JP7676009B2 - Method for inducing bile duct lumen formation - Google Patents

Description

The present invention relates to a cell culture substrate, specifically to a cell culture substrate having a claudin protein layer formed on the substrate surface. The present invention also relates to a method for inducing bile duct lumen formation on the surface of the cell culture substrate of the present invention, and a method for evaluating drug metabolism and/or membrane transport in liver-derived cells using the cell culture substrate of the present invention.

The liver is the largest organ in humans and plays a major role in maintaining the internal environment of the body. It also plays an important role in pharmacokinetics, including drug metabolism and biliary excretion.

Bile excretion is one of the mechanisms responsible for the excretion of highly lipid-soluble compounds from the body. Drugs taken up by passive diffusion from the vascular membrane of hepatic parenchymal cells or by transporter-mediated transport are metabolized and conjugated, and then excreted into bile by drug efflux transporters localized in the luminal membrane of the bile. Transporters localized in the luminal membrane of the bile include ABC (ATP-binding cassette) transporters such as P-glycoprotein (P-gp), bile salt export pump (BSEP), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2). These transporters play an important role in excreting bile acids and endogenous metabolic products into the bile, in addition to xenobiotics such as various administered drugs.

Evaluation of biliary excretion is extremely important in drug research and development, and is currently evaluated in vivo in laboratory animals, or in vitro using primary cultured human or rodent hepatocytes or established cultured hepatocyte lines. Sandwich-cultured human hepatocytes are also used as an in vitro method for evaluating biliary excretion (Non-Patent Documents 1-4).

Bile secretion and excretion of drugs and other substances into the bile are carried out via the bile lumen formed in the cell membrane of hepatocytes. The formation of the bile lumen in hepatocytes requires the formation of tight junctions (TJs) that are responsible for adhesion between hepatocytes. Tight junctions involve transmembrane proteins such as occludin, claudins, and JAMs, scaffolding proteins such as zonula occludens (ZO) proteins (ZO-1, 2, 3), and polarity signaling molecules such as Par-3 and aPKC, of which it has been reported that the claudin family plays a major role (Non-Patent Document 5).

Swift B. et al., Drug Metab Rev., 42: 446-471, 2010 Nakanishi T. et al., Toxicology and Applied Pharmacology 263 (2012)244-250 Matsunaga N. et al., Drug Metab Dispos 46:680-691, May 2018 Wada S. et al., Drug metabolism and Pharmacokinetics 35 (2020) 432-440 Gunzel & Yu, Physiol Rev. 93 : 525-569, 2013

In conventional in vitro methods, because bile duct lumens are formed between hepatocytes, compounds excreted in bile cannot be collected and directly measured, and indirect evaluations are performed using fluorescent compounds or the collapse of the bile duct lumen. Furthermore, because cell culture is time-consuming and measurements are required both with and without the collapse of the bile duct lumen, many tests must be performed, making it impossible to use this method as a rapid and quantitative method for evaluating bile excretion. Therefore, there is a strong demand for an evaluation system that can easily and accurately predict the bile excretion of drugs.

The inventors considered that the above problem could be solved by inducing the bile duct lumen of hepatocytes to the basal side of the cultureware and establishing a permeability test-type evaluation system. If permeability tests could be performed by inducing the formation of the bile duct lumen to the basement membrane side, it would be possible to quantitatively analyze compounds excreted into the bile lumen over time, and it would be possible to construct a highly accurate prediction system for bile excretion in humans.

The inventors conducted various studies aimed at establishing an effective evaluation system as described above, and were surprised to find that by using claudin proteins, they were able to induce bile duct lumen formation on the surface of a culture substrate. The inventors further investigated various conditions, and as a result, they were able to complete the present invention.

That is, the present invention provides the following.
(1) A cell culture device having a claudin protein layer formed on the surface of the device.
(2) The cell culture substrate according to (1) above, which has a permeable membrane on its surface.
(3) The cell culture equipment according to (1) or (2) above, comprising one or more selected from the group consisting of claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26 and 27 expressed in the human liver.
(4) A kit for evaluating drug excretion into bile, comprising the cell culture equipment according to any one of (1) to (3) above and liver-derived cultured cells.
(5) A method for producing a culture substrate according to any one of (1) to (3) above, characterized in that the surface of a permeable substrate is coated with a layer containing a claudin protein.
(6) A method for preparing an evaluation system for drug excretion into bile, which comprises forming a liver-derived cell layer on the culture vessel according to any one of (1) to (3) above.
(7) A method for forming a liver-derived cell layer on the culture vessel according to any one of (1) to (3) above, and inducing bile duct lumen formation at the site of contact between the cells and the surface of the vessel.
(8) A method for evaluating drug metabolism and/or membrane transport in liver-derived cells, comprising forming a liver-derived cell layer on the culture vessel according to any one of (1) to (3) above, and evaluating a drug excreted from the liver-derived cells through the culture vessel.

The present invention makes it possible to construct a permeability test system using hepatocytes by coating an insert membrane capable of permeability testing with claudins and culturing hepatocytes.

The number of bile canalicular lumen formations in HepG2 cells expressing 15 types of claudin proteins is shown by the number of MRP2 stainings. Results are shown as mean ± standard deviation (n = 6). *: P ≤ 0.05. The results of co-culture of HeLa cells expressing claudins 1, 2, 3, and 9 with HepG2 cells are shown. A: Staining results of WGA (plasma membrane), GFP (HeLa cells), and MRP2 (bile canaliculi) are shown. B: Schematic showing the induction of bile canalicular formation by co-culture. C: A significant increase in the number of MRP2 staining cells was observed by co-culture with HeLa cells expressing claudins. The results of detection of purified claudin-1 samples by CBB staining (A) and Western blotting (B) are shown. The results of detection of claudin 2 purified samples by CBB staining (A) and Western blotting (B) are shown. The results of detection of purified claudin 3 samples by CBB staining (A) and Western blotting (B) are shown. The results of detection of purified claudin-9 samples by CBB staining (A) and Western blotting (B) are shown. This shows that coating the surface of the device with claudin-1 protein induced bile canalicular formation in HepG2 cells at the contact surface with the device. A: Cellmatrix + claudin-1, B: claudin-1 only, C: Cellmatrix only. The figures show bile duct lumen formation when claudins 1, 2, 3, and 9 were coated onto the surface of a device together with Cellmatrix. A: xz plane, B: yz plane, C: xy plane, D: schematic diagram of observation. This shows the formation of bile duct lumen when the surface of the device was coated with only Cellmatrix. A: xz plane, B: yz plane, C: xy plane, D: schematic diagram of observation. The figures show bile duct lumen formation when claudins 1, 2, 3, and 9 were coated onto the surface of a device together with Matrigel. A: xz plane, B: yz plane, C: xy plane, D: schematic diagram of observation. The figures show bile duct lumen formation when the surface of the device was coated with only Matrigel (A: xz plane, B: yz plane, C: xy plane, D: schematic diagram of observation). This shows that bile canalicular lumen formation was significantly increased when claudins 1, 2, 3, and 9 (CLDN1, 2, 3, 9) were coated compared to the control. Results are shown as mean ± standard deviation (n = 4). **: p ≤ 0.01. 1 is a schematic diagram showing a drug permeation test using the culture substrate of the present invention.

The present invention provides a cell culture device having a claudin protein layer formed on the device surface.

<Claudin Protein>
As mentioned above, claudins are known to be major proteins involved in the formation of tight junctions, a type of intercellular junction. To date, 27 types of claudin proteins have been reported in humans and mice, each of which is a transmembrane protein with a molecular weight of 20 to 27 kDa.

The claudin proteins used in the present invention are used to create a system for evaluating hepatocyte function and drug metabolism, so although there are no particular limitations, it is preferable that they are derived from the same animal species as the hepatocytes used for evaluation are derived from.

Animal species include any animal with a liver, including mammals, birds, amphibians, reptiles, and fish, and include, but are not limited to, mammals, such as dogs, cats, rats, mice, rabbits, cows, horses, goats, monkeys, and humans. For example, for metabolic studies in humans, it is preferable to use claudins of human origin.

Twenty-six types of claudins (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, and 27) are expressed in humans, and it has been reported that 11 of these, claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, and 14, are expressed in the liver. It is also known that claudin molecules exert their functions by binding not only between the same molecules but also between different claudin molecules (Gunzel & Yu, 2013 Physiol Rev. 93: 525-569; D'Souza et al., 2009 J Gerontol A Biol Sci Med Sci. 64: 1146-1153; Yang et al., 2015 Oncol Rep. 34: 1415-1423).

Therefore, in the present invention, any of the 26 types of claudins whose expression has been confirmed in humans can be used alone or in combination. Preferably, the 11 types of claudins whose expression has been confirmed in the human liver can be used alone or in combination. That is, in the present invention, one or more types of claudin proteins can be used from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, and 27. Preferably, in the present invention, one or more types of claudin proteins can be used from 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, and 14. The present inventors have confirmed that, of the above, claudins 1, 2, 3, and 9 can be suitably used in the present invention. Therefore, in a preferred embodiment of the present invention, one or more types of claudin proteins selected from the group consisting of claudins 1, 2, 3, and 9 can be used.

The amino acid sequence of human claudin 1 (sometimes referred to as "CLDN1" in this specification) and the nucleotide sequence encoding it are available, for example, from the sequence database of the National Center for Biotechnology Information (NCBI) under accession number NP_066924 and Gene ID: 9076, respectively.

The amino acid sequence of human claudin 2 (sometimes referred to as "CLDN2" in this specification) and the nucleotide sequence encoding it are available, for example, from the NCBI sequence database under accession number NP_065117 and Gene ID: 9075, respectively.

The amino acid sequence of human claudin 3 (sometimes referred to as "CLDN3" in this specification) and the nucleotide sequence encoding it are available, for example, from the NCBI sequence database under accession number NP_001297 and Gene ID: 1365, respectively.

The amino acid sequence of human claudin 9 (sometimes referred to as "CLDN9" in this specification) and the nucleotide sequence encoding it are available, for example, from the NCBI sequence database under accession number NP_066192 and Gene ID: 9080, respectively.

The amino acid sequences and the base sequences encoding the amino acid sequences of other human claudins and claudin proteins derived from other animal species can also be obtained in a similar manner.

In the present invention, claudin proteins can be isolated and purified from animals for use. However, by synthesizing the desired claudin protein based on the above sequence information, the formation of a protein layer free of contaminants can be more easily achieved. The synthesis of claudins, which are transmembrane proteins, can be suitably performed by cell-free synthesis using lipid/detergent mixed micelles, although this is not limited thereto (see, for example, Shinoda T. et al., Sci Rep., 6: 30442, 2016).

The present invention is characterized in that a claudin protein layer is formed on the surface of the device. The term "claudin protein layer" refers to a state in which the claudin protein is uniformly coated on the surface of the device. However, the amount of claudin protein present on the device surface does not necessarily have to be uniform, as long as it is an amount that can bind to physiological claudin protein localized in the seeded cell membrane. As an example, it is preferable that the claudin protein is present on the device surface at a density in the range of 0.6 to 0.8 μg/ ^cm2 .

<Equipment>
In this specification, the term "apparatus" refers to a type of cell culture apparatus commonly used in the art, and is not limited to a particular shape or material. For example, a cell culture insert, slide, dish, plate, or multi-well plate made of glass or plastic can be used as appropriate.

For the purpose of evaluating drug permeation through a claudin protein layer, the surface of the device on which the claudin protein layer is formed must be permeable. Here, "permeable" means that gases, liquids, solutes, ions, etc. secreted from cells can pass through, and porous bodies, meshes, cell culture inserts, etc. with pore sizes of 100 nm or more can be used. For example, the insert membrane of Corning's Transwell (registered trademark) is an example of a device with permeability that can be suitably used. Note that the material used only needs to be permeable to substances, so it is also possible to use pore sizes of 100 nm or less.

The claudin protein layer on the surface of the device is formed by coating the surface of the device with claudin protein. The claudin protein coating can be performed, without particular limitation, by applying a solution of claudin protein at a concentration of 10 μg/ml to the surface of the device in an amount of 100 μL/ ^cm2 , leaving the solution at room temperature for 60 minutes, removing the solution, and drying the surface.

Claudin proteins can be coated alone on the above-mentioned surfaces of the substrate. Alternatively, claudin proteins can be coated in combination with extracellular matrix proteins such as collagen. In the art, products that contain multiple extracellular matrix proteins and form gels are provided for coating substrate surfaces for effective cell culture, and these may be combined with claudin proteins. For example, but not limited to, Cellmatrix Type I-C (Nitta Gelatin, Osaka, Japan) and Matrigel (Corning, NY, USA) are suitable examples, and these may be mixed with claudin proteins for coating, or may be coated prior to claudin proteins.

In this specification, the term "surface of the device" or "bottom surface of the device" refers to the surface of the device that comes into contact with the cells, particularly hepatocytes, that are cultured in the device. In the case of permeable devices, both surfaces of the device may be coated.

By using the cell culture equipment of the present invention, the formation of bile duct lumens is induced on the surface side of the equipment in cultured cells, particularly hepatic cells, and by using a permeable equipment, substances secreted from cells can be easily collected through the equipment having a claudin protein layer.

<Kit>
The present invention also provides a kit for evaluating drug excretion into bile, comprising the above-mentioned cell culture substrate of the present invention and liver-derived cultured cells.
In the present invention, "liver-derived cultured cells" (sometimes referred to as "cultured cells" or "hepatocytes" in the present specification) are not particularly limited, but examples thereof include commercially available liver-derived cultured cells, iPS-derived hepatocytes, cultured cells derived from liver cancer, and cultured cells derived from healthy individuals or individuals (humans or animals) with liver disease.

When normal hepatocytes or hepatocytes derived from a healthy individual are used, the kit of the present invention can be used to evaluate drug metabolism in a normal liver, for example, to evaluate the metabolism of a specific drug (compound).

Alternatively, when using cultured cells derived from liver cancer or liver cells derived from an individual with a liver disease such as liver cancer, the kit of the present invention can evaluate changes in drug metabolism, such as the presence or absence of a decrease in metabolic function, in comparison with a normal liver.

<Manufacturing method of culture equipment>
The present invention also provides a method for producing the above-mentioned culture substrate, which comprises coating a surface of a permeable substrate with a layer containing a claudin protein. The method comprises coating the surface of the substrate with the claudin protein alone or in combination with other components used in coating the culture substrate.
As described above, the culture device obtained by the method of the present invention can be used in a system for evaluating drug excretion into bile.

<Method of preparing a system for evaluating drug excretion into bile>
The present invention also provides a method for preparing a system for evaluating drug excretion into bile, which comprises forming a liver-derived cell layer on the above-mentioned culture substrate of the present invention.
The liver-derived cell layer is cultured as a monolayer, with the cells in close contact with each other and low intercellular permeability. The cell density to be seeded varies depending on the cell type and cannot be specified in general, but for example, primary human hepatocytes can be seeded over the entire surface of the cultureware when seeded at 2.0 x ¹⁰⁵ cells/ ^cm2 . However, this is affected by the cell survival rate and adhesion rate, so optimization for each lot is required.

Method for inducing bile duct lumen formation
The present invention also provides a method for forming a liver-derived cell layer on the above-mentioned culture substrate of the present invention and inducing bile ductular lumen formation at the contact site between the cells and the surface of the substrate.
The culture substrate of the present invention induces the formation of bile ductal lumens in liver-derived cells on the surface in contact with the culture substrate due to the presence of a claudin protein layer.

The formation of bile duct lumen can be confirmed, for example, by detecting the expression of MRP2 protein, which is selectively expressed in the bile duct luminal membrane. The expression of MRP2 can be detected using an antibody against MRP2. Commercially available antibodies against MRP2 can be suitably used. For example, the expression of MRP2 can be visualized by immunostaining using an anti-MRP2 mouse monoclonal antibody as the primary antibody and a fluorescently labeled goat anti-mouse antibody as the secondary antibody, and the number of bile duct lumens can be counted by fluorescence.

By using the method of the present invention, the number of bile duct lumens on the surface of the culture equipment (0.89 bile duct lumens per cell) can be increased by 1.5 times or more, preferably by 2 times or more, and more preferably by 3 times or more, compared to when the culture equipment of the present invention is not used.

The formation of the bile duct lumen can be confirmed by the expression of MRP2 as well as occludin, JAM, zonula occluden (ZO) proteins (ZO-1, 2, 3), etc.

Methods for assessing drug metabolism and/or membrane transport
The present invention also provides a method for evaluating drug metabolism and/or membrane transport in liver-derived cells, which comprises forming a liver-derived cell layer on the culture tool of the present invention described above, and evaluating drugs excreted from the liver-derived cells through the culture tool.

Figure 13 shows a schematic example of a drug permeation test using the cultureware of the present invention. In this example, it is assumed that a cell culture insert such as Corning's Transwell (registered trademark) is coated with claudin proteins.

By using the culture device of the present invention, hepatocytes are seeded and cultured on a device coated with claudin protein, and a bile duct lumen is formed on the surface that comes into contact with the device, and drugs can migrate from this bile duct lumen through the permeable plate (insert).
Therefore, by sampling the liquid outside the culture vessel (lower compartment), it is possible to easily recover the permeated drug and determine the type and concentration of the drug.

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[Example 1] Induction of bile duct lumen formation by expression of claudins
HepG2 cells (a human hepatoma-derived cell line obtained from the American Type Culture Collection) were cultured in _DMEM containing 10% FBS, 100 units/mL Penicillin G Potassium, 100 μg/mL Streptomycin, and 1% NEAA in an incubator at 37°C and 5% CO2. When the cells were 70-80% confluent, they were washed with PBS, and then harvested and passaged by treatment with 0.1% trypsin-EDTA/PBS.

Genes encoding claudins 1, 2, 3, 4, 5, 7, 8, 9, 10a, 12, 14, 15, 19, 23, and 25 were transfected into 0.75× ¹⁰⁵ HepG2 cells.

In the functional bile duct lumen of the resulting claudin-expressing cells, ZO-1, the supporting protein for claudin proteins, and MRP2, which excretes compounds into the bile duct lumen, are thought to colocalize. Therefore, the number of colocalized MRP2/ZO-1 cells was calculated, with the number in the non-transfected case (mock) set at 100.

As a result, as shown in Figure 1, bile canalicular lumen formation was increased in cells transfected with claudins 1, 2, 3, and 9 (significant for claudins 1, 2, and 9, p=0.07 for claudin 3).

[Example 2] Induction of bile duct lumen formation by co-culture of claudin-expressing cells and hepatocytes.
We investigated whether bile canalicular lumen formation is induced in hepatocytes when claudin-expressing cells are co-cultured with hepatocytes.

HeLa cells (a human cervical cancer-derived cell line obtained from the American Type Culture Collection) were cultured in _DMEM containing 10% FBS, 100 units/mL Penicillin G Potassium, and 100 μg/mL Streptomycin in an incubator at 37°C and 5% CO2. When the cells were 70-80% confluent, they were washed with PBS, and then harvested and passaged by treatment with 0.1% trypsin-EDTA/PBS.

HeLa cells were seeded at 5.0 × ¹⁰⁴ cells/ ^cm2 in a Nunc 6-well plate (obtained from Thermo Fisher Scientific), and the medium was removed after 24 hours, and the lentivirus solution-containing DMEM prepared as follows was added. After another 24 hours, the medium was replaced with normal culture medium.

Lentivirus-containing DMEM
DMEM for culture (FBS, antibiotics included) 37.5%
Lentivirus solution (CLDN-1) 12.5%
Lentivirus solution (CLDN-2) 12.5%
Lentivirus solution (CLDN-3) 12.5%
Lentivirus solution (CLDN-9) 12.5%
Lentivirus solution (EGFP) 12.5%

To prepare the lentivirus solution, HEK293T cells (provided by Professor Atsushi Hirao, Cancer Research Institute, Kanazawa University) were seeded on a nunc 6-well plate at 0.95 x ¹⁰⁵ cells/ ^cm2 the day before the plasmid was introduced. The culture medium was DMEM containing 110 mg/L sodium pyruvate and 10% FBS. 1.0 μg of FG12/hCLDNs, 0.5 μg of pCMC-VSV-G (obtained from Addgene), and 1.0 μg of psPAX2 (obtained from Addgene) were each introduced using Lipofectamine3000 (obtained from Thermo Fisher Scientific). The medium was replaced 6-12 hours after gene introduction. 72 hours after introduction, the medium supernatant was collected in a 15 mL tube and centrifuged at 150 x g for 5 minutes, and the supernatant was collected and used as the lentivirus solution.

On the other hand, HepG2 cells passaged in the same manner as in Example 1 were co-cultured with the above-mentioned claudin-expressing HeLa cells at a density of 0.3 × ¹⁰⁵ cells/well in DMEM containing 10% FBS, 100 units/mL Penicillin G Potassium, 100 μg/mL Streptomycin, and 1% NEAA at 37°C for 4 days.

As a result, as shown in Figure 2, when the above-mentioned gene-transfected HeLa cells and HepG2 cells were co-cultured, it was shown that the formation of bile ductal lumen was induced not only between HepG2 cells but also between HeLa cells and HepG2 cells.

[Example 3: Synthesis of claudin proteins]
1. Cell-free synthesis of claudins 1, 2, 3, and 9 proteins
Since claudins are membrane proteins, maintaining the transmembrane structure is important. Therefore, we performed in vitro protein synthesis using liposomes in a cell-free protein synthesis system as described in Shinoda T. et al., Sci Rep., 6: 30442, 2016.

Specifically, cell-free synthesis of claudins 1, 2, 3, and 9 proteins was performed using the reconstituted cell-free synthesis kit PUREfrex (GeneFrontier, Kashiwa, Japan). A claudin template with a T7 promoter and ribosomal binding site (RBS) sequence attached to the N-terminus and lipid/detergent mixed micelles were added to the cell-free synthesis reaction solution, and the reaction was carried out at 37°C for 4 hours to synthesize claudins 1, 2, 3, and 9 proteins.

First, to synthesize claudin-1 protein, three-step PCR was performed using pcDNA3.1(+)/hCLDN1 myc-tag plasmid (SEQ ID NO: 1) as a template and the following primers (Table 1) and PrimeSTAR Max DNA Polymerase (TaKaRa Bio, Inc, Shiga, Japan) to prepare a claudin-1 template for in vitro synthesis with a T7 promoter sequence, ribosomal binding site (RBS), and His tag sequence added to the N-terminal region. The PCR reaction conditions were thermal denaturation reaction (98°C, 10 seconds), annealing (55°C, 5 seconds), and extension reaction (72°C, 5 seconds), with 30 cycles performed.

Mixed micelles were prepared from 6.7 mg/mL lipid [5% (w/w) cholesterol and 95% (w/w) egg yolk phosphatidylcholine] and 10.0 mg/mL digitonin by sonication until the solution was clear.

Similarly, for claudins 2, 3, and 9 proteins, templates were prepared by performing three-step PCR using the primers shown in Tables 2 to 4, respectively, using plasmids (hCLDN2 myc-tag plasmid (SEQ ID NO: 2), hCLDN3 myc-tag plasmid (SEQ ID NO: 3), and hCLDN9 myc-tag plasmid (SEQ ID NO: 4)) having the sequences shown in SEQ ID NOs: 2 to 4.

2. Purification of claudins 1, 2, 3, and 9 The synthesized claudins 1, 2, 3, and 9 proteins were purified using Dynabeads His-tag Isolation & Pulldown (Thermo Fisher Scientific).

The reaction mixture for synthesis of claudins 1, 2, 3, and 9 proteins was mixed and added to 1 × Binding/Wash buffer to make a volume of 700 μL. After that, it was shaken together with magnetic beads at room temperature for 5 minutes, and the supernatant was collected (flow-through). The magnetic beads were mixed with 300 μL of 1 × Binding/Wash buffer, left to stand at room temperature for 2 minutes, and the supernatant was collected. This procedure was repeated four times (Washing solutions 1-4). The magnetic beads were mixed with 100 μL of His-Elution buffer, shaken at room temperature for 5 minutes, and the supernatant was collected. This procedure was repeated twice (Elution solutions 1 and 2).

3. Detection of claudins 1, 2, 3, and 9 by Western blotting. Claudin 1, 2, 3, and 9 protein purification samples (flow-through, washing solution, elution solution 1 and 2) were diluted with 4 × SDS loading buffer and separated on a 14% polyacrylamide gel containing a 3% stacking gel. Molecular weights were determined using BlueStar Prestained Protein Marker (Nihon Genetics, Tokyo, Japan).

Proteins were transferred to a PVDF membrane using a Trans-BlotSD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA) at 100 mA for 30 min. The membrane was blocked for 1 h with PBS-T containing 0.1% Tween-20 and 2% skim milk. After washing with PBS-T, the sections were incubated overnight with primary antibodies (anti-claudin 1 antibody, mouse monoclonal (sc-81796, Santa Cruz Biotechnology), anti-claudin 2 antibody, rabbit polyclonal (ab53032, Abcam), anti-claudin 3 antibody, rabbit monoclonal (ab214487, Abcam), anti-claudin 9 antibody, rabbit polyclonal (sc-398836, Santa Cruz Biotechnology)) at 4°C, and then incubated with secondary antibodies (fluorescently labeled goat anti-mouse antibody or fluorescently labeled goat anti-rabbit antibody, Thermo Fisher Scientific) at room temperature for 2 hours. Luminescence was detected using ImmunoStarZeta (Fujifilm Wako Pure Chemical).

4. Detection of claudins 1, 2, 3, and 9 by CBB staining After separating the samples on a polyacrylamide gel, the gel was immersed in CBB fixative and shaken for 30 minutes. The fixative was discarded, and the gel was immersed in CBB staining solution and shaken for 40 minutes. The staining solution was discarded, and the gel was immersed in CBB destaining solution for destaining, and the bands were confirmed.

A calibration curve was created from the BSA bands of each mass by densitometry analysis using the ImageJ application (National Institutes of Health, Bethesda, MD, USA), and the synthesis and purification amounts were estimated from the intensity of the bands of claudins 1, 2, 3, and 9. The synthesis amount was estimated by subtracting the band of the synthesis reaction solution without DNA addition [DNA(-)] from the band of the synthesis reaction solution with DNA of each claudin added [DNA(+)].

As a result, as shown in Figures 3 to 6, claudin 1 (23 kDa), claudin 2 (25 kDa), claudin 3 (23 kDa), and claudin 9 (22 kDa) were each purified as a single band. The purification rates were all in the range of 15 to 26%.

[Example 4] Confirmation of coating of claudin protein on culture equipment
As culture equipment, ibidi 8 well plates (Nihon Genetics, Tokyo, Japan) were used either as is or coated with collagen I to confirm whether claudin proteins could be coated.

To prepare collagen-coated devices, Cellmatrix Type I-C (Nitta Gelatin, Osaka, Japan) diluted with hydrochloric acid (pH 3.0) to 300 μg/ml was added to an ibidi 8-well plate at 100 μl/well and left to stand for 15 minutes. The solution was then removed and the plate was air-dried at room temperature for 1 hour. Immediately before use, the plate was washed twice with PBS.

An ibidi 8-well plate or an ibidi 8-well plate previously coated with Cellmatrix was coated with a solution of claudin 1 protein (final concentration 44 μg/ml) synthesized in Example 3, and then fixed with PBS solution containing 4% PFA for 10 minutes. After blocking with PBS containing 2% BSA for 60 minutes, the primary antibody (anti-claudin 1 antibody, mouse monoclonal (sc-81796, Santa Cruz Biotechnology)) was reacted overnight at 4°C, and then the secondary antibody (fluorescently labeled goat anti-mouse antibody, Thermo Fisher Scientific) was reacted at room temperature for 1 hour. Fluorescence was observed with an HS all-in-one fluorescence microscope (BZ-9000, KEYENCE, Osaka, Japan).

As a result, uniform fluorescence was observed in the claudin-1-coated wells, regardless of whether they were coated with Cellmatrix, indicating that claudin-1 was uniformly coated on the equipment (data not shown).

[Example 5 Preparation of claudin-1, -2, -3, -9 coated plates]
A solution of Cellmatrix Type IC (final concentration 300 μg/mL) and the claudin-1 synthesized in Example 3 (final concentration 43.9 μg/mL) was mixed at a ratio of 1:1 to prepare a coating solution.

In the same manner, Cellmatrix Type I-C (final concentration 300 μg/ml) or Matrigel (final concentration 42 μg/ml) was mixed 1:1 with the mixed solution of claudins 1, 2, 3, and 9 synthesized in Example 3 to prepare a coating solution (final concentrations of claudins 1, 2, 3, and 9 were 22.0, 9.55, 17.8, and 10.25 μg/mL, respectively). Then, a coating plate was prepared according to the coating method for each coating agent.

[Example 6] Induction of bile canalicular lumen formation in human hepatoma cells on claudin-1-coated plates
HepG2 cells were seeded at 0.75 × ¹⁰⁵ cells/ ^cm2 on the claudin-1 coated plate prepared in Example 5, and cultured in DMEM containing 10% FBS, 100 units/mL Penicillin G Potassium, 100 μg/mL Streptomycin, and 1% NEAA for 72 hours. After changing the medium, the cells were further cultured for a total of 4 days.

After 96 hours of culture from seeding, HepG2 cells were fixed in PBS containing 4% PFA for 10 minutes, and the cell membrane was stained with wheat germ agglutinin (WGA) (5 μg/mL, obtained from Thermo Fisher Scientific), and then permeabilized with PBS containing 0.2% Triton X-100. After blocking for 60 minutes with PBS containing 2% BSA, the cells were incubated with a primary antibody (anti-MRP2 mouse monoclonal antibody: 100x) at room temperature for 2 hours, and then with a secondary antibody (Goat anti-Mouse Alexa flour 594: 200x) at room temperature for 1 hour. Nuclei were stained with DRAQ5 (5 μM, room temperature, 30 minutes), and fluorescence was observed under a confocal microscope (LSM710, Carl-Zeiss).

As a result, as shown in Figure 7A, a tendency for MRP2 staining to be induced toward the bottom side of the device was observed. Furthermore, this induction was not affected by the presence or absence of Cellmatrix coating (Figure 7B). On the other hand, when HepG2 cells were cultured in wells coated with Cellmatrix only, MRP2 staining was observed between adjacent hepatocytes (Figure 7C).

[Example 7] Induction of bile canalicular lumen formation in human primary hepatocytes on claudin-1, -2, -3, -9 coated plates
Frozen primary human hepatocytes (Lot: Hu1663, Thermo Fisher Scientific) were thawed in a 37°C water bath and suspended in CHRM at 37°C, then centrifuged at 100×g for 10 minutes. The supernatant was discarded by decanting, and Williams' Medium E (obtained from Thermo Fisher Scientific) containing Primary Hepatocyte Thawing and Maintenance Supplements (obtained from Thermo Fisher Scientific) was added to a concentration of 1× ¹⁰⁶ cells/ml, and the number of cells was counted by trypan blue staining.

Then, primary human hepatocytes were seeded at a seeding density of 4.0 × ¹⁰⁵ cells/ ^cm2 on the plate coated with claudins 1, 2, 3, and 9 together with Cellmatrix Type IC in Example _5. After 4 hours of incubation at 37°C and 5% CO2, the medium was replaced with ice-cold Matrigel and Williams' Medium E containing Primary Hepatocyte Maintenance Supplements (obtained from Thermo Fisher Scientific). The medium was further replaced with Williams' Medium E containing Primary Hepatocyte Maintenance Supplements every 24 hours, and the cells were used for experiments 72 hours after seeding.

72 hours after seeding, primary human hepatocytes were fixed in PBS containing 4% PFA for 10 minutes, and the cell membrane was stained with wheat germ agglutinin (WGA) (5 μg/mL, Thermo Fisher Scientific), followed by permeabilization with PBS containing 0.2% Triton. After blocking with PBS containing 2% BSA for 60 minutes, the cells were incubated with a primary antibody (anti-MRP2 mouse monoclonal antibody: 100x) at room temperature for 2 hours, and then with a secondary antibody (Goat anti-Mouse Alexa flour 594: 200x) at room temperature for 1 hour. Nuclei were stained with DRAQ5 (5 μM, room temperature, 30 minutes), and fluorescence was observed under a confocal microscope (LSM710, Carl-Zeiss).

As a result, as shown in Figure 8, a tendency for MRP2 staining to be induced toward the bottom side of the device was observed. On the other hand, when HepG2 cells were cultured in wells coated with Cellmatrix only, MRP2 staining was observed between adjacent hepatocytes (Figure 9).

Similar results were observed when plates coated with claudins 1, 2, 3, and 9 together with Matrigel were used in Example 5 (Figures 10 and 11).

[Example 8] Measurement of bile ductal lumen of human primary hepatocytes induced on the culture substrate side
In Example 7, the bile ductular lumina of human primary hepatocytes induced on the culture substrate in the presence of claudins 1, 2, 3, and 9 were measured by visually counting the number of stained spots of MRP2, a bile ductular marker, using the Z-stack function of a confocal microscope (LSM710, Carl-Zeiss).

As a result, as shown in Figure 12, in both the cases where Cellmatrix and Matrigel were used, the number of MRP2 stainings on the device side increased by more than three-fold when cultured on plates coated with claudins 1, 2, 3, and 9.

The present invention provides a new culture equipment that allows evaluation of drug metabolism and/or membrane transport through permeation tests.

Claims (11)

A hepatocyte culture device with a layer of claudin protein formed on the permeable surface of the device. The hepatocyte culture equipment according to claim 1 , comprising one or more selected from the group consisting of claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26 and 27 expressed in the human liver. A kit for evaluating drug excretion into bile, comprising a cell culture device having a claudin protein layer formed on the surface of the device , and liver-derived cultured cells. 4. The kit according to claim 3, wherein the surface of the cell culture substrate is permeable. The kit according to claim 3 or 4, wherein the cell culture equipment comprises one or more selected from the group consisting of claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26 and 27 expressed in the human liver. 3. The method for producing a hepatocyte culture substrate according to claim 1 or 2 , characterized in that the surface of a substrate having permeability is coated with a layer containing a claudin protein. A method for preparing an evaluation system for drug excretion into bile, comprising forming a layer of liver-derived cells on a cell culture substrate having a claudin protein layer formed on the substrate surface . A method for inducing bile duct lumen formation at the site of contact between the cells and the surface of a cell culture device having a claudin protein layer formed on the surface of the device and forming a layer of liver-derived cells on the cell culture device. A method for evaluating drug metabolism and/or membrane transport in liver-derived cells, comprising forming a liver-derived cell layer on a cell culture vessel having a claudin protein layer formed on the surface of the vessel , and evaluating drugs excreted from the liver-derived cells through the culture vessel. The method according to any one of claims 7 to 9, wherein the surface of the cell culture substrate has permeability. The method according to any one of claims 7 to 10, wherein the cell culture equipment comprises one or more selected from the group consisting of claudins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26 and 27 expressed in the human liver.