.

[0094] In a preferred embodiment, a porous membrane separates the top-side compartment from the outer substrate compartment. More preferably, the porous membrane separating the top-side compartment from the outer substrate compartment is in the form of a cell culture well insert. Those skilled in the art will recognize and be able to obtain suitable cell culture well inserts according to their specific needs.

[0095] Porous membranes enable the partitioning of the cellular microenvironment in vitro. Examples of porous membranes suitable for use in this invention include, but are not limited to, polycarbonate (PC), polyethylene terephthalate (PET), and polytetrafluoroethylene (PTFE). Porous membranes can have various pore sizes, including but not limited to 0.4 μm, 1 μm, 3 μm, and 8 μm. Preferably, a porous membrane with a pore size of 0.4 μm is used. Porous membranes can also have pore sizes including, but not limited to, <0.85 × 10⁻⁶ pores. 8 <1.70×10 6 and <0.85×10 5 (holes / cm) 2The pore density is within a certain range. Those skilled in the art will be able to select the appropriate pore size and pore density according to their specific needs. The material that can pass through the pores of the membrane depends on the pore size used.

[0096] In some embodiments, at least one compartment comprises a 3D scaffold. Preferably, the liver compartment comprises a 3D scaffold. In some embodiments, the liver compartment comprises a porous collagen-coated scaffold and / or the top compartment comprises a biomimetic scaffold.

[0097] Preferably, the apical compartment contains primary jejunal stem cells / progenitor cells, and the liver compartment contains primary hepatocytes. More preferably, the primary hepatocytes and primary intestinal cells are human.

[0098] The system according to the present invention is a microphysiological system. Also known as organ-on-a-chip technology, a microphysiological system is a multi-channel 3D microfluidic cell culture integrated circuit that simulates the activity, mechanical, and physiological responses of an entire organ or organ system.

[0099] The apical compartment of the system according to the invention represents the internal lumen of the intestine. In one embodiment, this is established by seeding primary intestinal cells in the apical compartment (preferably on a biomimetic scaffold over a porous membrane as described above).

[0100] In one embodiment, primary intestinal cells in the apical compartment are exposed to differentiation signals. These differentiation signals include, but are not limited to, growth factors, hormones, and cytokines.

[0101] In one implementation, primary intestinal cells form a polarized barrier between the apical lateral compartment and the basal lateral compartment.

[0102] This invention is suitable for scaling up to allow multiple experiments to be performed in parallel on a single system, or simultaneously using multiple systems. Attached Figure Description

[0103] Figure 1 - The example system disclosed in this paper for co-culturing primary liver cells and primary intestinal cells has an example experimental timeline.

[0104] Figure 2 - Metabolic rates of primary liver cell cultures after 4 and 7 days in liver-only medium, serum-free (SF) intestinal differentiation medium (intestinal DM SF) with liver medium at a ratio of 25:75, intestinal DM SF with liver medium at a ratio of 50:50, and intestinal DM SF medium only.

[0105] Figure 3-Intestinal barrier strength (measured by transepithelial electrical resistance) of primary intestinal cells cultured in the following medium:

[0106] Culture medium 1 - WEM + Mixture B (cocktail B) + 500 nM hydrocortisone (HC) (negative control)

[0107] Culture medium 2 - Intestinal DM SF (positive control)

[0108] Culture medium 3 - WEM + Mixture B + 500 nM hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 mM N-acetylcysteine ​​(NAC)

[0109] Culture medium 4 - Intestinal DM SF + 1 uM hydrocortisone (HC)

[0110] Culture medium 5 - WEM + Mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01 + 1.25 mM N-acetylcysteine ​​(NAC)

[0111] Culture medium 6 - Advanced DMEM / F12 + Mixture B + 500 nM hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 mM N-acetylcysteine ​​(NAC)

[0112] Culture medium 7 - Advanced DMEM + Mixture B + 500 nM hydrocortisone (HC) + 5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0113] Culture medium 8 - WEM + Mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01.

[0114] Figure 4 - CYP3A4 metabolism in primary liver cells co-cultured with primary intestinal cells using the following culture media:

[0115] Culture medium 1 - (Liver culture medium) WEM + Mixture B + 500 nM hydrocortisone (HC)

[0116] Culture medium 2 - Advanced DMEM + 4% v / v mixture B + 500 nM hydrocortisone (HC) + 2.5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0117] Culture medium 3 - Advanced DMEM + 4% v / v mixture B + 500 nM hydrocortisone (HC) + 5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0118] Culture medium 4 - Advanced DMEM + 4% v / v mixture B + 500 nM hydrocortisone (HC) + 10 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0119] Culture medium 5 - System volume (liver compartment and basal lateral compartment) - AdvancedDMEM + Mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0120] Top-side compartment - Advanced DMEM + Mixture B + 500 nM hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0121] For media 2-4, the same media are used in the systemic areas (liver compartment and basal lateral compartment) and the apical compartment.

[0122] Figure 5 -Intestinal barrier strength (measured by transepithelial electrical resistance) of primary intestinal cells cultured in the following media:

[0123] Culture medium 1 (liver culture medium) - WEM + Mixture B + 500 nM hydrocortisone (HC)

[0124] Culture Medium 2 - Advanced DMEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC) + 2.5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0125] Culture medium 3 - Advanced DMEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC) + 5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0126] Culture medium 4 - Advanced DMEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC) + 10 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0127] Culture medium 5 - Systemic zone (liver compartment and basal lateral compartment) - Advanced DMEM + 4 Mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0128] Top-side compartment - Advanced DMEM + Mixture B + 500 nM hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0129] For media 2-4, the same media are used in the systemic areas (liver compartment and basal lateral compartment) and the apical compartment.

[0130] Figure 6 - A schematic diagram of the culture system and fluid pathways; showing the apical compartment, basal lateral compartment, and liver compartment. Independent fluid recirculation exists within the basal lateral compartment and the liver compartment, and the fluid pathways between the basal lateral compartment and the liver compartment can be connected via an interconnecting pump.

[0131] Definition List

[0132] The subject matter is defined using the following definitions in this specification and claims. Other terms not referenced below should have their meanings as generally accepted in the art.

[0133] Primary hepatocytes are cells directly isolated from liver tissue. The liver contains various cell types, including but not limited to hepatocytes, Kupffer cells, sinusoidal endothelial cells, and stellate cells. Hepatocytes are the most abundant cell type in the liver and perform a range of functions, including but not limited to the metabolism of carbohydrates, lipids, and proteins, protein synthesis, and detoxification of harmful substances. Kupffer cells are resident macrophages in the liver. Their primary function is phagocytosis to remove foreign particles and microorganisms. Kupffer cells also produce inflammatory cytokines in response to infection. Sinusoidal endothelial cells line the surface of the hepatic sinusoids and help regulate the exchange of substances between the blood and the liver. Stellar cells are located in the perisinusoidal space (or Disse space) (the space between hepatocytes and sinusoidal epithelial cells) and are resident fibroblasts in the liver, responsible for storing vitamin A and producing collagen. Stellar cells can be activated in response to injury, leading to increased collagen production and causing liver fibrosis. In the context of this invention, primary liver cells may include one or more cell types selected from hepatocytes, Kupffer cells, sinusoidal endothelial cells, stellate cells, or any other cell type found in the liver.

[0134] "Primary intestinal cells" are cells directly isolated from intestinal tissue. The intestine comprises various cell types, including but not limited to: intestinal epithelial cells, goblet cells, Panette cells, enteroendocrine cells, and stem cells. Intestinal epithelial cells are the most abundant cell type in the small intestine and are responsible for absorbing compounds ingested orally. Goblet cells secrete mucus, which helps protect the intestinal lining. Panette cells secrete antimicrobial substances, helping to protect against infection. Enteroendocrine cells secrete hormones that regulate other processes such as digestion. Stem cells are located at the base of the intestinal crypts and are responsible for supplementing other cell types found in the intestine. In the context of this invention, primary intestinal cells may include one or more cell types selected from intestinal epithelial cells, goblet cells, Panette cells, enteroendocrine cells, or any other cell type found in the intestine.

[0135] "Cell culture" refers to the process of culturing cells outside a living organism under controlled conditions (such as temperature, pH, nutrient levels, and waste levels). Both eukaryotic and prokaryotic cells can be cultured.

[0136] "Primary cell culture" refers to the culture of cells obtained directly from multicellular organisms.

[0137] "Co-culture" refers to a cell culture setup in which at least two cell populations are cultured in contact with each other to some extent. Cell co-culture is fundamental to the study of cell-cell interactions. Two forms of co-culture are known in this field: direct co-culture and indirect co-culture.

[0138] "Direct co-culture" refers to the culture in which at least two cell populations are in direct physical contact with each other.

[0139] "Indirect co-culture" refers to the co-culturing of at least two cell populations, but in which the different cell populations do not come into direct contact with each other. In one embodiment, the present invention is used for the indirect co-culture of primary liver cells and primary intestinal cells.

[0140] As used in this specification, "fluid-connected" means that fluid can flow from one compartment to another. In the context of this invention, fluid can flow between the basal lateral compartment and the liver compartment.

[0141] "Bioavailability" is the amount of a compound (such as a drug or metabolite) that enters the circulation.

[0142] Oral bioavailability refers to the amount of a compound that enters the systemic circulation after oral ingestion. Oral bioavailability can be calculated using the following formula:

[0143] Detailed Implementation

[0144] While the foregoing disclosure provides a general description of the subject matter covered within the scope of the invention, including methods and best modes of making and using the invention, the following embodiments are provided to further enable those skilled in the art to practice the invention and to provide its complete written description. However, those skilled in the art should understand that the details of these embodiments should not be construed as limiting the invention, and the scope of the invention should be understood from the appended claims and their equivalents. Various other aspects and embodiments of the invention will be apparent to those skilled in the art in light of this disclosure.

[0145] As used herein, “and / or” means a specific disclosure of each of two specified features or components, regardless of the presence of the other. For example, “A and / or B” should be understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein.

[0146] Unless the context otherwise requires, the description and definition of the features listed above are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all described aspects and embodiments.

[0147] The invention will now be described by way of the following non-limiting embodiments.

[0148] Example 1 - A Proposed Experimental Design

[0149] Figure 1An experimental design for a non-limiting embodiment of the use of the method and system of the present invention is shown. According to this embodiment, primary intestinal cells are first cultured for 8 days on conventional static cell culture plates in expansion medium, and then transferred to differentiation medium. On day 7, primary hepatocytes (PHH) are seeded into the liver compartment of the system of the present invention. After 14 days, the primary intestinal cells are transferred to the top compartment of the system of the present invention. Once the primary intestinal cells and primary hepatocytes are respectively in the top compartment and liver compartment, a treatment agent can be applied, and samples can be collected from each compartment for analysis over a period of time (48 hours in this embodiment).

[0150] Figure 6 A schematic diagram of a system used for such experiments is shown. A top-side compartment, a basal-lateral compartment, and a liver compartment are shown. Independent fluid recirculation exists in the basal-lateral compartment and the liver compartment, and the fluid pathway between the basal-lateral compartment and the liver compartment can be connected by an interconnecting pump. For experiments simulating an oral dosing regimen, at least one treatment agent is added to the top-side compartment. For experiments simulating an IV dosing regimen, at least one treatment agent is added to the liver compartment in a system independent of the system simulating the oral dosing regimen.

[0151] Example 2 - Intestinal differentiation medium (serum-free) is harmful to the metabolic function of primary liver cells.

[0152] Human primary liver cells were co-cultured with human primary intestinal cells. When this was performed using liver cell culture medium, the intestinal barrier function was impaired, and when this was performed using intestinal cell culture medium, the metabolic function of the liver cells was eliminated.

[0153] To verify that the presence of intestinal cell culture medium led to the elimination of function in human primary hepatocytes rather than the presence of human primary intestinal cells themselves, human primary hepatocytes (PHH) were cultured under isolated conditions using liver culture medium, intestinal differentiation medium, or a mixture of both. The liver culture media used included William's E medium (WEM), 4% Mixture B, for example, available from Thermo Fisher (which includes 0.5% penicillin-streptomycin, ITS+ (6.25 μg / ml insulin, 6.25 μg / ml transferrin, 6.25 ng / ml selenium complex, 1.25 mg / ml BSA, and 5.35 μg / ml linoleic acid), and 2 mM GlutaMAX. TMThe serum-free intestinal differentiation medium (intestinal DM SF) used contained Advanced DMEM F12, 50 ng / ml EGF, 500 nM A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thioamide) and 1.25 mM NAC.

[0154] To measure the metabolic function of PHH, the metabolic rate of CYP3A4 was measured. CYP3A4 is an important enzyme found primarily in the liver, which metabolizes a wide range of molecules (e.g., drugs). The threshold for the metabolic rate of CYP3A4 is 1 pmol / min / million cells. If the metabolic activity is below this, it is insufficient and indicates that the metabolic function of PHH is impaired. After 4 days of culture, the metabolic rate was high in all media combinations tested, approximately 4 pmol / min / million cells, although PHH cultured in intestinal differentiation medium alone showed the lowest metabolic rate. After 7 days of culture, the metabolic rate of PHH cultured in liver-only medium was approximately 2 pmol / min / million cells. However, as... Figure 2 As shown, in PHH cultured with intestinal differentiation medium and liver medium at a ratio of 25:75, PHH cultured with intestinal differentiation medium and liver medium at a ratio of 50:50, and PHH cultured with intestinal differentiation medium alone, the metabolic rate decreased to only about 0.5 pmol / min / million cells after 7 days of culture. This indicates that intestinal differentiation medium eliminates the metabolic function of PHH. Furthermore, it is surprising that the detrimental effect of intestinal differentiation medium was not concentration-dependent, as the metabolic rate did not increase in PHH cultured with a lower percentage of intestinal differentiation medium.

[0155] Example 3 - EGF is crucial for the barrier function of primary intestinal cells

[0156] It is evident that serum-free intestinal differentiation medium impairs the metabolic function of primary hepatocytes and is therefore unsuitable for co-culturing these two cell types. Therefore, the inventors investigated which components of serum-free intestinal differentiation medium are important for intestinal barrier function. To test this, primary human intestinal cells were cultured in media with different compositions (detailed below), and transepithelial electrical resistance (TEER) was measured as an indicator of intestinal barrier strength.

[0157] Culture medium 1 - WEM + Mixture B + 500 nM hydrocortisone (HC) (negative control)

[0158] Culture medium 2 - Intestinal DM SF (positive control)

[0159] Culture medium 3 - WEM + Mixture B + 500 nM hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 mM N-acetylcysteine ​​(NAC)

[0160] Culture medium 4 - Intestinal DM SF + 1 uM hydrocortisone (HC)

[0161] Culture medium 5 - WEM + Mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01 + 1.25 mM N-acetylcysteine ​​(NAC)

[0162] Culture medium 6 - Advanced DMEM / F12 + Mixture B + 500 nM hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 mM N-acetylcysteine ​​(NAC)

[0163] Culture medium 7 - Advanced DMEM + Mixture B + 500 nM hydrocortisone (HC) + 5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0164] Culture medium 8 - WEM + Mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01

[0165] Figure 3 The results show that the TEER was stable in media 3, 4, 6, and 7 after 8 days of culture. Notably, all these media contained EGF, either added independently or as part of the intestinal DM SF medium (whose composition is given in Example 2). Meanwhile, media 1, 2, 5, and 8 showed TEER collapse after 8 days of culture. Media 1, 5, and 8 did not contain EGF, while medium 2 contained EGF but not FBS (fetal bovine serum). This data indicates that EGF is crucial for the barrier function of primary intestinal cells in serum-free media and also suggests that hydrocortisone can compensate for the lack of FBS and help maintain the barrier function of primary intestinal cells. This illustrates that the TEER collapsed on medium 2 (intestinal DM SF) on day 8, but not on medium 4 (intestinal DM SF and 1 μM hydrocortisone), where the TEER remained stable after 8 days of culture.

[0166] Example 4 - Selection of Culture Medium for Co-culturing Primary Liver Cells and Primary Intestine Cells

[0167] To determine how to co-culture primary hepatocytes and primary intestinal cells without impairing hepatocyte metabolic function or intestinal barrier function, co-culture was performed in the dual-organ plate described in this paper using five different culture media:

[0168] Culture medium 1 - (Liver culture medium): WEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC)

[0169] Culture Medium 2 - Advanced DMEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC) + 2.5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0170] Culture medium 3 - Advanced DMEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC) + 5 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0171] Culture medium 4 - Advanced DMEM + 4% v / v Mixture B + 500 nM hydrocortisone (HC) + 10 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0172] Culture medium 5 -

[0173] Systemic zone (hepatic compartment and basolateral compartment) - Advanced DMEM + 4% v / v mixture B + 500 nM hydrocortisone (HC) + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0174] Top-side compartment - Advanced DMEM + 4% v / v Mixture B + 500 nM Hydrocortisone (HC) + 50 ng / ml EGF + 500 nM A83-01 + 1.25 uM N-acetylcysteine ​​(NAC)

[0175] Note that for media 2-4, the same media are used in the systemic zone (liver compartment and basal lateral compartment) and the apical compartment. However, for media 5, two different types of media are used in both the systemic zone and the apical compartment; the apical compartment media and the systemic zone media are both made from the same matrix medium, but only the apical compartment media contains EGF.

[0176] Figure 4 The results showed that only the liver culture medium control (medium 1) and medium 5 were suitable for maintaining the metabolic function of primary hepatocytes. This was demonstrated by the CYP3A4 metabolic rate, which remained above the threshold of 1 pmol / min / million cells in both media. Meanwhile, on day 6 of culture in media 2, 3, and 4, the CYP3A4 metabolic rate decreased to below the threshold; media 2, 3, and 4 all contained EGF. These data indicate that EGF impairs the metabolic function of primary hepatocytes.

[0177] Figure 5 This demonstrates the intestinal barrier strength maintained by media 2, 3, 4, 5, and intestinal differentiation medium when primary intestinal cells are co-cultured with primary hepatocytes. Figure 4 The data clearly show that only medium 5 was suitable for the co-culture of primary hepatocytes and primary intestinal cells, and both the metabolic function of primary hepatocytes and the barrier function of primary intestinal cells were maintained. Ultimately, this indicates that the presence of EGF on the apical surface of primary intestinal cells is crucial for barrier function, and the EGF... Missing It is necessary to maintain the metabolic function of primary liver cells.

Claims

1. A method for co-culturing primary liver cells and primary intestinal cells, the method comprising: a. A system is provided comprising at least one three-compartment group, the three-compartment group including a liver compartment, a lateral compartment and a basolateral compartment, wherein the system includes a first fluid circulation pathway thereby fluidly connecting the basolateral compartment to the liver compartment, the first fluid circulation pathway being selectively interruptible to fluidly isolate the liver compartment from the basolateral compartment; b. Inoculate primary liver cells into liver compartments; c. Add primary intestinal cells to the top side compartment, wherein the primary intestinal cells form a barrier between the top side compartment and the basal lateral compartment; d. Culture medium without EGF is circulated through the first fluid circulation pathway; e. Add EGF-containing culture medium to the top side compartment.

2. The method of claim 1, wherein the system further comprises a second fluid circulation pathway, wherein fluid in the liver compartment is recirculated.

3. The method of any one of claims 1 or 2, wherein the system further comprises a third fluid circulation passage, wherein fluid in the outer compartment of the substrate is recirculated.

4. The method of any of the preceding claims, wherein the primary liver cells are seeded onto a 3D scaffold and / or the primary intestinal cells are seeded onto a biomimetic scaffold.

5. The method of any one of claims 1 to 4, wherein the primary liver cells are seeded onto a porous collagen-coated scaffold.

6. The method of any of the preceding claims, wherein the primary intestinal cells are seeded onto a biomimetic scaffold.

7. The method of any of the preceding claims, further comprising adding at least one treatment agent to at least one compartment.

8. The method of claim 7, further comprising adding at least one treatment agent to the top side compartment and adding at least one treatment agent to the liver compartment.

9. The method of any one of claims 7 or 8, wherein the treatment agent is selected from at least one of a drug, nutrient, vitamin, metabolite, cytokine, chemokine, carbohydrate, lipid, nucleic acid, hormone or peptide.

10. The method of any one of claims 7 to 9, further comprising periodically collecting culture medium samples from at least one compartment and measuring the concentration of the at least one treatment agent in the samples.

11. The method of claim 10, further comprising calculating the oral bioavailability of at least one treatment agent using the following formula: 。 12. The method of any of the preceding claims, wherein the primary liver cells and primary intestinal cells are human.

13. A method for determining bioavailability, the method comprising: a. Providing at least one system having at least two three-compartment groups, the three-compartment groups comprising a liver compartment, a lateral parietal compartment, and a lateral basal compartment, wherein the system comprises: A first fluid circulation pathway, thereby fluidly connecting the basal lateral compartment to the liver compartment; A second fluid circulation pathway, through which the fluid in the liver compartment is recirculated; A third fluid circulation pathway allows the fluid in the outer compartment of the substrate to be recirculated. b. In the first three-compartment group, primary hepatocytes are seeded in the liver compartment and primary intestinal cells are added to the top side compartment, wherein the primary intestinal cells form a barrier between the top side compartment and the basal lateral compartment; c. In the second and third compartment group, primary liver cells are seeded in the liver compartment without adding primary intestinal cells to the top compartment; d. The culture medium without EGF is circulated through the first fluid circulation pathway, the second fluid circulation pathway, and the third fluid circulation pathway of the first three-compartment group and the second three-compartment group; e. Add a culture medium that does not contain EGF to the top compartment of the first three-compartment group; f. Add at least one treatment agent to the top side compartment of the first three-compartment group, and add at least one treatment agent separately to the liver compartment of the second three-compartment group; g. Periodically collect samples from the top side compartment and the liver compartment of the first and second three-compartment groups; h. Measure the concentration of at least one treatment agent in the sample; i. Calculate bioavailability using the following formula: 。 14. A system for co-culturing primary hepatocytes and primary enterocytes, wherein the system comprises at least one three-compartment group, the three-compartment group comprising a liver compartment containing primary hepatocytes, an apical compartment containing primary enterocytes, and a basolateral compartment, wherein the primary enterocytes form a barrier between the apical compartment and the basolateral compartment, and the system includes a first fluid circulation pathway thereby fluidly connecting the basolateral compartment to the liver compartment, the first fluid circulation pathway being selectively interruptible to fluidly isolate the liver compartment from the basolateral compartment; the three-compartment group may further include a second fluid circulation pathway thereby recirculating fluid in the liver compartment, and / or a third fluid circulation pathway thereby recirculating fluid in the basolateral compartment, and only the culture medium in the apical compartment contains EGF.

15. The system of claim 14, wherein the culture medium does not flow from the top side compartment to the liver compartment.

16. The system according to any one of claims 14 or 15, wherein the porous membrane separates the top-side compartment from the outer compartment of the substrate.

17. The system according to any one of claims 14 to 16, wherein at least one compartment comprises a 3D support.

18. The system of claim 17, wherein the liver compartment comprises a 3D scaffold and / or the top compartment comprises a biomimetic scaffold.

19. The system according to any one of claims 17 or 18, wherein the liver compartment comprises a porous collagen-coated scaffold.

20. The system according to any one of claims 14 to 19, wherein the top side compartment comprises primary jejunal stem cells / progenitor cells.

21. The system according to any one of claims 14 to 20, wherein the liver compartment comprises primary hepatocytes.

22. The system according to any one of claims 14 to 21, wherein the primary liver cells and primary intestinal cells are human.

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