Method for producing culture substrate coated with membrane protein

Abstract

When a culture substrate is coated with a membrane protein, performing a washing operation after the coating with the protein together with a lipid and a surfactant may result in aggregation of the protein, resulting in the inability to perform uniform coating. Therefore, performing more uniform coating on the culture substrate has been an issue. Provided is a method for producing a culture substrate coated with a membrane protein, the method comprising: a step for synthesizing a membrane protein in vitro; a step for forming a lipodisc that contains the synthesized membrane protein, a lipid, and a lipid bilayer membrane stabilizing polymer; and a step for coating the surface of the culture substrate with the lipodisc.

Description

Method for manufacturing culture equipment coated with membrane proteins

[0001] The present invention relates to a method for producing culture equipment coated with membrane proteins, and more particularly to a method in which the membrane proteins are cell adhesion proteins. The present invention also relates to culture equipment produced by the above method, a method for inducing bile duct lumen formation on the surface of the culture equipment, and a method for evaluating drug metabolism and / or membrane transport in liver-derived cells using the culture equipment.

[0002] The liver is the largest organ in the human body by volume 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 bile excretion.

[0003] In drug research and development, evaluating biliary excretion is extremely important. For this purpose, in vivo studies using experimental animals, or in vitro evaluations using primary cultured human or rodent hepatocytes or established hepatocyte lines, are known, and sandwich cultured human hepatocytes are also used. However, in vitro methods have been difficult to use as a rapid and quantitative method for evaluating biliary excretion.

[0004] Bile secretion and the bile excretion of drugs and other substances occur through the bile duct lumen formed in the cell membrane of hepatocytes. The present inventors' group previously created cell culture equipment with a claudin protein layer formed on its surface, and using this, established a permeation test type evaluation system that can easily and accurately predict drug bile excretion by inducing the bile duct lumen of hepatocytes to the basal surface of the culture equipment (Patent Document 1, Non-Patent Document 1). This enables the temporal and quantitative analysis of compounds excreted into the bile duct lumen, and has established a highly accurate predictive system for bile excretion in humans.

[0005] On the other hand, numerous proteins involved in intercellular adhesion not only in hepatocytes but also in various epithelial cells have been reported, and several models have been proposed for the function and interactions of these proteins that constitute tight junctions (for example, Non-Patent Documents 2-5).

[0006] Furthermore, techniques for analyzing the function of membrane proteins in a state similar to that of nature include cell-free protein synthesis methods (e.g., Non-Patent Document 6) and methods for preparing lipodiscs (e.g., Non-Patent Document 7).

[0007] Japanese Patent Publication No. 2022-103781

[0008] Arakawa H. et al., Commun Biol., 6(1):866, Aug 22, 2023Takai Y. et al., Journal of Cell Science, 116(1), 17-27, 2003Nomme J. et al., Journal of Biological Chemistry, Vol.286, No.50, 43352-43360, December 16, 2011Steinbacher T. et al., Cellular and Molecular Life Sciences, 75: 1393-1409, 2018Beutel O. et al., Cell, 179, 923-936, 2019Shinoda T. et al., Scientific Reports, 6:30442, 2016, DOI: 10.1038 / srep30442Dorr JM et al., Eur Biophys. J. (2016) 45:3-21

[0009] One problem in the preparation of culture equipment previously reported by the present inventors was that after coating the synthesized claudin protein with lipids and surfactants, the surfactant was removed during the washing process, which sometimes caused the protein to aggregate and prevent a uniform coating. Therefore, improving the process to achieve a uniform coating on culture equipment was a challenge.

[0010] As a result of various studies to solve the above problems, the inventors discovered that a very uniform coating can be achieved by encapsulating the claudin protein in a polymer-based lipodisk after synthesis and then coating the culture equipment with it. Furthermore, they confirmed that this method can be similarly applied to various membrane proteins that require coating of culture equipment, and thus completed the present invention.

[0011] In other words, the present invention provides the following: 1. A method for producing a culture device coated with a membrane protein, comprising the steps of: synthesizing a membrane protein in vitro; forming a lipodisc containing the synthesized membrane protein, a lipid, and a lipid bilayer stabilizing polymer; and coating the lipodisc on the surface of the culture device. 2. The method according to 1, wherein the membrane protein is a cell adhesion protein. 3. The method according to 2, wherein the membrane protein is selected from claudin, junction adhesion molecule-A (JAM-A), and combinations thereof. 4. The method according to 3, wherein the lipodisc is coated on the surface of the culture device together with a ZO protein. 5. The method according to item 1 above, wherein the polymer is selected from styrene-maleic acid (SMA) copolymer, diisobutylene maleic acid (DIBMA) copolymer, and as an SMA derivative, styrene-maleic acid-ethanolamine (SMA-EA), ethylenediamine-modified styrene-maleic anhydride copolymer modified with Ethylene Diamine (SMA-ED and SMAd-A), and styrene-maleimide quaternary ammonium (SMA-QA) copolymer. 6. Cell culture equipment manufactured by any of the methods described in items 1 to 5 above. 7. Cell culture equipment having a JAM-A protein layer formed on its surface. 8. Cell culture equipment having a layer of a complex composed of claudin protein, JAM-A protein, and ZO protein formed on its surface. 9. A protein complex comprising claudin protein or fragments thereof containing claudin extracellular loops 1 and 2, JAM-A protein or fragments thereof containing the JAM-A extracellular domain, and ZO-1 protein or fragments thereof containing the PDZ1 to SH3 domain of ZO-1.10. A method for producing culture equipment, characterized by coating the surface of the culture equipment with a layer containing the protein complex described in 9 above. 11. A kit for evaluating drug efflux into bile, comprising the culture equipment described in 6 above and liver-derived cultured cells. 12. A method for producing a drug efflux into bile evaluation system, characterized by forming a liver-derived cell layer on the culture equipment described in 6 above. 13. A method for inducing bile duct lumen formation at the contact site between the cells and the surface of the equipment, by forming a liver-derived cell layer on the culture equipment described in 6 above. 14. A method for evaluating drug metabolism and / or membrane transport in liver-derived cells, characterized by forming a liver-derived cell layer on the culture equipment described in 6 above and evaluating the drug effluxed from the liver-derived cells through the culture equipment. 15. A kit for evaluating drug efflux into bile, comprising the culture equipment described in 7 or 8 above and liver-derived cultured cells. 16. A method for producing a drug efflux into bile evaluation system, characterized by forming a liver-derived cell layer on the culture equipment described in 7 or 8 above. 17. A method for inducing the formation of a bile duct lumen at the contact site between the cells and the surface of the culture device, wherein a layer of liver-derived cells is formed on the culture device described in 7 or 8 above. 18. A method for evaluating drug metabolism and / or membrane transport in liver-derived cells, characterized by inducing the formation of a layer of liver-derived cells on the culture device described in 7 or 8 above and evaluating the drug discharged from the liver-derived cells through the culture device. This specification encompasses the disclosures of Japanese Patent Application No. 2024-159370, which forms the basis of the priority of this application.

[0012] This invention makes it possible to uniformly coat culture materials with membrane proteins. For example, by coating a cell adhesion protein onto an insert membrane capable of permeability testing and culturing hepatocytes, a permeability testing system using hepatocytes can be constructed.

[0013] This document outlines cell-free protein synthesis and membrane protein lipodiscs using lipid bilayer solubilizing polymers. It shows the detection results of claudin 1 purified samples by CBB staining (A) and Western blotting (B). It also shows the detection results of claudin 1 purified samples extracted using lipid bilayer solubilizing polymers (SMA, SMA-EA, and DIBMA) by CBB staining. Immunofluorescence staining of claudin 1 confirms the coating of various claudin 1-containing lipodiscs onto cell culture equipment. A: Coating surface of claudin 1-containing lipodiscs extracted using lipid bilayer solubilizing polymers (SMA, SMA-EA, and DIBMA). B: Comparison of the amount of claudin 1-containing lipodiscs and claudin 1 extracted by the conventional method (S-MF method) coating cell culture equipment. C: Quantitative comparison of fluorescence intensity on the claudin 1-containing lipodiscs and the claudin 1-coated surface extracted by the conventional method (S-MF method) (n = 1). The results of detection of purified JAM-A samples by CBB staining (A) and Western blotting (B) are shown. Immunofluorescence staining of JAM-A confirms the coating of JAM-A-containing lipodiscs on cell culture equipment. An overview of the oriented coating method of adhesion proteins claudin 1 and JAM-A using the membrane-backed protein ZO-1 is shown. A: Schematic diagram of the inter-cell trans-interaction of adhesion proteins. B: Schematic diagram showing the necessity of oriented coating of adhesion proteins on cell culture equipment. C: Constituent domains of ZO-1. D: Schematic diagram of the ZO-1, claudin 1, and JAM-A complex coated on cell culture equipment. The results of detection of purified ZO-1 samples by CBB staining (A) and Western blotting (B) are shown. Immunofluorescence staining of ZO-1, claudin 1, and JAM-A confirms the co-coating of claudin 1 and ZO-1 (A), and JAM-A and ZO-1 (B) on cell culture equipment. This shows that co-immunoprecipitation experiments confirmed the binding of ZO-1 to claudin 1 and ZO-1 to JAM-A. A: Western blotting detection results for ZO-1, claudin 1, and JAM-A.B: Quantitative comparison of band intensities of Western blotting for claudin 1 and JAM-A in the immunoprecipitation fraction (n = 1). Shows the localization of adhesion proteins in PXB-cells derived from human liver chimeric mice. A: Immunostaining images of claudin 1, B: JAM-A, and C: ZO-1. Shows that the formation of an open bile canaliculus in PXB-cells was induced when claudin 1, JAM-A, and ZO-1 were coated onto cell culture equipment. A: Proposal of an induced open-form bile canaliculus hepatocyte (icHep) and immunostaining images of the open bile canaliculus in PXB-cells. B: Calculation results of the bile canaliculus opening rate of icHep when various adhesion proteins are coated onto cell culture equipment (n = 3). *: P ≤ 0.05. Shows that drug excretion in bile can be evaluated by permeability testing using icHep constructed according to the present invention. A: Schematic diagram of the icHep drug permeability test system constructed according to the present invention. B, C: Results of calculating the permeability clearance of the paracellular transport marker TD4 (B) and the MRP2 substrate E217βG (C) using the icHep permeability test system constructed by coating cell culture equipment with various adhesion proteins (n ​​= 3). *: P ≤ 0.05. Results of drug permeability tests using icHep constructed under various coating conditions (A: permeability of paracellular transport marker TD4, B: permeability of MRP2 substrate E217βG). E-Cad-Fc: E-cadherin-Fc. *: P ≤ 0.05.

[0014] <Method for Manufacturing Culture Equipment> The present invention provides a method for manufacturing culture equipment coated with membrane proteins, comprising the steps of: synthesizing membrane proteins in vitro; forming lipodiscs containing the synthesized membrane proteins, lipids, and lipid bilayer stabilizing polymers; and coating the surface of the culture equipment with the lipodiscs.

[0015] In this specification, "membrane protein" refers to a protein that may have a lipophilic portion embedded in the lipid bilayer of the cell membrane or organelle membrane in animal, plant, and microbial cells, as is commonly understood, and a hydrophilic portion located outside the lipid bilayer. Membrane proteins present in the cell membrane may have hydrophilic portions extracellularly, intracellularly, or both. Membrane proteins that have hydrophilic portions both extracellularly and intracellularly are also called transmembrane proteins.

[0016] Membrane proteins include both native proteins and proteins synthesized in vitro or in vivo. Synthesized proteins may have the same or modified constituent amino acids, their sequences, and modifications by sugars, etc., as native proteins.

[0017] The method of the present invention is applicable to all of the above-mentioned membrane proteins, but in one preferred embodiment, the membrane protein is a cell adhesion protein that can be involved in cell-cell adhesion. Examples of cell adhesion proteins include claudin proteins, junction adhesion molecule-A (also referred to herein as JAM-A) proteins, and E-cadherin proteins.

[0018] In one preferred embodiment, the membrane protein is a claudin protein or a JAM-A protein. Claudin proteins or JAM-A proteins can also be used in combination. Depending on the desired function, the membrane protein may also be used in the form of a fragment, or a complex of the fragment with a protein that promotes adhesion to the culture medium (such as an IgG-Fc domain), provided that its function is not impaired.

[0019] The method of the present invention includes the step of synthesizing membrane proteins in vitro. Methods for synthesizing membrane proteins in vitro are well known in the field, for example, a method is known in which an expression plasmid containing a polynucleotide encoding a membrane protein is introduced into a suitable host cell to synthesize the membrane protein. Another known cell-free protein synthesis method is in which a membrane protein expression plasmid is reacted in a test tube with a mixed micelle consisting of appropriate lipids and surfactants, along with the amino acids and enzymes necessary for protein synthesis (Shinoda T. et al., Scientific Reports, 6:30442, 2016, DOI: 10.1038 / srep30442). Due to its ease of operation and the advantage of being able to obtain the required membrane protein in a state with few impurities, the cell-free protein synthesis method is preferred in the present invention.

[0020] Furthermore, cell-free protein synthesis methods include a method (P-MF method) that uses mixed micelles with a relatively low surfactant ratio to obtain proteoliposomes / large membrane fragments precipitated by centrifugation at 100,000 × g and requires solubilization for purification, and a method (S-MF method) that uses mixed micelles with a relatively high surfactant ratio to obtain small membrane fragments contained in the supernatant by centrifugation at 100,000 × g and does not require solubilization. Both methods can be suitably used in the present invention.

[0021] When using the cell-free protein synthesis method described above, in the step of synthesizing membrane proteins, proteoliposomes or micelles containing membrane proteins can be obtained and used in the next step together with lipids and surfactants.

[0022] The method of the present invention then includes the step of forming a lipodisc containing the synthesized membrane protein, lipids, and lipid bilayer stabilizing polymer. Here, since the membrane protein synthesized in the above step is present together with the lipid bilayer or in lipid / surfactant mixed micelles, this step involves contacting the membrane protein present with the lipids with the lipid bilayer stabilizing polymer.

[0023] Suitable lipids include phospholipids similar to those that make up natural cell membranes, such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and cholesterol. For example, phosphatidylcholine derived from egg yolk can be used.

[0024] While not limited to specific surfactants, it is preferable to use digitonin, which can form relatively large micelles for reasons such as being difficult to remove by dialysis, or steroidal surfactants such as cholic acid or 3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). While not limited to specific surfactants, it is preferable to use a ratio of 0.025:1 to 2:1 by weight.

[0025] Examples of lipid bilayer stabilizing polymers, though not limited to them, include styrene-maleic acid (SMA) copolymer, diisobutylene maleic acid (DIBMA) copolymer, and SMA derivatives such as styrene-maleic acid-ethanolamine (SMA-EA), ethylenediamine-modified styrene-maleic anhydride copolymer modified with Ethylene Diamine (SMA-ED and SMAd-A), and styrene-maleimide quaternary ammonium (SMA-QA) copolymer. Each of these polymers has different surface charge, pH stability, and cationic stability, and can be appropriately selected depending on the membrane protein to be isolated. These polymers are commercially available, for example, from Funakoshi Co., Ltd., and those with a molecular weight of approximately 7.8 to 10.4 kDa are preferably used.

[0026] The extraction of membrane proteins using lipid bilayer stabilizing polymers is sometimes referred to as the SMA copolymer method, after the names of representative polymers, and is described in publications such as Dorr JM et al., Eur Biophys. J. (2016) 45:3-21; Gulamhussein AA et al., BBA-Biomembranes 1862 (2020) 183281; and Orekhov PS et al., Nanomaterials, 2022, 12, 361.

[0027] In this specification, "lipodisk" refers to a disc-shaped structure in which a membrane fragment containing membrane proteins within a lipid bilayer is surrounded by a lipid bilayer stabilizing polymer as described above, and is sometimes called a nanodisk. The size of the formed lipodisk varies depending on the polymer used; for example, it is about 10 nm for SMA copolymer and about 25 nm for DIBMA copolymer. Since the lipodisk formation efficiency differs depending on the type of polymer used, it is preferable to optimize the lipodisk formation conditions.

[0028] In the present invention, the use of SMA copolymer is preferred due to its high lipodisc formation efficiency and higher stability. In one preferred embodiment of the present invention, lipodiscs are formed using an SMA copolymer with a molecular weight of approximately 10.4 kDa. When using an SMA polymer, a more uniform coating can be achieved by pre-coating with poly-L-lysine.

[0029] When membrane proteins are solubilized using surfactants, the surfactant is removed during the washing process after coating the culture equipment, leading to liposome formation. This can result in the extracellular loop not being exposed, potentially leading to a decrease in bile duct lumen opening.

[0030] In contrast, when using lipodiscs, most residual surfactants are removed in the synthesis washing step, and coating can be performed under surfactant-free conditions (buffer solution) (it can be done without using surfactants), so membrane proteins can remain stable even after washing after coating. Furthermore, lipodiscs are stable at 4°C for 2-3 weeks and can withstand at least 5 freeze-thaw cycles, thus providing stability for storage of lipodiscs before coating and for storage of culture equipment after coating. Free lipid bilayer stabilizing polymers that have not been lipodiscized can be removed before coating by affinity purification or ultrafiltration.

[0031] The method of the present invention then includes the step of coating the surface of the culture equipment with the lipodisk formed in the above step. Prior to coating the lipodisk, poly-L-lysine or collagen may be coated first. Since collagen has been found to act in a manner that suppresses the opening of the bile duct lumen, it is preferable not to coat with collagen if collagen is not required for the adhesion of cells such as hepatocytes to the culture equipment.

[0032] In this field, products containing multiple extracellular matrix proteins to form gels are available for coating the surface of equipment for effective cell culture, and these may be used in combination in the present invention. For example, but are not limited to, Cellmatrix Type IC (Nitta Gelatin, Osaka, Japan) and Matrigel (Corning, NY, USA) are examples of products that can be suitably used.

[0033] In this specification, "culture equipment" refers to equipment commonly used as cell culture equipment in this field, and does not particularly limit its shape or material. For example, glass or plastic equipment such as cell culture inserts, slides, dishes, plates, multiwell plates, and microfluidic devices can be used as appropriate.

[0034] For the purpose of evaluating the permeation of drugs through culture equipment, the surface of the equipment needs to be permeable. Here, "permeability" means that gases, liquids, solutes, ions, etc. secreted from cells can pass through, and porous bodies, meshes, cell culture inserts, etc. having a pore diameter of 100 nm or more can be used. For example, the insert membrane of Transwell (registered trademark) of Corning is an example of a permeable equipment that can be preferably used. In addition, since the material to be used only needs to be able to permeate substances, the use of a pore diameter of 100 nm or less is also considered possible.

[0035] Specifically, the coating on the surface of the equipment can be carried out by placing a solution containing lipodisks on the surface of the equipment, for example, leaving it standing at room temperature for 60 minutes, performing a fixing operation as necessary, and then drying it. Although not particularly limited, for example, the concentration of membrane protein can be made to be in the range of 1.5 to 3.0 μg / cm 2 on the surface of the equipment.

[0036] In addition, in this specification, "equipment surface" or "equipment bottom surface" refers to the surface of the equipment that contacts the cells to be cultured, particularly hepatocytes, in the equipment. In the case of a permeable equipment, both surfaces of the equipment may be coated.

[0037] By using the culture equipment of the present invention, the formation of bile duct lumens on the surface side of the equipment is induced in the cultured cells, particularly hepatocytes, and by using a permeable equipment, substances secreted from the cells can be easily recovered through the equipment.

[0038] <Claudin protein> An example of a protein that can be preferably used in the method of the present invention is claudin protein. Claudin is a transmembrane protein known to be a major protein involved in the formation of tight junctions, which are one type of cell-cell junctions. Twenty-seven types of claudin proteins have been reported so far in humans and mice, and they are all transmembrane proteins with a molecular weight of 20 to 27 kDa.

[0039] Claudin proteins are intended for use in systems that evaluate hepatocyte function and drug metabolism, and therefore are not particularly limited in their type, but it is preferable that they be derived from the same animal species as the hepatocytes used in the evaluation.

[0040] The animal species can include any animal with a liver, such as mammals, birds, amphibians, reptiles, and fish, and are not limited to these. However, mammals such as dogs, cats, rats, mice, rabbits, cattle, horses, goats, monkeys, and humans are particularly noteworthy. For example, for metabolic studies in humans, it is preferable to use claudins derived from humans.

[0041] In humans, 26 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, 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. Furthermore, it is known that claudin molecules can exert their functions not only through bonding between identical 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).

[0042] Therefore, in the present invention, any of the 26 claudins whose expression has been confirmed in humans can be used individually or in combination. Preferably, 11 claudins whose expression has been confirmed in the human liver can be used individually or in combination. That is, in the present invention, one or more 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 claudin proteins can be used from 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, and 14. The inventors have confirmed that among the above, claudins 1, 2, 3, and 9 are suitably usable in the present invention. Therefore, in a preferred embodiment of the present invention, one or more claudin proteins selected from the group consisting of claudins 1, 2, 3, and 9 can be used.

[0043] The amino acid sequence of human claudin 1 (sometimes referred to as "CLDN1" herein) and the nucleotide sequence encoding it can be obtained, for example, from the sequence database of the National Center for Biotechnology Information (NCBI) under accession number NP_066924 and Gene ID: 9076, respectively. Sequence ID 5 shows an example of the nucleotide sequence encoding the human claudin 1 protein.

[0044] The amino acid sequence of human claudin 2 (sometimes referred to as "CLDN2" herein) and the nucleotide sequence encoding it can be obtained, for example, from the NCBI sequence database under accession number NP_065117 and Gene ID: 9075, respectively.

[0045] The amino acid sequence of human claudin 3 (sometimes referred to as "CLDN3" herein) and the nucleotide sequence encoding it can be obtained, for example, from the NCBI sequence database under accession number NP_001297 and Gene ID: 1365, respectively.

[0046] The amino acid sequence of human claudin 9 (sometimes referred to as "CLDN9" herein) and the nucleotide sequence encoding it can be obtained, for example, from the NCBI sequence database under accession number NP_066192 and Gene ID: 9080, respectively.

[0047] The amino acid sequences and encoding sequences of other human claudins and claudin proteins derived from other animal species can be obtained in the same manner.

[0048] In this invention, claudin proteins can be isolated and purified from animals before use. However, synthesizing the target claudin protein based on the sequence information described above makes it easier to form a protein layer free of impurities.

[0049] The claudin protein may be a full-length protein or a functional fragment thereof, such as a fragment containing claudin extracellular loops 1 and 2. The claudin protein binds to the PDZ1 domain of ZO-1 at its C-terminus (Nomme et al., J. Biol. Chem., 290(27): 16595-16606 (2015)). Therefore, in order to maintain binding affinity to the ZO protein, the fragment is preferably one that does not have a C-terminus deletion. For example, an expression plasmid having the nucleotide sequence shown in SEQ ID NO: 1 can be suitably used to synthesize the claudin protein.

[0050] Preferably, when using claudin 1, it is preferable to use the S205 mutant, for example, the S205Y mutant, in order to enhance its binding affinity to ZO-1. The nucleotide sequence shown in SEQ ID NO: 5 encodes the S205Y mutant.

[0051] <JAM-A Protein> Another example of a protein that can be suitably used in the method of the present invention is the JAM-A protein. Junctional adhesion molecule-A (JAM-A, also known as JAM1, CD321, and F11R) is a member of the immunoglobulin superfamily and is known to play an important role in the assembly of tight junctions in epithelial cells (Konopka G. et al., Journal of Biological Chemistry, Vol.282, No.38, 28137-28148, September 21, 2007; Kakogiannos N., Circulation Research, 127, 1056-1073, September 25, 2020).

[0052] JAM-A is a single-pass transmembrane protein that, along with claudin, plays a crucial role in the formation of tight junctions. Furthermore, it has been reported that JAM-A trans-interacts with adjacent JAM-A on the cell membrane prior to claudin (Steinbacher T. et al., Cell Mol Life Sci, 75(8): 1393-1409, 2018), and that knockout of JAM-A leads to the disappearance of the bile duct lumen in hepatocytes (Konopka G. et al., J Biol Chem, 282(38): 28137-48, 2007), suggesting that JAM-A may be important for early cell adhesion and bile duct lumen formation. Additionally, JAM-A may play a role equivalent to that of adhesion junctions, as it accumulates the polarity protein Par3-Par6-aPKC complex and claudin at adhesion sites.

[0053] JAM-A proteins are not particularly limited, but examples include mammals, birds, amphibians, reptiles, and fish, which are animals with livers, and especially mammals such as dogs, cats, rats, mice, rabbits, cattle, horses, goats, monkeys, and humans. For example, for metabolic studies in humans, it is preferable to use human-derived JAM-A. Furthermore, when used in combination with claudin proteins, it is preferable to use those from the same species.

[0054] The amino acid sequence of the human JAM-A protein and the nucleotide sequence encoding it can be obtained, for example, from the sequence database of the National Center for Biotechnology Information (NCBI) as accession number 058642.1 and Gene ID: 50848, respectively. Sequence ID 7 shows an example of the nucleotide sequence encoding the human JAM-A protein.

[0055] The JAM-A protein may be a full-length protein, or it may be a functional fragment, such as the JAM-A extracellular domain, or a complex with a protein that promotes adhesion between the functional fragment and culture media (e.g., the IgG-Fc domain). The fragment is preferably one that does not have a C-terminus deletion in order to maintain its binding affinity to the ZO protein. For example, an expression plasmid having the nucleotide sequence shown in Sequence ID No. 6 can be suitably used to synthesize the JAM-A protein.

[0056] <ZO Protein> When using claudin protein, JAM-A protein, or both as membrane proteins, the method of the present invention described above may also involve coating the culture material with ZO family proteins together with the lipodisc described above.

[0057] Tight junction proteins such as claudin 1 and JAM-A form tight junctions through trans interactions between extracellular domains located on adjacent hepatocyte membranes (Tsukita S. et al., Nat Rev Mol Cell Biol, 2(4):285-93, 2001) (Figure 7A). Therefore, it is hypothesized that controlling the orientation of tight junction proteins coated on cell culture equipment can improve trans interactions between the equipment and hepatocytes (Figure 7B).

[0058] In vivo, the membrane-backed protein ZO-1 binds to adhesion proteins such as claudin 1 and JAM-A, determining the orientation of these adhesion proteins on the cell membrane (Beutel O. et al., Cell, 179: 923-936, 2019) (Figure 7C). Based on these findings, it was hypothesized that co-coating ZO-1, which has an IgG-Fc domain with high binding affinity to hydrophobic surfaces fused to its C-terminus, with claudin 1 and JAM-A would enable the in vitro construction of a tightly bound protein complex with extracellular domain orientation (Figure 7D). Therefore, ZO-1 protein was synthesized using a cell-free synthesis method, and a complex was formed between ZO-1 and claudin 1 and JAM-A.

[0059] For tight junctions between cells to form, the extracellular loops of adjacent claudin proteins must interact with each other. Therefore, in order to form tight junctions between culture equipment and hepatocytes, the extracellular loops of claudin proteins coated on the surface of the equipment must be oriented toward the hepatocytes.

[0060] The inventors also confirmed that co-coating claudin protein with JAM-A protein and ZO protein further improves the orientation of claudin protein and increases the bile duct lumen opening rate.

[0061] The ZO (Zonula Occludens) family of proteins is known to constitute tight junctions and function as supporting proteins for intercellular junctions (Nomme J. et al., Journal of Biological Chemistry, Vol.286, No.50, 43352-43360, December 16, 2011). There are three types of ZO proteins: ZO-1, ZO-2, and ZO-3, all of which possess a PDZ domain and a Src homology 3 (SH3) domain. Although not limited to these, ZO-1 is particularly preferred for use in the present invention due to its high versatility.

[0062] While the ZO protein is not particularly limited, it is preferable that it be derived from the same species as the claudin protein and JAM-A protein, as they are used in combination.

[0063] The amino acid sequence of the human ZO-1 protein and the nucleotide sequence encoding it can be obtained, for example, from the sequence database of the National Center for Biotechnology Information (NCBI) under accession number 003257.5 and Gene ID: 7082, respectively. Information on the human ZO-2 protein and the human ZO-3 protein can be obtained in the same manner.

[0064] The ZO protein may be a full-length protein or a functional fragment, such as a fragment from the PDZ1 domain to the SH3 domain. The fragment must retain the PDZ1 domain to maintain binding affinity to claudin proteins, and the PDZ3 and SH3 domains to maintain binding affinity to JAM-A proteins. On the other hand, it has been reported that the U6 domain in the ZO-1 protein suppresses clustering necessary for tight junction formation (Beutel O. et al., Cell, 179, 923-936, 2019). Therefore, it is preferable for ZO-1 to not contain the U6 domain for complex formation. Sequence ID 9 is an example of a nucleotide sequence encoding a fragment of the human ZO protein from the PDZ1 domain to the SH3 domain.

[0065] Furthermore, for binding to the surface of equipment, a ZO protein with an IgG-Fc domain attached to its C-terminus (referred to as ZO-1-Fc in this specification) is suitably used in the present invention. For example, an expression plasmid having the nucleotide sequence shown in SEQ ID NO: 8 can be suitably used to synthesize the ZO-1 protein.

[0066] The inventors confirmed that ZO-1-Fc protein alone can provide a uniform coating on the surface of equipment (data not shown). Coating conditions are not limited, but for example, 1.5–3.0 μg / cm³. 2 It is preferable to use the ZO-1-Fc protein. Since both claudin and JAM-A bind to ZO-1, a more uniform coating can be expected by forming a complex with them before coating.

[0067] <Culture Equipment> The present invention provides cell culture equipment manufactured by the coating method of the present invention described above. The present invention also provides cell culture equipment in which a JAM-A protein layer is formed on the surface of the equipment, regardless of the coating method. Furthermore, the present invention provides cell culture equipment in which a layer of a complex composed of claudin protein, JAM-A protein, and ZO protein is formed on the surface of the equipment, regardless of the coating method. As described above, the culture equipment of the present invention can be used in a drug efflux evaluation system into bile.

[0068] <Protein Complex> The present invention provides a protein complex composed of claudin protein, JAM-A protein, and ZO-1 protein. The ratio of claudin protein, JAM-A protein, and ZO-1 protein in the protein complex is not limited, but since each protein binds in a 1:1 ratio, it is preferable to have a ratio of approximately 1:1:1.

[0069] The synthesis of the complex can be carried out in various ways, though not limited to these methods. For example, by adding lipodisk solutions of claudin protein and JAM-A protein to culture equipment pre-coated with ZO-1-Fc, or by adding a mixed solution of each protein to the culture equipment. The membrane proteins, claudin protein and JAM-A protein, are used in lipodisk solutions, while the supporting protein, ZO protein, is used in solution without lipodisks to carry out the complex formation reaction.

[0070] A preferred embodiment of the coating method of the present invention involves preparing a complex of claudin protein, JAM-A protein, and ZO-1 protein, and then performing the coating. Accordingly, the present invention provides a method for producing culture equipment, characterized by coating the surface of the culture equipment with a layer containing the above-mentioned protein complex.

[0071] <Kit> The present invention also provides a kit for evaluating drug efflux into bile, comprising the cell culture equipment of the present invention described above and liver-derived cultured cells. In the present invention, "liver-derived cultured cells" (which may also be referred to as "cultured cells" or "hepatocytes" in this specification) are not particularly limited, but examples include commercially available liver-derived cultured cells, iPS-derived hepatocytes, liver cancer-derived cultured cells, and cultured cells derived from healthy individuals or individuals with liver disease (humans or animals).

[0072] When using normal hepatocytes or hepatocytes derived from a healthy individual, 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).

[0073] Alternatively, when using cultured cells derived from liver cancer, or hepatocytes derived from individuals with liver diseases such as liver cancer, the kit of the present invention can evaluate changes in drug metabolism, such as the presence or absence of decreased metabolic function, compared to a normal liver.

[0074] <Method for preparing a drug efflux evaluation system into bile> The present invention also provides a method for preparing a drug efflux evaluation system into bile, characterized by forming a liver-derived cell layer on the culture equipment of the present invention described above. The liver-derived cell layer is cultured as a monolayer, with cells closely packed together 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, in primary human hepatocytes, it is 2.0 × 10⁶ 5 pieces / cm 2 This method of seeding can sometimes result in seeding liver cells across the entire culture medium. However, it is affected by cell viability and adhesion rates, so optimization for each batch is necessary.

[0075] <Method for Inducing Bile Duct Lumen Formation> The present invention also provides a method for inducing bile duct lumen formation at the contact site between the cells and the surface of the culture device described above, by forming a liver-derived cell layer on the culture device of the present invention. In the culture device of the present invention, the presence of a claudin protein layer induces the formation of bile duct lumen in liver-derived cells at the surface in contact with the culture device.

[0076] The formation of bile duct lumens can be confirmed, for example, by detecting the expression of the MRP2 protein, which is selectively expressed on the bile duct luminal membrane. MRP2 expression can be detected using an antibody against MRP2. Commercially available antibodies against MRP2 can be suitably used. For example, MRP2 expression 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. The method of the present invention can form an equivalent or greater number of bile duct lumens compared to the case using a coating method that does not use lipodiscs. In addition to MRP2 expression, the formation of bile duct lumens can also be confirmed by the expression of occludin and other proteins.

[0077] <Method for evaluating 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, characterized by forming a layer of liver-derived cells on the culture material of the present invention described above, and evaluating the drugs excreted from the liver-derived cells through the culture material.

[0078] By using the culture equipment of the present invention and seeding and culturing hepatocytes on equipment coated with cell adhesion proteins, a bile duct lumen is formed on the surface in contact with the equipment, and drugs can move through the equipment from this bile duct lumen. Therefore, by collecting the liquid outside the culture equipment, it is possible to easily recover the permeated drugs and determine the type and concentration of the drugs.

[0079] In drug permeability studies, MRP2 transport activity can be observed by suitably using exemplary drugs such as estradiol-17β-d-glucuronide (E217βG), an MRP2 substrate, and TRITC-dextran 4 (TD4), an intercellular transport marker.

[0080] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0081] [Example 1: Preparation of Claudin 1-Containing Lipodiscs] <1-1 Cell-Free Protein Synthesis and Lipodisc Formation of Claudin 1> An overview of this example is shown in Figure 1. Since Claudin 1 is a membrane protein, maintaining its transmembrane structure is important. Therefore, in vitro synthesis of Claudin 1 was performed using a cell-free protein synthesis system (Cell-Free-Kun, Taiyo Nippon Sanso, Tokyo, Japan) and a cell-free protein synthesis method (P-MF method) (Shinoda T. et al., Sci Rep., 6: 30442, 2016).

[0082] Lipid surfactant mixed micelles were prepared by sonicating a mixture of 50 mg / ml lipids (47.5 mg / ml egg yolk PC and 2.5 mg / ml cholesterol) and 30 mg / ml digitonin until the solution became clear. Next, 5% (v / v) 50 ng / μl Claudin 1 template DNA (SEQ ID NO: 1), fused with a modified natural polyhistidine affinity tag (N11, MKDHLIHNHHKEEHAHAHNKDYDIPTTHHAHHST, SEQ ID NO: 3) and a TEV protease recognition sequence at its N-terminus, and 10% (v / v) lipid surfactant mixed micelles were reacted by the P-MF method at 30°C for 6 hours in the presence of S30 extract, T7 RNA polymerase, NTPs, and an amino acid mixture to synthesize Claudin 1-containing proteoliposomes.

[0083] The reaction solution was centrifuged at 15,000 × g for 30 minutes at 4°C, and the synthesized claudin 1-containing proteoliposomes were recovered in the precipitate. The precipitate was washed with PBS / 10 mM EDTA solution (pH 7.6) (synthesis washing fraction). Next, the weight of the precipitate was measured and suspended in proteoliposome buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.6) to a concentration of 40 mg precipitate / ml.

[0084] Subsequently, lipid bilayer solubilizing polymers (SMA, SMA-EA, and DIBMA, manufactured by Funakoshi Co., Ltd.) were added to the precipitate in a ratio of 1:0.4 (w / w) and reacted at 25°C for 16 hours to convert claudin 1 into lipodiscs. The reaction mixture was centrifuged at 15,000 × g for 30 minutes at 4°C to recover the claudin 1-containing lipodiscs in the supernatant (SMA-soluble fraction).

[0085] <1-2 Purification of Claudin 1-containing lipodiscs> The Claudin 1-containing lipodiscs synthesized in 1-1 were purified using Ni Sepharose 6 Fast Flow affinity resin (Cytiva, Tokyo, Japan).

[0086] A lipodisc solution containing Claudin 1 was shaken with Ni resin at 4°C for 16 hours to bind Claudin 1 to the Ni resin. The mixture was then centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was collected (Ni pass-through fraction). The Ni resin was suspended in proteoliposome buffer and centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was collected (Ni washing fraction). The Ni resin was suspended in elution buffer (50mM Tris-HCl, 500mM NaCl, 500mM imidazole, 10% glycerol, pH 7.6) and centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was collected. This procedure was repeated four times (Ni elution fractions 1-4).

[0087] Subsequently, the Ni-eluted fraction was dialyzed against proteoliposome buffer at 4°C for 16 hours using a snakeskin dialysis tubing (Thermo Fisher Scientific, Waltham, MA, USA) to remove imidazole (dialysis fraction).

[0088] <1-3 Detection of Claudin 1 by CBB Staining> Samples from each fraction obtained in 1-1 and 1-2 were separated on 14% polyacrylamide gels containing 3% stacking gel. The gels were then immersed in CBB staining solution and shaken for 60 minutes. The staining solution was discarded, and the gels were destained by immersion in CBB destaining solution, and the bands were observed. The molecular weight was determined using a BlueStar Prestained Protein Marker (Nippon Genetics, Tokyo, Japan). As a result, it was confirmed that claudin 1 (28.7 kDa) was purified as a uniform band in the dialysis fraction (Figure 2A).

[0089] In addition, when claudin 1 was liposcillated using various lipid bilayer solubilizing polymers (SMA, SMA-EA, and DIBMA), a claudin 1 band was observed in the soluble fraction under all conditions, confirming that claudin 1 liposcillation was independent of the polymer type (Figure 3).

[0090] <1-4 Detection of Claudin 1 by Western Blotting> The claudin 1 dialyzed fraction obtained in 1-2 was separated on a 14% polyacrylamide gel containing a 3% stacking gel. The molecular weight was determined using a BlueStar Prestained Protein Marker (Nippon Genetics). The protein was transferred to a PVDF membrane using a Mini Trans-Blot Cell (Bio-Rad, Hercules, CA) at 100V for 1 hour. The PVDF membrane was blocked for 1 hour with TBS-T containing 0.1% Tween-20 and 0.3% skim milk.

[0091] After washing with TBS-T, the primary antibody [anti-claudin-1 antibody, mouse monoclonal (sc-81796, Santa Cruz Biotechnology, Santa Cruz, CA, USA)] was reacted at 4°C for 16 hours, followed by the reaction with a secondary antibody (HRP-labeled goat anti-mouse antibody, Thermo Fisher Scientific) at room temperature for 2 hours. Luminescence was detected using ImmunoStarZeta (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) and LAS-4000 (Fujifilm, Tokyo, Japan). As a result, a claudin-1 specific band was confirmed in the dialysis fraction (Figure 2B).

[0092] [Example 2: Evaluation of coating of Claudin 1-containing lipodiscs onto cell culture equipment] An ibidi 18-well plate (Nippon Genetics) was used as the cell culture equipment, and it was confirmed whether the Claudin 1-containing lipodiscs prepared in Example 1 could be coated onto the equipment.

[0093] ibidi 18 well plate (bottom area: 0.3cm 2 ) contains a claudin 1-containing lipodisc solution (dialysis fraction in Example 1, 3 μg / cm³). 2 After adding the ibidi plate coating agent and coating at 4°C for 16 hours, the plate was fixed in a PBS solution containing 4% PFA at room temperature for 15 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 at 4°C for 16 hours, 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 using a confocal microscope (LSM710, Carl-Zeiss, Oberkochen, Germany). The fluorescence intensity of the ibidi plate coating surface was quantified by densitometry analysis using the ImageJ application (National Institutes of Health, Bethesda, MD, USA).

[0094] As a result, a uniform staining pattern was observed on one surface of the ibidi plate for claudin 1 lipodiscized with various lipid bilayer solubilizing polymers (SMA, SMA-EA, and DIBMA). On the other hand, no staining pattern was observed on the ibidi plate under conditions where only the SMA polymer solution was coated (Figure 4A).

[0095] In addition, when claudin 1 protein synthesized using a cell-free membrane protein synthesis method with surfactants (S-MF method: conventional method) (Shinoda T. et al., Sci Rep., 6: 30442, 2016) was coated as a comparative measure, heterogeneous staining of claudin 1 was observed on the ibidi plate compared to the condition in which a claudin 1-containing lipodisc was coated (Figure 4B).

[0096] When the fluorescence intensity of the coated surface was quantified under both coating conditions, a 26.6-fold increase in fluorescence intensity was observed under the Claudin 1-containing lipodisc coating condition compared to the conventional method (Figure 4C).

[0097] [Example 3: Preparation of JAM-A-containing lipodiscs] <3-1 Cell-free protein synthesis and lipodisc formation of JAM-A> In vitro synthesis of JAM-A was carried out in the same manner as in Example 1, using a cell-free protein synthesis system (Cell-free-kun, Taiyo Nippon Sanso) and the cell-free protein synthesis method (P-MF method) (Shinoda T. et al., Sci Rep., 6: 30442, 2016).

[0098] Lipid surfactant mixed micelles were prepared by sonicating a mixture of 50 mg / ml lipids (47.5 mg / ml egg yolk PC and 2.5 mg / ml cholesterol) and 30 mg / ml digitonin until the solution became clear. Next, 5% (v / v) 50 ng / μl JAM-A template DNA (SEQ ID NO: 6), fused with a modified natural polyhistidine affinity tag (N11, MKDHLIHNHHKEEHAHAHNKDYDIPTTHHAHHST, SEQ ID NO: 3) and a TEV protease recognition sequence at the N-terminus, and 10% (v / v) lipid surfactant mixed micelles were reacted by P-MF at 30°C for 6 hours in the presence of S30 extract, T7 RNA polymerase, NTPs, and an amino acid mixture to synthesize JAM-A-containing proteoliposomes.

[0099] The reaction solution was centrifuged at 15,000 × g for 30 minutes at 4°C, and the synthesized JAM-A-containing proteoliposomes were recovered in the precipitate. The precipitate was washed with PBS / 10 mM EDTA solution (pH 7.6) (synthesis washing fraction). Next, the weight of the precipitate was measured and suspended in proteoliposome buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.6) to a concentration of 40 mg precipitate / ml. Then, lipid bilayer solubilizing polymer (SMA) was added to the precipitate at a ratio of precipitate:polymer = 1:0.4 (w / w), and the mixture was reacted at 25°C for 16 hours to condense JAM-A into lipodiscs. The reaction solution was centrifuged at 15,000 × g for 30 minutes at 4°C, and the JAM-A-containing lipodiscs were recovered in the supernatant (SMA-soluble fraction).

[0100] <3-2 Purification of JAM-A-containing lipodiscs> The JAM-A-containing lipodiscs synthesized in 3-1 were purified using Ni Sepharose 6 Fast Flow affinity resin (Cytiva). The JAM-A-containing lipodisc solution was shaken with Ni resin at 4°C for 16 hours to bind JAM-A to the Ni resin. Then, the solution was centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was collected (Ni pass-through fraction). The Ni resin was suspended in proteoliposome buffer and centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was collected (Ni washing fraction). The Ni resin was suspended in elution buffer (50mM Tris-HCl, 500mM NaCl, 500mM imidazole, 10% glycerol, pH 7.6) and centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was collected. This procedure was repeated four times (Ni elution fractions 1-4). Subsequently, the Ni elution fractions were dialyzed against proteoliposome buffer at 4°C for 16 hours using a snakeskin dialysis tubing (Thermo Fisher Scientific) to remove imidazole (dialysis fraction).

[0101] <3-3 Detection of JAM-A by CBB Staining> Samples from each fraction obtained in 3-1 and 3-2 were separated using 12% polyacrylamide gels containing 3% stacking gel. The gels were then immersed in CBB staining solution and shaken for 60 minutes. The staining solution was discarded, and the gels were destained by immersion in CBB destaining solution, and the bands were observed. The molecular weight was determined using a BlueStar Prestained Protein Marker (Nippon Genetics). As a result, it was confirmed that JAM-A (35.1 kDa) was purified as a uniform band in the dialysis fraction (Figure 5A).

[0102] <3-4 Detection of JAM-A by Western Blotting> The JAM-A dialysis fraction obtained in 3-2 was separated using a 12% polyacrylamide gel containing a 3% stacking gel. The molecular weight was determined using a BlueStar Prestained Protein Marker (Nippon Genetics). The protein was transferred to a PVDF membrane using a Mini Trans-Blot Cell (Bio-Rad) at 100V for 1 hour. The PVDF membrane was blocked for 1 hour with TBS-T containing 0.1% Tween-20 and 0.3% skim milk.

[0103] After washing with TBS-T, the primary antibody [anti-CD321(FR11) antibody, mouse monoclonal (14-3219-82, Thermo Fisher Scientific)] was reacted at 4°C for 16 hours, and then the secondary antibody (HRP-labeled goat anti-mouse antibody, Thermo Fisher Scientific) was reacted at room temperature for 2 hours. Luminescence was detected using ImmunoStarZeta (Fujifilm Wako Pure Chemical Industries) with LAS-4000 (Fujifilm). As a result, a JAM-A specific band was confirmed in the dialysis fraction (Figure 5B).

[0104] [Example 4: Evaluation of coating of JAM-A-containing lipodiscs onto cell culture equipment] An ibidi 18-well plate (Nippon Genetics) was used as the cell culture equipment, and it was confirmed whether the JAM-A-containing lipodiscs prepared in Example 3 could be coated onto the equipment.

[0105] ibidi 18 well plate (bottom area: 0.3 cm 2 ) contains JAM-A-containing lipodisc solution (dialysis fraction in Example 3) (3 μg / cm³ 2After adding ( ) and coating at 4°C for 16 hours, the samples were fixed in a PBS solution containing 4% PFA at room temperature for 15 minutes. After blocking in PBS containing 2% BSA for 60 minutes, the primary antibody [anti-CD321(FR11) antibody, mouse monoclonal (14-3219-82, Thermo Fisher Scientific)] was reacted at 4°C for 16 hours, 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 using a confocal microscope (LSM710, Carl-Zeiss).

[0106] As a result, a uniform staining pattern was observed on one surface of the ibidi plate for JAM-A lipodiscs formed using SMA polymer (Figure 6), confirming that this lipodiscs method is applicable to various membrane proteins.

[0107] [Example 5 Formation of tightly bound protein complexes using membrane-backed protein ZO-1] <5-1 Cell-free protein synthesis of ZO-1> In vitro synthesis of ZO-1 was carried out using a cell-free protein synthesis method with a cell-free protein synthesis system (Cell-Free-kun, Taiyo Nippon Sanso), similar to Example 1.

[0108] ZO-1 was synthesized by reacting a 5% (v / v) 50 ng / μl ZO-1 template DNA (SEQ ID NO: 8), fused with a modified natural polyhistidine affinity tag (N11, MKDHLIHNHHKEEHAHAHNKDYDIPTTHHAHHST, SEQ ID NO: 3) and a TEV protease recognition sequence at its N-terminus, with S30 extract, T7 RNA polymerase, NTPs, and an amino acid mixture at 30°C for 6 hours. The reaction solution was centrifuged at 15,000 × g for 30 minutes at 4°C, and the synthesized ZO-1 was collected in the supernatant (synthesized soluble fraction).

[0109] <5-2 Purification of ZO-1> The ZO-1 synthesized in 5-1 was purified using TARON Cobalt (Co) resin (TaKaRa Bio, Inc., Shiga, Japan). The ZO-1 solution was shaken with the Co resin at 4°C for 16 hours to bind the ZO-1 to the Co resin. Then, the mixture was centrifuged at 700×g for 5 minutes at 4°C, and the supernatant was collected (Co unpermeable fraction). The Co resin was suspended in proteoliposome buffer and centrifuged at 700×g for 5 minutes at 4°C, and the supernatant was collected (Co washing fraction). The Co resin was suspended in elution buffer (50mM Tris-HCl, 500mM NaCl, 150mM imidazole, 10% glycerol, pH 7.6) and centrifuged at 700×g for 5 minutes at 4°C, and the supernatant was collected. This procedure was repeated three times (Co elution fractions 1-3). Subsequently, the Co-eluted fraction was dialyzed against proteoliposome buffer at 4°C for 16 hours using a snakeskin dialysis tubing (Thermo Fisher Scientific) to remove imidazole (dialysis fraction).

[0110] <5-3 Detection of ZO-1 by CBB staining> Samples from each fraction obtained in 5-1 and 5-2 were separated using a 6% polyacrylamide gel containing a 3% stacking gel. The gels were then immersed in CBB staining solution and shaken for 60 minutes. The staining solution was discarded, and the gels were destained by immersion in CBB destaining solution, and the bands were observed. The molecular weight was determined using a BlueStar Prestained Protein Marker (Nippon Genetics). As a result, a band derived from ZO-1 (90.7 kDa) was confirmed in the dialysis fraction. On the other hand, the dialysis fraction also contained bands that appeared to be degradation products of ZO-1 (Figure 8A).

[0111] <5-4 Detection of ZO-1 by Western blotting> The ZO-1 dialysis fraction obtained in 5-2 was separated on a 6% polyacrylamide gel containing a 3% stacking gel. The molecular weight was determined using BlueStar Prestained Protein Marker (Nippon Genetics). The protein was transferred to a PVDF membrane using a Mini Trans-Blot Cell (Bio-Rad) at 100 V for 1 hour. The PVDF membrane was blocked with TBS-T containing 0.1% Tween-20 and 0.3% skim milk for 1 hour. After washing with TBS-T, the primary antibody [anti-ZO-1 antibody, rabbit monoclonal (D6L1E, Cell Signaling Technology, Danvers, MA, USA)] was reacted at 4 °C for 16 hours, and then the secondary antibody (HRP-labeled goat anti-rabbit antibody, Thermo Fisher Scientific) was reacted at room temperature for 2 hours. Luminescence was detected using ImmunoStarZeta (FUJIFILM Wako Pure Chemical Corporation) with a LAS-4000 (FUJIFILM). As a result, a ZO-1-specific band was confirmed in the dialysis fraction (Figure 8B).

[0112] [Example 6 Evaluation of co-coating of ZO-1, claudin 1, and JAM-A-containing lipid disks on cell culture equipment] As cell culture equipment, an ibidi 18 well plate (Nippon Genetics) was used to confirm whether the claudin 1, JAM-A-containing lipid disks, and ZO-1 prepared in Examples 1, 3, and 5, respectively, could be co-coated on the equipment.

[0113] An ibidi 18 well plate (bottom area: 0.3 cm 2 ) was added with a ZO-1 solution (dialysis fraction, 3 μg / cm 2 ) and coated at 4 °C for 16 hours. Then, a claudin 1-containing lipid disk solution (dialysis fraction, 3 μg / cm 2 ) or a JAM-A-containing lipid disk solution (dialysis fraction, 3 μg / cm 2 ) was added and coated at 4 °C for 16 hours. Then, it was fixed with a PBS solution containing 4% PFA at room temperature for 15 minutes.

[0114] Next, after blocking with PBS containing 2% BSA for 60 minutes, the primary antibody [anti-claudin-1 antibody, mouse monoclonal (sc-81796, Santa Cruz Biotechnology), anti-CD321 (FR11) antibody, mouse monoclonal (14-3219-82, Thermo Fisher Scientific), or anti-ZO-1 antibody, rabbit monoclonal (D6L1E, Cell Signaling Technology)] was reacted at 4°C for 16 hours, followed by a reaction with a secondary antibody (fluorescently labeled goat anti-mouse or rabbit antibody, Thermo Fisher Scientific) at room temperature for 1 hour. Fluorescence was observed using a confocal microscope (LSM710, Carl-Zeiss).

[0115] As a result, co-stained images of Claudin 1 and ZO-1 (Figure 9A) and JAM-A and ZO-1 (Figure 9B) were confirmed across the entire surface of the equipment, demonstrating that the prepared ZO-1, Claudin 1, and JAM-A-containing lipodiscs can be co-coated onto the equipment.

[0116] [Example 7: Evaluation of binding affinity of ZO-1, Claudin 1, and JAM-A] To evaluate whether ZO-1 prepared in Example 5 can bind to the Claudin 1 and JAM-A-containing lipodiscs prepared in Examples 1 and 3, a binding test was performed by co-immunoprecipitation.

[0117] After mixing a ZO-1 (dialysis fraction, 0.5 μg) solution with Dynabeads Protein G (Thermo Fisher Scientific), the mixture was shaken at room temperature for 10 minutes to bind ZO-1 to the Dynabeads. Subsequently, the ZO-1-bound Dynabeads or unbound Dynabeads were mixed with a Claudin 1-containing lipodisc (dialysis fraction, 0.5 μg) solution or a JAM-A-containing lipodisc (dialysis fraction, 0.5 μg) solution and shaken at room temperature for 1 hour to bind ZO-1 to Claudin 1 and JAM-A.

[0118] Subsequently, Dynabeads were mixed with SDS sample buffer [48 mM Tris-HCl, 1.6% (w / v) SDS, 8% (v / v) glycerol, 0.02% (w / v) bromophenol blue, 4% (v / v) 2-mercaptoethanol, pH 6.8], and the binding protein was eluted (immunoprecipitation fraction).

[0119] The immunoprecipitation fraction was separated on a 12% polyacrylamide gel containing a 3% stacking gel. Molecular weight was determined using a BlueStar Prestained Protein Marker (Genetics Japan). Proteins were transferred to a PVDF membrane using a Mini Trans-Blot Cell (Bio-Rad) at 100V for 1 hour. The PVDF membrane was blocked for 1 hour with TBS-T containing 0.1% Tween-20 and 0.3% skim milk. After washing with TBS-T, primary antibodies [anti-claudin-1 antibody, mouse monoclonal (sc-81796, Santa Cruz Biotechnology), anti-CD321 (FR11) antibody, mouse monoclonal (14-3219-82, Thermo Fisher Scientific), anti-ZO-1 antibody, rabbit monoclonal (D6L1E, Cell Signaling Technology)] were reacted at 4°C for 16 hours. Subsequently, secondary antibodies (HRP-labeled goat anti-mouse or rabbit antibody, Thermo Fisher Scientific) were reacted at room temperature for 2 hours, and luminescence was detected using ImmunoStarZeta (Fujifilm Wako Pure Chemical Industries) with LAS-4000 (Fujifilm).

[0120] As a result, compared to the condition in which claudin 1 or JAM-A was applied to Dynabeads without ZO-1 binding, the band intensity of claudin 1 and JAM-A in the immunoprecipitation fraction of Dynabeads with ZO-1 binding increased by 6.64 times and 23.0 times, respectively, when claudin 1 or JAM-A was applied (Figures 10A and 10B). Therefore, the binding of the prepared lipodiscs containing ZO-1, claudin 1, and JAM-A was confirmed.

[0121] [Example 8: Localization of tight junction proteins in human liver chimeric mouse PXB-cells] To verify whether plates coated with claudin 1, JAM-A, and ZO-1 (tight junction protein) prepared in Examples 2, 4, and 6 can interact with hepatocyte-derived tight junction proteins, the cellular localization of claudin 1, JAM-A, and ZO-1 in human liver chimeric mouse PXB-cells was evaluated.

[0122] T25 flasks seeded with PXB cells were purchased from PhoenixBio (Hiroshima, Japan). The flasks were treated with 0.25% trypsin-EDTA / PBS at 37°C for 15 minutes, and the PXB cells were recovered from the T25 flasks and suspended in 2% DMSO-supplemented hepatocyte clonal growth medium (dHCGM) (manufactured by PhoenixBio Co., Ltd.). The recovered cell suspension was centrifuged at 100×g for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in fresh dHCGM. Cell counts were determined by Trypan blue staining. PXB cells (0.63×10⁶) 5 The suspension of cells was centrifuged at 500×g for 2 minutes at 4°C and pelletized. The cells were then fixed in PBS solution containing 4% PFA at room temperature for 15 minutes.

[0123] Next, the cells were blocked for 60 minutes with PBS containing 2% BSA and 0.2% Triton X-100. Then, the cells were reacted with primary antibodies [anti-claudin-1 antibody, mouse monoclonal (sc-81796, Santa Cruz Biotechnology), anti-CD321 (FR11) antibody, mouse monoclonal (14-3219-82, Thermo Fisher Scientific), or anti-ZO-1 antibody, rabbit monoclonal (D6L1E, Cell Signaling Technology)] at 4°C for 16 hours. Subsequently, secondary antibodies (fluorescently labeled goat anti-mouse or rabbit antibody, Thermo Fisher Scientific) were reacted at room temperature for 1 hour. Fluorescence was observed using a confocal microscope (LSM710, Carl-Zeiss).

[0124] As a result, it was confirmed that claudin 1 is localized in the cytoplasm of PXB cells and is concentrated at cell adhesion sites on the cell membrane only when adhering to adjacent cells (Figure 11A). On the other hand, JAM-A is localized in both the cell membrane and cytoplasm of PXB cells (Figure 11B), and ZO-1 is localized in the cytoplasm directly beneath the cell membrane (Figure 11C). Therefore, it was suggested that claudin 1 and JAM-A expressed in PXB cells can interact with the tightly bonded coated plate that was prepared.

[0125] [Example 9: Induction of open bile duct lumen formation in PXB-cells on a tightly bound protein-coated plate] Claudin 1, JAM-A, and coated plates (0.3 cm) prepared in Examples 2, 4, and 6, along with ZO-1. 2 ) Place PXB-cells on top of 2.13 × 10 5 cells / cm 2 Seeds were seeded at the specified density. After incubation at 37°C under 5% CO2 for 24 hours, the medium was changed to ice-cold Matrigel-containing dHCGM and cultured for a further 2 days. On day 3 of culturing the PXB-cells, the cells were fixed in PBS solution containing 4% PFA at room temperature for 15 minutes.

[0126] Next, the cells were blocked for 60 minutes with PBS containing 2% BSA and 0.2% Triton X-100. Then, primary antibodies [anti-MRP2 antibody, mouse monoclonal (GTX23373, Gene Tex, Irvine, CA, USA), anti-ZO-1 antibody, rabbit monoclonal (D6L1E, Cell Signaling Technology)] were reacted at 4°C for 16 hours, followed by secondary antibodies (fluorescently labeled goat anti-mouse or rabbit antibody, Thermo Fisher Scientific) reacted at room temperature for 1 hour. Fluorescence was observed using a confocal microscope (LSM710, Carl-Zeiss).

[0127] As a result, staining of the bile duct lumen marker MRP2, which was open on the equipment side, was confirmed, accompanied by concentration of the tightly bound marker ZO-1 at the adhesion surface between PXB-cells and the equipment. The inventors have decided to simply call this hepatocyte culture system with an open bile duct lumen (induced open-form bile canaliculus hepatocyte) "icHep". On the other hand, when PXB-cells were cultured on a collagen-coated plate, the bile duct lumen was formed as a closed system between adjacent cells (Figure 12A).

[0128] Next, the bile duct lumen opening rate was calculated for icHep constructed using plates coated with various combinations of Claudin 1, JAM-A, and ZO-1. The bile duct lumen opening rate was calculated using the Z-stack function of a confocal microscope (LSM710, Carl-Zeiss), by visually determining the number of open bile duct lumens and dividing by the total number of bile duct lumens.

[0129] As a result, the bile duct lumen opening rate of icHep constructed by coating with various tight-binding proteins was significantly increased compared to the normal collagen-coated conditions. Furthermore, icHep constructed using the coated plates prepared in Examples 2, 4, and 6 showed a bile duct lumen opening rate equivalent to or better than that of icHep constructed by the conventional method (S-MF method). On the other hand, under conditions where tight-binding proteins and collagen were co-coated, the bile duct lumen opening rate tended to decrease compared to conditions without collagen (Figure 12B).

[0130] [Example 10 Evaluation of drug biliary excretion by permeability test using icHep] icHep forms an open bile duct lumen at the interface between hepatocytes and tight-junction protein-coated culture media, making it suitable for applications in drug biliary excretion studies using permeability tests (Figure 13A). Therefore, we evaluated the biliary excretion of E217βG, a typical substrate of the biliary excretion transporter MRP2, by a permeability test.

[0131] Claudine 1 or JAM-A coated Transwell (0.3cm) prepared in the same manner as in Examples 2 and 4. 2 ) Place PXB-cells on top of 2.13 × 10 5 cells / cm 2 icHep was constructed by seeding at the specified density. After incubation for 24 hours at 37°C under 5% CO2, the medium was changed to ice-cold Matrigel-containing dHCGM and cultured for a further 2 days. icHep on day 3 of culture was used for permeability testing. icHep was pre-incubated with 10 mM HEPES-containing HBSS at 37°C for 15 minutes.

[0132] To calculate the permeation clearance of the test compound, 1 μM E217βG and 25 μM TD4 were added to the blood chamber to initiate the permeation test. icHep was incubated in a 37°C water bath, and permeation samples were collected from the bile chamber after 30, 60, 90, and 120 minutes, and the same amount of 10 mM HEPES-containing HBSS was added.

[0133] The permeation clearance was calculated by dividing the permeation rate of the test compound into the bile chamber by the initial concentration of the test compound in the blood chamber. The concentration of TD4 was measured using a fluorescence plate reader (1420 ARVO MX / Light, PerkinElmer, Waltham, MA, USA) at an excitation wavelength of 550 nm and an fluorescence wavelength of 572 nm. The concentration of E217βG was measured using LC / MS-MS (LCMS-8050, Shimadzu, Kyoto, Japan).

[0134] The degree of intercellular barrier formation in icHep constructed using plates coated with various combinations of Claudin 1 and JAM-A was evaluated by permeability tests of the intercellular transport marker TD4.

[0135] As a result, all icHep structures constructed under different conditions showed equivalent TD4 permeability to the bile chamber, indicating that they possess equivalent barrier functions (Figure 13B). Next, bile excretion of E217βG, a typical substrate of the bile excretion transporter MRP2, was evaluated using icHep through a permeability test.

[0136] As a result, compared to culture conditions coated only with collagen, a significant increase in E217βG permeation clearance into the bile chamber was observed in icHep constructed under all conditions. The E217βG permeation clearance of icHep was reduced to a level comparable to that of the collagen-coated condition by treatment with the MRP2 inhibitor (benzbromarone). Therefore, it was demonstrated that drug excretion into the bile can be evaluated by permeation tests using icHep constructed with the culture equipment of the present invention. Furthermore, icHep constructed with the culture equipment of the present invention showed E217βG permeation clearance equivalent to or better than icHep constructed by the conventional method (S-MF method) (Figure 13C).

[0137] [Example 11 Evaluation of icHep constructed under different coating conditions] In the same manner as in Examples 9 and 10, transmission tests for TD4 and E217βG were performed on icHep constructed under various coating conditions. Transmission coefficient (P app ) is the permeation clearance of the test compound to the bile-side chamber, measured by the surface area of ​​the Transwell insert membrane (0.3 cm²). 2The result was calculated by dividing by ). As a result, the degree of intercellular transport was similar under various coating conditions (Figure 14A). On the other hand, under conditions coated with scaffold protein ZO-1, claudin, and JAM-A, an improvement in MRP2 activity of approximately three times was observed compared to conditions coated with collagen (Figure 14B). This suggests that the scaffolding action of ZO-1 increased the total number of claudin-1 and JAM-A capable of interacting with hepatocytes, thereby promoting the formation of an open bile duct lumen.

[0138] The present invention enables the uniform coating of membrane proteins onto culture materials for the evaluation of drug metabolism and / or membrane transport by permeability testing, thereby providing a more useful evaluation system. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for producing culture equipment coated with a membrane protein, comprising the steps of: synthesizing a membrane protein in vitro; forming a lipodisc containing the synthesized membrane protein, lipids, and a lipid bilayer stabilizing polymer; and coating the lipodisc onto the surface of the culture equipment.

2. The method according to claim 1, wherein the membrane protein is a cell adhesion protein.

3. The method according to claim 2, wherein the membrane protein is selected from claudin, junction adhesion molecule-A (JAM-A), and combinations thereof.

4. The method according to claim 3, wherein the lipodisc is coated onto the surface of the culture equipment together with the ZO protein.

5. The method according to claim 1, wherein the polymer is selected from styrene-maleic acid (SMA) copolymer, diisobutylene maleic acid (DIBMA) copolymer, and as an SMA derivative, styrene-maleic acid-ethanolamine (SMA-EA), ethylenediamine-modified styrene-maleic anhydride copolymer modified with Ethylene Diamine (SMA-ED and SMAd-A), and styrene-maleimide quaternary ammonium (SMA-QA) copolymer.

6. Cell culture equipment manufactured by the method described in any one of claims 1 to 5.

7. Cell culture equipment with a JAM-A protein layer formed on its surface.

8. Cell culture equipment having a layer of a complex composed of claudin protein, JAM-A protein, and ZO protein formed on its surface.

9. A protein complex comprising claudin protein or fragments thereof containing claudin extracellular loops 1 and 2, JAM-A protein or fragments thereof containing the JAM-A extracellular domain, and ZO-1 protein or fragments thereof containing the PDZ1 to SH3 domain of ZO-1.

10. A method for producing culture equipment, characterized by coating the surface of the culture equipment with a layer containing the protein complex described in claim 9.

11. A kit for evaluating drug efflux into bile, comprising the culture equipment described in claim 6 and liver-derived cultured cells.

12. A method for preparing a drug efflux evaluation system into bile, characterized by forming a liver-derived cell layer on the culture material described in claim 6.

13. A method for forming a liver-derived cell layer on a culture device according to claim 6, and inducing bile duct lumen formation at the contact site between the cells and the surface of the device.

14. A method for evaluating drug metabolism and / or membrane transport in liver-derived cells, characterized by forming a layer of liver-derived cells on the culture material described in claim 6, and evaluating the drug released from the liver-derived cells through the culture material.

15. A kit for evaluating drug efflux into bile, comprising the culture equipment described in claim 7 or 8 and liver-derived cultured cells.

16. A method for preparing a drug efflux evaluation system into bile, characterized by forming a liver-derived cell layer on the culture material according to claim 7 or 8.

17. A method for forming a liver-derived cell layer on a culture device according to claim 7 or 8, and inducing bile duct lumen formation at the contact site between the cells and the surface of the device.

18. A method for evaluating drug metabolism and / or membrane transport in liver-derived cells, characterized by forming a liver-derived cell layer on a culture material according to claim 7 or 8, and evaluating the drug discharged from the liver-derived cells through the culture material.

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