Method for culturing hepatocytes, method for forming liver tissue, and liver tissue using same

Abstract

A hepatocyte culture method for culturing hepatocytes using a liquid medium and a substrate on which a fusion protein of E-cadherin and an Fc region of an immunoglobulin is immobilized, the hepatocyte culture method being characterized by the application of a flow velocity to the liquid medium.

Description

Hepatocyte culture method, liver tissue formation method, and liver tissue using the same

[0001] The present invention relates to a method for culturing hepatocytes, a method for forming hepatic tissue, and hepatic tissue using the same, and in particular to a method for culturing hepatocytes and forming hepatic tissue on a substrate, and hepatic tissue formed by these methods.

[0002] Conventionally, culture substrates and cell culture methods for culturing cells on a substrate have been known (see, for example, Patent Document 1). The cell culture substrate described in Patent Document 1 is a substrate having a fusion protein (E-Cad-Fc) of E-cadherin and the Fc region of immunoglobulin immobilized on its surface. Patent Document 1 describes a method in which primary cultured hepatocytes are isolated from the liver tissue of male ICR mice (6 to 8 weeks, SLC) using this substrate, and the isolated cells are then spread at a density of 3 × 10 cells on the surface of a substrate made of polystyrene douche coated with the E-Cad-Fc at a concentration of 7.5 μg / ml. ４ cells / cm ２ The method disclosed involves seeding the cells at a density of 1000×1000×1000 and removing the medium after 4 hours to remove non-adherent cells and dead cells.

[0003] JP 2013-126405 A

[0004] However, the cell culture substrate and culture method described in Patent Document 1 have a problem in forming a monolayer of hepatocytes without aggregation and without gaps. Therefore, there has been a demand for a hepatocyte culture method, a method for forming liver tissue, and liver tissue using the same that can form a monolayer of hepatocytes without aggregation and without gaps.

[0005] To achieve the above object, the present invention provides the following inventions [1] to [8], which relate to a method for culturing hepatocytes, a method for forming hepatic tissue, and hepatic tissue using the same. [1] A method for culturing hepatocytes using a substrate on which a fusion protein of E-cadherin and the Fc region of immunoglobulin is immobilized and a liquid medium, the method comprising applying a flow rate to the liquid medium. [2] The method for culturing hepatocytes described in [1], characterized in that the flow rate is 50 μL / h or more. [3] The method for culturing hepatocytes described in [1] to [2], characterized in that the coating concentration when immobilizing the fusion protein on the substrate is adjusted to 0.3 to 5 μg / ml. [4] A method for forming hepatic tissue, characterized in that after hepatocytes are seeded on a substrate on which a fusion protein of E-cadherin and the Fc region of immunoglobulin is immobilized, a flow rate is applied to the liquid medium starting four days later. [5] The method for forming hepatic tissue described in [4], characterized in that the flow rate is 50 μL / h or more. [6] The method for forming liver tissue according to [4] to [5], wherein the coating concentration when the fusion protein is immobilized on a substrate is adjusted to 0.3 to 5 μg / ml. [7] Liver tissue formed by the method according to any one of [4] to [6]. [8] A method for producing liver tissue, comprising the steps of culturing hepatocytes using a substrate on which a fusion protein of E-cadherin and an immunoglobulin Fc region is immobilized and a liquid medium, and applying a flow rate to the liquid medium.

[0006] According to the present invention, as described above, it is possible to prevent the occurrence of gaps and aggregation in the hepatocyte layer formed on the surface of a substrate, thereby preventing the decline in liver tissue function. This allows the formation of liver tissue with three important functions: metabolism, detoxification, and bile production and secretion. Furthermore, in fields such as toxicity testing, particularly in non-clinical drug safety evaluations using animal experiments and primary human stem cells (PHH), it is possible to overcome problems in the reconstruction of liver tissue, such as differences in drug metabolism between experimental animals and humans, differences between PHH lots, and the difficulty of long-term culture.

[0007] 1 is a diagram showing the configuration of an apparatus for imparting a flow rate to a culture medium in the hepatocyte culture method of the present invention.

[0033] FIG. 1 is a cross-sectional view of a container used in a method for immobilizing a fusion protein of E-cadherin and the Fc region of immunoglobulin on a substrate used in the hepatocyte culture method of the present invention.

[0034] FIG. 2 is a diagram showing an embodiment of the hepatocyte culture method of the present invention.

[0035] FIG. 3 is a micrograph showing the state of hepatocytes depending on the coating concentration of the fusion protein in the hepatocyte culture method of the present invention.

[0036] FIG. 4 is a graph showing the results of ELISA assays for sinusoidal transporters (NTCP), biliary transporters (ABCB1, ABCC3, and ABCG2), and cell-cell adhesion (E-cadherin and ZO1) in liver tissue formed by the method for forming liver tissue of FIG. 4.

[0037] FIG. 5 is a diagram showing the results for mature hepatocyte markers and drug-metabolizing enzymes in liver tissue formed by the embodiment shown in FIG. 3.

[0038] FIG. 6 is a micrograph showing the state of HepG2 cells when cultured on a 35 mm dish coated with E-cadherin-Fc at concentrations of 0 to 10 μg / ml. Figure 1 shows micrographs of HepG2 cells cultured on 35-mm dishes coated with E-cadherin-Fc at concentrations of 0 to 10 μg / ml, as well as the results of real-time PCR (RT-qPCR) measurement of E-Cadherin and ZO-1 expression levels. Figure 2 shows the results of confirming the polarity (sinusoidal and bile canalicular sides) of hepatocytes in liver tissue by visual inspection and intensity measurement of fluorescence intensity using CDF-DA.

[0008] The present invention relates to a method for culturing hepatocytes using a liquid medium and a substrate on which a fusion protein of E-cadherin and the Fc region of immunoglobulin is immobilized, and the method is characterized in that a flow rate is applied to the liquid medium. Specific embodiments of the present invention are described below.

[0009] [Definitions] As used herein, the term "liquid medium" includes all liquid media that can be applied to conventional methods for co-culturing hepatocytes or hepatocytes with other cells.

[0010] [Method for Culturing Hepatocytes] The method for culturing hepatocytes of the present invention will be described.

[0011] The cell culture substrate is not particularly limited as long as it is a material capable of culturing cells, but examples include dishes (also called culture plates), petri dishes and plates (microtiter plates with 6, 24, 48, 96, 384, or 9600 holes, microplates, deep-well plates, etc.), flasks, chamber slides, tubes, cell factories, roller bottles, spinner flasks, phorofibers, microcarriers, beads, etc. These substrates may be made of either inorganic materials such as glass or organic materials such as polystyrene. However, materials with high adsorption properties for proteins, peptides, etc., such as polystyrene, polyethylene, or polypropylene, or materials that have been treated to enhance adsorption, such as hydrophilic or hydrophobic treatments, are preferred. Furthermore, the substrate may be solid or gel-like. Because hepatocytes consume a large amount of oxygen, it is preferable to use an oxygen-permeable material.

[0012] Furthermore, it is preferable that the substrate is made of a material that is sterilizable, heat-resistant, and water-resistant. An example of a suitable substrate for this purpose is a polystyrene dish and / or plate that has not been specially treated for cell culture (hereinafter referred to as an untreated polystyrene plate), which is frequently used mainly for culturing Escherichia coli and the like, and such a culture substrate is generally commercially available.

[0013] The fusion protein immobilized on the surface of the substrate is a fusion protein of E-cadherin and the Fc region of immunoglobulin (hereinafter referred to as the fusion protein). Cadherin is a Ca-cadherin that binds to the Fc region of an immunoglobulin called an adherens junction. ２＋Cadherin is an adhesion molecule involved in cell-cell adhesion and junction dependent on the cellular cellular fate, and the E (epithelial) type is used in the present invention. This cadherin molecule is a membrane-bound glycoprotein molecule consisting of 700 to 750 amino acid residues, and its extracellular region contains five repeating structures of approximately 110 amino acid residues, known as extracellular cadherin (EC) domains. For example, in the case of human E-cadherin (the amino acid sequence of which is shown in SEQ ID NO: 1), the EC1, EC2, EC3, EC4, and EC5 domains correspond to residues 157-262, 265-375, 378-486, 487-595, and 596-700, respectively (the numbers represent the residue numbers in the amino acid sequence shown in SEQ ID NO: 1). In the case of mouse E-cadherin (the amino acid sequence of which is shown in SEQ ID NO: 2), the EC1, EC2, EC3, EC4, and EC5 domains correspond to 159-264, 267-377, 380-488, 489-597, and 598-702, respectively (the numbers indicate the residue numbers in the amino acid sequence shown in SEQ ID NO: 2). These EC domains share homology among different cadherin molecular species, with the N-terminal domains (EC1 and EC2) being particularly highly homologous.

[0014] E-cadherin (also known as cadherin-1) is widely expressed in parenchymal cells of visceral organs such as the liver, kidney, and lung, and in epithelial cells such as keratinocytes, and is known to be an important adhesion molecule responsible for cell-cell adhesion (for reviews, see Mareel et al., Int. J. Dev. Biol. 37:227, 1993; Mays et al., Cord Spring Herb. Symp. Quant. Biol. 60:763, 1995; El-Bahrawy & Pignatelli, Microsc. Res. Tech. 43:224, 1998; Nollet et al., Mol. Cell. Biol. Res. Commun. 2:77, 1999). Furthermore, E-cadherin is also strongly expressed in undifferentiated mouse ES cells, and it is known that ES cells in which the expression of the E-cadherin gene has been genetically deleted exhibit significantly impaired cell-cell adhesion (Larue et al., Development 122:3185, 1996). Furthermore, information registered in the public gene expression database of the U.S. National Center for Biotechnology Information (NCBI) confirms that the E-cadherin gene is also expressed in human ES cell lines.

[0015] The method for producing a protein belonging to the cadherin family is not particularly limited, but it is preferable to produce and purify a recombinant protein using molecular biological techniques and use the purified protein. Any other method that has a similar effect can be used. For example, a protein belonging to the cadherin family of pluripotent stem cells can be extracted from biological tissues or cells, purified, and used, or the peptide can be chemically synthesized and used.

[0016] Standard protocols for producing recombinant proteins of cadherin family proteins and for obtaining genes encoding such molecules have already been established, and practitioners can refer to the reference books listed above or a genetic engineering manual (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press (2012)), but are not limited thereto. Taking E-cadherin as an example, the E-cadherin gene has already been isolated and identified in animals such as humans (SEQ ID NO: 1), mice (SEQ ID NO: 2), and rats, and its nucleotide sequence is available in public DNA databases such as NCBI (Accession Nos.: (Human) NM_004360; (Mouse) NM_009864; (Rat) NM_031334). Therefore, those skilled in the art can obtain and use cDNA of the E-cadherin gene by designing primers or probes specific to the E-cadherin gene and using standard molecular biology techniques. Furthermore, E-cadherin gene cDNA can also be purchased from the RIKEN Gene Bank (Tsukuba, Japan), the American Type Culture Collection (ATCC), Invitrogen / ResGen, etc. The gene encoding a protein belonging to the cadherin family used is preferably derived from an animal of the same species as the species from which the pluripotent stem cells are derived. For example, when practicing the present invention using mouse ES cells, it is desirable to use mouse E-cadherin cDNA. However, E-cadherin cDNA derived from a different animal species, i.e., from humans, monkeys, cows, horses, pigs, sheep, birds (e.g., chickens), or amphibians (e.g., Xenopus laevis), can also be used.

[0017] One suitable method for producing a recombinant protein of a cadherin family protein involves introducing a gene encoding the molecule into mammalian cells such as COS cells, 293 cells, or CHO cells and expressing the gene. Preferably, the gene is linked to a nucleic acid sequence, i.e., a promoter sequence, that enables gene transcription and expression in a wide range of mammalian cells, in a manner that enables transcription and expression under the control of the promoter. Furthermore, the gene to be transcribed and expressed is preferably further linked to a poly(A) addition signal. Suitable promoters include promoters derived from viruses such as SV (Simian Virus) 40, cytomegalovirus (CMV), and Rous sarcoma virus, as well as the β-actin promoter and EF (Elongation Factor) 1α promoter.

[0018] The gene used to produce the above-described recombinant protein does not need to contain the full-length region of the gene encoding the molecule; even a partial gene sequence can be used, as long as the protein or peptide molecule encoded by the partial sequence has adhesive activity comparable to or greater than that of the original molecule. For example, in the case of E-cadherin, which is used in a preferred embodiment of the present invention, a recombinant protein produced from a partial sequence encoding the extracellular domain, comprising 690 to 710 amino acid residues from the N-terminus, i.e., a protein comprising the EC1 to EC5 domains, can be used. Furthermore, since the domain (EC1) located most N-terminally of a cadherin molecule generally determines the binding specificity, i.e., homophilicity, of the molecule (Nose et al., Cell 61:147, 1990), it is also possible to produce and use a protein molecule that contains at least EC1 but lacks one or more of the other domains. Furthermore, proteins that exhibit homology of 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more with the above-mentioned protein molecules at the amino acid level and have adhesive activity can also be used.

[0019] The above-mentioned recombinant proteins can also be prepared as fusion proteins with other proteins or peptides. For example, they can be prepared as fusion proteins with the Fc region of immunoglobulin, GST (Glutathione-S-Transferase) protein, MBP (Mannose-Binding Protein) protein, avidin protein, His (oligohistidine) tag, HA (HemAgglutinin) tag, Myc tag, or VSV-G (Vesicular Stromatitis Virus Glycoprotein) tag. Purification of the recombinant proteins can be easily and efficiently performed using a Protein A / G column or a specific antibody column. Fc fusion proteins are particularly suitable for implementing the present invention because of their enhanced ability to adsorb to culture substrates made from materials such as polystyrene.

[0020] Numerous genes encoding the Fc region of immunoglobulins have already been isolated and identified in mammals, including humans. Numerous nucleotide sequences have also been reported. For example, sequence information for the Fc regions of human IgG1, IgG2, IgG3, and IgG4 is available in public DNA databases such as the NCBI, and is registered under accession numbers AJ294730, AJ294731, AJ294732, and AJ294733, respectively. Therefore, those skilled in the art can design primers or probes specific to the Fc region and obtain and use cDNA encoding the Fc region portion using standard molecular biology techniques. In this case, the gene encoding the Fc region to be used is not particularly limited in terms of animal species or subtype, but genes encoding the Fc region of human IgG1 or IgG2, or mouse IgG2a or IgG2b, which have strong binding to Protein A / G, are preferred. Furthermore, a method for increasing binding affinity to Protein A by introducing a mutation into the Fc region is also known (Nagaoka et al., Protein Eng. 16:243, 2003), and Fc proteins genetically modified by this method can also be used.

[0021] In the case of E-cadherin, which is preferably used to carry out the present invention, reference can be made to, for example, Nagaoka et al., Biotechnol. Lett. 24:1857, 2002; Protein Eng. 16:243, 2003, as an example of a method for producing the recombinant protein.

[0022] Furthermore, a purified recombinant protein (Recombinant Human / Mouse E-cadherin-Fc Chimera; R&D Systems, Genzyme Techne) is commercially available, which is prepared by introducing a fusion gene into mouse cells, in which cDNA encoding the extracellular region of mouse or human E-cadherin is linked to cDNA encoding a sequence encoding the Fc region portion of human IgG and a His tag sequence, and expressing the fusion gene. This protein can also be used as a mouse- or human-derived E-cadherin protein (E-cad-Fc protein).

[0023] The fusion protein can be immobilized or coated on the solid surface of the substrate by physical methods such as adsorption or chemical methods such as covalent bonding, but adsorption is preferred due to its ease of operation. Adsorption can be achieved by contacting a solution containing the fusion protein with the substrate surface for a certain period of time, preferably several hours to one day and night, more preferably 1 hour to 12 hours. Alternatively, an antigenic molecule can be artificially attached and fused to the adhesive molecule in advance, and then binding of the antigenic molecule with a specific antibody can be utilized. In this case, the specific antibody must be immobilized or coated on the solid surface of the culture substrate in advance by physical methods such as adsorption or chemical methods such as covalent bonding.

[0024] In carrying out the method disclosed in the present invention, physical methods such as adsorption and chemical methods such as covalent bonding can be used to immobilize or coat the fusion protein on the solid surface of the culture substrate. However, adsorption is preferred due to its ease of operation. When the adhesive molecule is a proteinaceous or peptide molecule, the molecule can be easily adsorbed onto the solid surface of a culture substrate such as a plate by contacting the molecule solution with the surface and removing the solvent after a certain period of time. More specifically, for example, a solution of the adhesive molecule in a solvent such as distilled water or PBS can be filtered and sterilized, and then contacted with a culture substrate such as a plate. The resulting solution can be left for several hours to overnight to obtain a cell culture substrate on which the adhesive molecule is immobilized or coated. Preferably, the substrate is washed several times with distilled water or PBS, and then replaced with a balanced salt solution such as PBS before use.

[0025] Furthermore, when an antigenic molecule is artificially added and fused to an adhesive molecule in advance, binding with a specific antibody to the antigenic molecule can be utilized, which is more preferable because it allows the adhesive molecule to be efficiently modified onto the substrate surface. In this case, the specific antibody must be immobilized or coated onto the surface of the culture substrate in advance by a physical method such as adsorption or a chemical method such as covalent bonding. For example, in the case of a recombinant protein in which an IgG Fc region protein is fused to an adhesive molecule, an antibody that specifically recognizes the IgG Fc region can be used as the antibody to be pre-modified onto the culture substrate. In the case of a recombinant protein in which an adhesive molecule is fused to various proteins or tag sequence peptides, the culture substrate can be used by pre-modifying the culture substrate with an antibody specific to the fused molecule.

[0026] The concentration of the fusion protein solution needs to be determined appropriately depending on the adsorption amount and / or affinity of the protein, as well as the physical properties of the protein. In the case of a fusion protein that is a recombinant protein in which an Fc region is fused to the extracellular region of E-cadherin, the concentration ranges from about 0.3 to 10 μg / ml.

[0027] If the concentration of the fusion protein solution is lower than this range, the state of the substrate surface will be similar to that without the fusion protein immobilized, making it difficult to immobilize hepatocytes on the substrate surface in a monolayer. On the other hand, if the amount is higher than this range, aggregation may occur in the hepatocyte layer, which is undesirable. Therefore, from the perspectives of immobilizing hepatocytes on the substrate, forming a monolayer hepatocyte layer, and suppressing aggregation, the concentration of the fusion protein solution is preferably about 0.3 to 5 μg / ml, more preferably 0.5 to 3 μg / ml, and most preferably 0.8 to 2 μg / ml.

[0028] [Hepatocytes] The hepatocytes used in the present invention can be primary hepatocytes isolated from humans or mice, cell lines derived from liver cancer, or cells derived from pluripotent stem cells that have been induced to differentiate into hepatocytes.

[0029] Primary hepatocytes were isolated from the liver tissue of male ICR mice (6-8 weeks, SLC) by a two-step in situ collagenase perfusion method according to the method described in Seglen PO., Exp. Cell Res. 1973; 82: 391-398 and Ise H, Sugihara N, Negishi N, Nikaido T, Akaike T., Biochemical and biophysical research communications. 2001; 285: 172-182. Specifically, mice were anesthetized with pentobarbital, the portal vein was cannulated, and the liver was perfused with CaCl2 containing EGTA. ２＋ The tissue was perfused with a free pre-perfusion solution (pH 7.4) followed by a collagenase solution (pH 7.4) containing trypsin inhibitor (0.05 mg / mL, Wako) and collagenase (0.15 mg / mL, Wako). The perfused tissue was separated using Hank's solution and collected using a 100 μm nylon cell strainer (BD Falcon). Hepatocytes were purified by density gradient centrifugation (50 × g, 10 minutes, 4 °C) using 10% Percoll solution (GE Healthcare Bioscience).

[0030] Furthermore, the hepatocytes of the present invention may be cells derived from pluripotent stem cells and induced to differentiate into hepatocytes. Examples of the pluripotent stem cells include ES cells, ntES cells, iPS cells, and mesenchymal stem cells.

[0031] Taking the differentiation into hepatocytes using stem cells as an example, hepatocytes can be obtained by differentiating ES cells in the following order: mesendoderm (endodermal cells), definitive endoderm (endodermal cells), hepatic progenitor cells (liver precursor cells), and hepatocytes (liver cells). Differentiation into hepatocytes is carried out using differentiation-inducing factors such as Activin A, Nodal, bFGF (basic fibroblast growth factor), HGF (hepatocyte growth factor), OSM (oncostatin M), DEX (dexamthasone), EGF (epidermal growth factor), and TGF-α (transforming growth factor-α), but other factors may also be used according to techniques described in publicly known literature. Differentiation into cells other than hepatocytes, such as sinusoidal endothelial cells, can also be induced by using differentiation-inducing factors necessary for differentiation into the respective cells.

[0032] [Cell Culture Method] The cell culture method of the present invention is characterized by culturing and growing hepatocytes using the cell culture substrate and a liquid medium, and by applying a flow rate to the medium.

[0033] Hepatocytes can be cultured on the surface of a substrate immobilized or coated with a fusion protein using conventional culture methods. Specifically, an appropriate number of hepatocytes can be suspended in a commonly used liquid medium or cell culture solution and added to the substrate (i.e., seeding). Subsequent changes of the liquid medium and subcultures can be performed in the same manner as conventional methods. In this case, the present invention preferably performs the culture for approximately three days after cell seeding. The number of cells can be appropriately selected depending on the size of the substrate to be seeded and the intended use.

[0034] Furthermore, in the hepatocyte culture method of the present invention, after the culture by the above-mentioned method, it is necessary to impart a flow rate to the medium. The method for imparting a flow rate to the medium is not limited to perfusion culture in which the medium is pumped and then discharged, or in which the medium is allowed to flow only over the cell surface, but perfusion culture is preferred from the viewpoint of flow rate controllability.

[0035] Perfusion culture is a method in which the culture medium is supplied while the culture medium is withdrawn while leaving the cells, thereby maintaining a constant volume of the culture medium. This method allows for a high cell density while maintaining constant concentrations of nutrients and waste products. Furthermore, the medium can be supplied at a desired volume and flow rate using a liquid delivery device such as a pump. In the present invention, it is preferable to start adding a flow rate to the liquid medium four days after seeding hepatocytes on the substrate with the fusion protein immobilized thereon.

[0036] By culturing cells in a micro-flowing environment and applying shear stress, mechanical stimuli, biochemical gradients, etc. to the cells during culture, it is possible to culture cells in an environment that is closer to a living body than a static culture environment.

[0037] Furthermore, organ-on-a-chip, a type of perfusion culture, is a miniaturized in vitro human-type test system in which multiple human tissues are placed in the culture space of a microfluidic chip, allowing cells to be cultured in a perfusion environment. By preparing culture units for multiple organs and connecting and culturing them, it is possible to reproduce the interactions between organs in the human body in an in vitro culture vessel, making organ-on-a-chip a preferred method for application to the present invention.

[0038] The configuration for perfusion culture and for delivering culture medium to a microfluidic chip including an organ-on-a-chip will be described using the perfusion cell culture setup shown in Figure 1. Culture medium 103 is supplied to the substrate surface from pump 106a through valve 108a and is discarded as waste liquid 109 through valve 108b.

[0039] The flow rate of the culture medium 103 can be adjusted by controlling the pump 106a, and the flow rate of the culture medium is in the range of 10 μL / h to 300 μL / h. A flow rate slower than this range may cause hepatocyte aggregation or gaps to form in the hepatocyte layer, while a flow rate faster than this range may cause cells to detach or gaps to form in the hepatocyte layer, which is undesirable.

[0040] To inhibit hepatocyte aggregation and improve the density of the hepatocyte monolayer, the flow rate of the medium 103 is preferably in the range of 50 to 250 μL / h, more preferably 80 to 250 μL / h, and even more preferably 90 to 150 μL / h.

[0041] [Formation of liver tissue] In the present invention, liver-like tissue (hereinafter also referred to as liver tissue) can be formed by culturing hepatocytes and forming a sinusoidal endothelial cell layer based on the method for culturing hepatocytes. The present invention also provides a method for producing liver tissue, which includes the steps of culturing hepatocytes using the substrate and a liquid medium, and applying a flow rate to the liquid medium.

[0042] The method for forming and producing liver tissue of the present invention will be described with reference to FIG. 2 . The culture vessel shown in FIG. 2 comprises a culture vessel 60 having a silicone sheet 61 on its bottom surface and a culture vessel 50 having a polystyrene bottom surface 51a, allowing permeation of the vessel 50 and various components therein. The method for immobilizing or coating the fusion protein on the bottom surfaces 51a and 61b of this culture vessel can be performed in the same manner as the hepatocyte culture method described above. Furthermore, to form a sinusoidal endothelial cell layer, a scaffold for adhering cells to the surface of the bottom surface 61a of the vessel 60 can be used, such as collagen, or polymers commonly used as scaffolds, including laminin, fibronectin, and PVLA (poly-N-p-vinylbenzyl-D-lactonamide).

[0043] For example, the fixation or coating method for forming a sinusoidal endothelial cell layer involves adding atelocollagen to pH-adjusted sterile water on the surface of the silicone sheet 61a of the culture vessel 60, diluting the solution to the desired concentration, and then treating the surface for a time sufficient for fixation. After this, the surface can be washed with PBS or the like, yielding a vessel with a scaffold fixed to its surface. As with the fusion protein, physical methods such as adsorption and chemical methods such as covalent bonding can be used for fixation, but adsorption is preferred for ease of operation. When the adhesive molecule is a proteinaceous or peptide-based molecule, the molecule can be easily adsorbed by contacting a solution of the molecule with the solid surface of a culture substrate such as a plate and then removing the solvent after a certain period of time.

[0044] More specifically, a cell culture substrate on which the adhesive molecules are immobilized or coated can be obtained by simply filtering and sterilizing a solution of adhesive molecules in a solvent such as distilled water or PBS, contacting it with a culture substrate such as a plate, and leaving it for several hours to a day and night. Preferably, the substrate is washed several times with distilled water or PBS, and then replaced with a balanced salt solution such as PBS before use. When collagen is used in particular, the dilution concentration can be determined as desired. In the case of sinusoidal endothelial cells, a coating solution diluted to about 1 / 100 to 1 / 20, preferably 1 / 80 to 1 / 30, and more preferably 1 / 65 to 1 / 40 can be used to form a monolayer cell layer without clumping the adhered cells, and also to allow the formation of bile canaliculi.

[0045] [Cells] The hepatocytes used in the method for forming and producing liver tissue of the present invention can be the same as those used in the hepatocyte culture method. Furthermore, the sinusoidal endothelial cells can be primary sinusoidal endothelial cells or vascular endothelial cells isolated from humans or mice, cell lines derived from these cells, or cells derived from pluripotent stem cells induced to differentiate into sinusoidal endothelial cells. In addition to sinusoidal endothelial cells, other non-parenchymal hepatic cells, such as hepatic stellate cells and Kupffer cells, can also be used.

[0046] [Formation and Production of Liver Tissue] The method for forming and producing liver tissue of the present invention is characterized by culturing and growing hepatocytes and sinusoidal endothelial cells using the above-mentioned cell culture vessel and liquid medium, and by applying a flow rate to the medium.

[0047] The fusion protein is immobilized or coated on the bottom surfaces 51a and 61b of the containers 50 and 60. Furthermore, a scaffolding material such as collagen is immobilized or coated on the surface of the bottom surface 61a of the container 60. Conventional methods can be used to culture hepatocytes and sinusoidal endothelial cells. That is, an appropriate number of hepatocytes and sinusoidal endothelial cells are suspended in a commonly used liquid medium or cell culture solution, and the hepatocytes are added to the bottom surfaces 51a and 61b, and the sinusoidal endothelial cells are added to the bottom surface 61a (so-called seeding). Subsequent changes of the liquid medium and subculture can be performed as in conventional methods. In this case, in the present invention, it is preferable to perform this for approximately three days after cell seeding. The number of cells can be appropriately selected depending on the size of the substrate to be seeded and the intended use.

[0048] Furthermore, in the method for forming and producing liver tissue of the present invention, it is necessary to impart a flow rate to the medium after the culture by the above-mentioned method. The method for imparting a flow rate to the medium is not limited to perfusion culture, in which the medium is pumped and then discharged, or in which the medium is allowed to flow only over the cell surface, but perfusion culture is preferred from the viewpoint of flow rate controllability.

[0049] Perfusion culture is a method of maintaining a constant volume of culture medium by simultaneously supplying medium and withdrawing only the culture medium while leaving the cells. This method allows for high cell density while maintaining constant concentrations of nutrients and waste products. Furthermore, medium can be supplied at a desired volume and flow rate using a liquid delivery device such as a pump. In the present invention, as shown in Figure 3, it is preferable to seed hepatocytes onto a substrate immobilized with a fusion protein and sinusoidal endothelial cells onto a substrate immobilized with a desired scaffold, and then add a flow rate to the liquid medium starting four days later. In this case, the liquid medium can be introduced through containers 50a and 60a and discharged through containers 50b and 60b.

[0050] By culturing cells in a micro-flowing environment and applying shear stress, mechanical stimuli, biochemical gradients, etc. to the cells during culture, it is possible to culture cells in an environment that is closer to a living body than a static culture environment.

[0051] Furthermore, organ-on-a-chip, a type of perfusion culture, is a miniaturized in vitro human-type test system in which multiple human tissues are placed in the culture space of a microfluidic chip, allowing cells to be cultured in a perfusion environment. By preparing culture units for multiple organs and connecting and culturing them, it is possible to reproduce the interactions between organs in the human body in an in vitro culture vessel, making organ-on-a-chip a preferred method for application to the present invention.

[0052] The configuration for perfusion culture and for delivering culture medium to a microfluidic chip including an organ-on-a-chip will be described using the perfusion cell culture setup 1 shown in Figure 1. In the perfusion cell culture setup 1, culture medium 103 is supplied to the substrate surface from pump 106a through valve 108a and is discarded as waste liquid 109 from valve 108b.

[0053] The flow rate of the culture medium 103 can be adjusted by controlling the pump 106a, and the flow rate of the culture medium is in the range of 10 μL / h to 300 μL / h. A flow rate slower than this range may cause hepatocyte aggregation or gaps to form in the hepatocyte layer, while a flow rate faster than this range may cause cells to detach or gaps to form in the hepatocyte layer, which is undesirable.

[0054] To inhibit hepatocyte aggregation and improve the density of the hepatocyte monolayer, the flow rate of the medium 103 is preferably in the range of 50 to 250 μL / h, more preferably 80 to 250 μL / h, and even more preferably 90 to 150 μL / h.

[0055] According to the method for forming liver tissue of the present invention, the hepatic cell layer is a cobblestone-like layer with no aggregation and a high cell density, and a sinusoidal endothelial cell layer is formed on the hepatic cell layer, so that liver tissue that mimics biology can be obtained.

[0056] The effects of the present invention will be described below using examples and comparative examples, but the present invention is not limited to these examples. [Synthesis of a fusion protein of E-cadherin and the Fc region of immunoglobulin (hereinafter referred to as E-cadherin-Fc)] Expression and purification of E-cadherin-Fc were performed according to Nagaoka M, Akaike T., Protein Eng. 2003;16:243-245. In this example, extracellular domain cDNA obtained from full-length mouse E-cadherin (RIKEN BRC DNA Bank, code 1184) was ligated with mutated IgG1-Fc domain cDNA (T252M / T254S), and E-cadherin-Fc was expressed.

[0057] [Culture substrate] Fig. 2 shows a cross-sectional view of one embodiment of a vessel used for immobilizing a fusion protein on a substrate. A membrane-mediated microfluidic device having a two-layer structure consisting of a culture vessel 60 having a silicone sheet 61 on the bottom surface and a polystyrene culture vessel 50 shown in Fig. 2 was used.

[0058] [Observation of the substrate surface] From the fourth day, the states of hepatocytes cultured with and without adding flow to the medium were observed using an inverted fluorescence microscope (Olympus).

[0059] [Quantification by Real-Time PCR (RT-qPCR)] Cultured hepatocytes were harvested, and RNA was extracted using the Fast Gene RNA Basic Kit (Nihon Genetics). cDNA was synthesized using 5x RT Buffer, dNTP mixture (10 mM), Oligo (dT)20 (10 pmol / μl), and ReverseTra Ace reverse transcriptase (TOYOBO) and stored at -80°C until use. The primers used were as follows:

[0060] E-cad gene Forward primer: 5'-TACCCTGGTGGTTCAAGCTG-3' (SEQ ID NO: 3) Reverse primer: 5'-CCTGACCCTTGTACGTGGTG-3' (SEQ ID NO: 4) ZO-1 (TJP1) gene Forward primer: 5'-AGGAGTGAGAAGATTTGGCC-3' (SEQ ID NO: 5) Reverse primer: 5'-ATGGCTTGCCAATCGAAGAC-3' (SEQ ID NO: 6)

[0061] TBP gene Forward primer: 5'-TGAGTTGCTCATACCCTGCTGCTA-3' (SEQ ID NO: 7) Reverse primer: 5'-CCCTCAAACCAACTTGTCAACAGC-3' (SEQ ID NO: 8) ABCB1 gene Forward primer: 5'-AATGGATCTTGAAGGGGACC-3' (SEQ ID NO: 9) Reverse primer: 5'-TAAGTCGGGTGTTAAGCTCC-3' (SEQ ID NO: 10)

[0062] ABCC2 gene Forward primer: 5'-TCCGTGCCTTTGAGCACCAGC-3' (SEQ ID NO: 11) Reverse primer: 5'-ACTTTCCAGCTCCTGTCCTGC-3' (SEQ ID NO: 12) ABCG2 gene Forward primer: 5'-TGTGATGGGCACTCTGACGG-3' (SEQ ID NO: 13) Reverse primer: 5'-GCCTCACAGTGATAACCAGC-3' (SEQ ID NO: 14)

[0063] NTCP gene Forward primer: 5'-ATCGTCCTCAAATCCAAACG-3' (SEQ ID NO: 15) Reverse primer: 5'-CCATTGAGGCAGAAGAGAGC-3' (SEQ ID NO: 16) ALB gene Forward primer: 5'-AAGAGTGAGGTTGCTCATCG-3' (SEQ ID NO: 17) Reverse primer: 5'-CAGTGCACATCACATCAACC-3' (SEQ ID NO: 18)

[0064] HNF4A gene Forward primer: 5'-AACGACCGCCAGTATGACTC-3' (SEQ ID NO: 19) Reverse primer: 5'-CATCTGTCCGTTGCTGAGGT-3' (SEQ ID NO: 20) CEBPA gene Forward primer: 5'-GACCCTCAGCCTTGTTTGTACT-3' (SEQ ID NO: 21) Reverse primer: 5'-CTTGTCATAACTCCGGTCCCTC -3' (SEQ ID NO: 22)

[0065] AAT gene Forward primer: 5'-GGCCAAGAAACAGATCAACG-3' (SEQ ID NO: 23) Reverse primer: 5'-TCATCATAGGCACCTTCACG-3' (SEQ ID NO: 24) CYP2E1 gene Forward primer: 5'-GGATAGGCAAGAGATGCCCTAC-3' (SEQ ID NO: 25) Reverse primer: 5'-AGAGTTGGCACTACGACTGTG-3' (SEQ ID NO: 26)

[0066] CYP3A4 gene Forward primer: 5'-AGCCCAGCAAAGAGCAACAC-3' (SEQ ID NO: 27) Reverse primer: 5'-TCTGGGATGAGAGCCATCACTA-3' (SEQ ID NO: 28) RT-qPCR was performed using KAPA SYBR FAST qPCR Master Mix (KAPABIOSYSTEMS) according to the manufacturer's protocol. The amplification profile consisted of denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0067] For quantitative analysis of each gene's expression level, the ΔΔCT method was performed. A standard curve was constructed using an aliquot of each experimental sample. The expression level of each gene was normalized by the expression level of TBP as a housekeeping gene.

[0068] A 2 μg / ml CDF-DA solution (colorless and transparent) was introduced into the culture medium from the upstream channel of the liver tissue at a flow rate of 100 μl / h, and the CDF fluorescence was visually observed and measured in both the upstream and downstream channels using a Tecan Infinite M200PRO device.

[0069] (Example 1, Comparative Example 1) E-cadherin-Fc was adjusted to concentrations of 0.1 μg / ml, 1 μg / ml, and 10 μg / ml in PBS+, and the coating solution was poured onto the surface of a 35 mm polystyrene dish up to the top of the dish. The dish was incubated overnight at 4°C, and the coating solution was then removed from the dish. After washing the surface of the dish with PBS+, 1.2 × 10 HepG2 (hereinafter referred to as RH4), a hepatoma cell line that exhibits red fluorescence, was cultured. ４ The cells were seeded with 1000 ng / ml E-cadherin-Fc and cultured for 3 days. The state of the cells was observed by electron microscopy (Figures 7 and 8), and the expression of E-cadherin and ZO-1 in relation to cell-cell adhesion (Figure 8) was shown. Cells cultured on a culture dish not coated with E-cadherin-Fc (concentration: 0 μg / ml) served as a control.

[0070] The results in Figures 7 and 8 show that when the coating solution concentration was 1 μg / ml, no aggregation of RH4 cells was observed, and the cells were closely packed (like paving stones) over the entire surface of the 35 mm dish. In contrast, gaps and aggregation were observed between the cells in the 35 mm dishes coated with RH4 solutions at concentrations of 0, 0.1, and 10 μg / ml. Furthermore, the results in Figure 8 show that the expression levels of E-cadherin and ZO-1 in RH4 cells cultured with a coating solution concentration of 1 μg / ml were higher than those cultured on 35 mm dishes with other coating concentrations. This indicates that the surface of the 35 mm dish coated with a 1 μg / ml concentration had a high adhesion density of hepatocytes and no aggregation, forming a state similar to that of liver tissue in vivo.

[0071] Example 2 Preparation of a Culture Substrate The rear surface (61b) of a silicone sheet of a culture vessel 60 having a silicone sheet 61 on the bottom surface as shown in Figure 2 was immersed in a coating solution prepared by adjusting E-cadherin-Fc to a concentration of 1 μg / ml with PBS+, incubated overnight at 4°C, and then washed with PBS+. Next, the surface of the silicone sheet 61a of the culture vessel 60 was coated with a coating solution prepared by adding atelocollagen to sterilized water at pH 3 and diluting it 1 / 50. Further, the surface of the bottom surface 51a of a polystyrene culture vessel 50 was immersed in a coating solution prepared by adjusting E-cadherin-Fc to a concentration of 1 μg / ml with PBS+, incubated overnight at 4°C, and then the atelocollagen surface was washed with PBS- and the E-cadherin-Fc surface was washed with PBS+ to prepare a culture substrate.

[0072] [Cell seeding] HepG2 (hereinafter referred to as RH4), a hepatoma cell line that exhibits red fluorescence as hepatocytes on the surface of E-cadherin-Fc, was seeded at a cell count of 3 × 10 ５ EAhy926GFP (hereinafter referred to as GH7), a human endothelial cell line that exhibits green fluorescence, was seeded on the surface of the collagen at a cell count of 1 × 10 ５ The cells were seeded.

[0073] [Cell Culture] As shown in Figure 4, liquid medium was placed in each culture vessel and cultured for 3 days after cell seeding. From the 4th day, liquid medium was introduced from 50a and 60a at a flow rate of 200 µL / h, and the liquid was discharged from 50b and 60b. The liquid medium was cultured at a flow rate of 200 µL / h for up to 13 days.

[0074] Comparative Example 2 In Example 2, hepatocytes were cultured in the same manner as in Example 1, except that no flow rate was applied to the liquid medium even after the fourth day after cell seeding. The results in Figure 4 show that when a flow rate was applied to the liquid medium from the fourth day onward and cell morphology was examined 48 hours later, the hepatocytes underwent a morphological change to a paving-stone-like state. Furthermore, the results in Figure 5 show that gene expression of E-cadherin, a cell adhesion marker, increased with prolonged culture, with higher expression observed when a flow rate was applied than when no flow rate was applied. These results indicate that hepatocytes cultured in liquid culture with a flow rate were formed on the substrate surface without gaps, and no aggregation was observed in the hepatocyte layer, whereas when a flow rate was not applied, aggregation of hepatocytes was observed due to low intercellular adhesion of E-cadherin.

[0075] (Example 3) [Formation of liver tissue] As shown in Figure 3, liquid medium was placed in each culture vessel, and cells were seeded and cultured for 3 days. From the 4th day, liquid medium was introduced from 50a and 60a at a flow rate of 200 µL / h, and the liquid was discharged from 50b and 60b. Culture was continued for up to 13 days with the liquid medium flowing at a constant rate.

[0076] Comparative Example 3 In Example 3, hepatocytes were cultured in the same manner as in Example 1, except that no flow rate was applied to the liquid medium even after the fourth day after cell seeding. The results in Figure 4 show that when a flow rate was applied to the liquid medium from the fourth day onward and cell morphology was examined 48 hours later, the hepatocytes underwent a morphological change to a paving-stone-like state. Furthermore, the results in Figure 5 show that gene expression of E-cadherin, a cell adhesion marker, increased with prolonged culture, with higher expression observed when a flow rate was applied than when no flow rate was applied. These results indicate that hepatocytes cultured in liquid culture with a flow rate were formed on the substrate surface without gaps, and no aggregation was observed in the hepatocyte layer, whereas when a flow rate was not applied, aggregation of hepatocytes was observed due to low intercellular adhesion of E-cadherin.

[0077] Furthermore, the results in Figure 5 show that the sinusoidal transporter NTCP and the bile ductal transporters ABCB1 and ABCG2 are highly expressed by applying a flow rate. Therefore, it is presumed that applying a flow rate to the liquid medium changes cell morphology, forming stronger cell tight junctions, and co-culturing with sinusoidal endothelial cells to form sinusoids and bile canaliculi with blood flow improves hepatocyte function. This is also evident from the results in Figure 6, where the expression of mature hepatocyte markers ALB, HNF4A, CEBPA, and AAT is higher than that of the control without applying a flow rate, and further shows that the kinetics of CYP3A4 drugs is improved.

[0078] Furthermore, the polarity of hepatocytes (sinusoidal and bile canalicular sides) in liver tissue was confirmed, and the results are shown in Figure 9. It is known that in the liver of liver tissue, CDF-DA (5(6)-carboxy-2',7'-dichlorofluorescein diacetate; colorless) is absorbed by the OATP1B1 transporter localized on the sinusoidal side of hepatocytes, deacetated by esterases in the hepatocytes to form CDF (5(6)-carboxy-2',7'-dichlorofluorescein; green fluorescence), which is then excreted into the bile canaliculi by the ABCC2 transporter localized on the bile canaliculus side. The results are shown in Figure 9. In Figure 9, a CDF-DA solution (colorless and transparent) was introduced into the upstream flow path of the liver tissue through the culture medium inlet 70a, and the CDF fluorescence was quantified in the upstream flow path (culture medium outlet 70b) and downstream flow path (culture medium outlet 80b). The upstream flow path, i.e., the sinusoidal side, remained colorless, but green fluorescence was observed downstream.

[0079] The results of Figure 9 show that in the liver tissue of Example 3, CDF-DA (5(6)-carboxy-2',7'-dichlorofluorescein diacetate: colorless) is absorbed by the OATP1B1 transporter localized on the upstream sinusoidal endothelial cell side, deacetated by esterase in the hepatocytes to form CDF (5(6)-carboxy-2',7'-dichlorofluorescein: green fluorescence), and then excreted into the bile canaliculi by the ABCC2 transporter localized on the bile canaliculus side. This is clear from the fact that no fluorescence was detected in the upstream flow path for CDF-DA introduced from the upstream flow path, but fluorescence was quantified from the downstream flow path after 2 and 3 hours.

[0080] These results demonstrate that three-dimensional liver tissue mimicking the liver can be formed by forming each tissue using the method for culturing hepatocytes and the method for forming liver tissue of the present invention.

[0081] 1 Perfusion culture setup 2 Hepatocytes 3 Sinusoidal endothelial cells 50, 60 Culture vessel 50a, 60a, 70a, 80a Medium inlet 50b, 60b, 70b, 80b Medium outlet 51a, 61a, 61b Bottom surface 61 Bottom surface made of silicone sheet 80c Medium collection vessel 103 Medium 106a Pump 108a, 108b Valve 109 Waste liquid

[0082] SEQ ID NOs: 3 to 28: synthetic DNA

Claims

1. A method for culturing hepatocytes using a liquid medium and a substrate on which a fusion protein of E-cadherin and the Fc region of immunoglobulin is immobilized, the method comprising applying a flow rate to the liquid medium.

2. The method for culturing hepatocytes according to claim 1, characterized in that the flow rate is 50 μL / h or more.

3. The method for culturing hepatocytes according to claim 1, wherein the coating concentration of the fusion protein when immobilized on the substrate is adjusted to 0.3 to 10 μg / ml.

4. A method for forming liver tissue, comprising seeding hepatocytes onto a substrate onto which a fusion protein of E-cadherin and the Fc region of immunoglobulin has been immobilized, and then applying a flow rate to the liquid medium starting from day 4.

5. The method for forming liver tissue according to claim 4, characterized in that the flow rate is 50 μL / h or more.

6. The method for culturing hepatocytes according to claim 4, wherein the coating concentration of the fusion protein when immobilized on the substrate is adjusted to 0.3 to 10 μg / ml.

7. Liver tissue formed by the method according to any one of claims 4 to 6.

8. A method for producing liver tissue, comprising the steps of culturing hepatocytes using a substrate on which a fusion protein of E-cadherin and the Fc region of immunoglobulin is immobilized and a liquid medium, and applying a flow rate to the liquid medium.

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