Method for producing cell support complex, method for producing drug evaluation device, drug evaluation method, and drug evaluation device

Abstract

A method for producing a cell support complex (10) includes culturing kidney cells in an aggregate state, separating the aggregate into individual cultured cells (16), inoculating the cultured cells (16) on a substrate (12) at an inoculation density of 5 * 104 pieces / cm < 2 > to 18 * 104 pieces / cm < 2 >, culturing the cultured cells (16) on the substrate (12) for a period of 12 hours to 48 hours, and separating the cultured cells (16) from the substrate (12). A single-layer film of the cultured cell (16) is formed.

Description

Technical Field

[0001] The present invention relates to a method for producing a cell-supporting complex, a method for producing a drug evaluation device, a drug evaluation method, and a drug evaluation device. Background Art

[0002] After the drug administered to the organism exerts its effect in the organism, it is excreted from the blood into the urine through the kidneys. The transport of the drug from the blood to the urine is carried out by transporters in kidney cells. Therefore, kidney cells are susceptible to the nephrotoxicity of the drug. In particular, proximal tubular epithelial cells (RPTEC) tend to be most affected by drugs in the kidney. Therefore, using kidney cells to evaluate the dynamics and nephrotoxicity of drugs is important in clarifying the effects of drugs.

[0003] In developing renal cells to evaluate the dynamics of drugs and analysis of renal toxicity, a large number of renal cells are required. In contrast, for example, Patent Document 1 discloses a technique for culturing a sufficient number of proximal tubular epithelial cells by inhibiting gene expression of cell cycle regulators.

[0004] [Prior art literature]

[0005] [Patent Document]

[0006] Patent Document 1: Japanese Patent Application Publication No. 2011-50358 Summary of the invention

[0007] [Problems to be solved by the invention]

[0008] In the existing culture technology, there is a problem that the physiological function of kidney cells is reduced during culture. Therefore, it is difficult to perform high-precision drug evaluation in drug evaluation analysis using cells cultured by the existing method.

[0009] The present disclosure has been made in view of such circumstances, and one of its objects is to provide a technology for improving the accuracy of drug evaluation analysis using cells.

[0010] [Technical solutions for solving technical problems]

[0011] One embodiment of the present invention is a method for producing a cell-supporting complex. The method comprises the steps of culturing kidney cells in an aggregate state, separating the aggregate into individual cultured cells, and 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2The cultured cells are seeded on the substrate at the following seeding density, and the cultured cells are cultured on the substrate for a period of 12 hours to 48 hours to form a monolayer film of the cultured cells.

[0012] Another aspect of the present invention is a method for manufacturing a drug evaluation device. The manufacturing method includes the following steps: placing a cell-supporting complex manufactured by the manufacturing method of the cell-supporting complex of the above aspect on a support portion, or placing a substrate on a support portion and inoculating cultured cells on the substrate by the manufacturing method of the cell-supporting complex of the above aspect to manufacture a cell-supporting complex, and forming a first chamber and a second chamber, wherein the first chamber and the second chamber are arranged with the cell-supporting complex interposed therebetween and at least one of the first chamber and the second chamber contains a drug to be evaluated.

[0013] Another aspect of the present invention is a method for evaluating a drug, including the step of evaluating the transport of a drug by cultured cells included in a cell-supporting complex using the drug evaluation device obtained by the method for producing the drug evaluation device of the above aspect.

[0014] Another aspect of the present invention is a drug evaluation device. The device comprises: a cell-supporting complex manufactured by the method for manufacturing a cell-supporting complex of the above aspect; a support portion for supporting the cell-supporting complex; and a first chamber and a second chamber, the first chamber and the second chamber being arranged across the cell-supporting complex and at least one of which contains a drug to be evaluated.

[0015] Furthermore, arbitrary combinations of the above-described constituent elements and embodiments in which constituent elements and expressions in methods, apparatuses, systems, and the like of the present invention are mutually replaced may also be additional modes of the present invention.

[0016] [Effects of the invention]

[0017] According to the present invention, it is possible to improve the accuracy of drug evaluation analysis using cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram schematically showing a cell-supporting complex according to an embodiment.

[0019] Figure 2 (A)~ Figure 2 (C) is a process diagram of a method for producing a cell-supporting complex and a method for producing a drug evaluation device according to an embodiment.

[0020] Figure 3 (A)~ Figure 3 (C) is a process diagram of a method for producing a cell-supporting complex and a method for producing a drug evaluation device according to an embodiment.

[0021] Figure 4 (A)~ Figure 4 (E) is a diagram schematically showing an example of application of the cell-supporting complex.

[0022] Figure 5 This is a graph showing the temporal changes in gene expression levels in cells constituting aggregates.

[0023] Figure 6 (A)~ Figure 6 (C) is a graph showing the temporal changes in gene expression levels in cells constituting aggregates.

[0024] Figure 7 (A)~ Figure 7 (C) is a graph showing the temporal changes in gene expression levels in cells constituting aggregates.

[0025] Figure 8 (A)~ Figure 8 (C) is a graph showing the temporal changes in gene expression levels in cells constituting aggregates.

[0026] Fig. 9 This is an optical microscope image of cells separated from the aggregates taken the day after reseeding.

[0027] Fig.10 It is a graph showing TEER at each elapsed time from reinoculation.

[0028] Fig.11 (A) and Fig.11 (B) is a graph showing the temporal changes in TEER at each seeding density.

[0029] Fig.12 This is a graph showing the temporal changes in the expression level of the OAT1 gene.

[0030] Fig.13 (A)~ Fig.13 (F) is a graph showing the temporal changes in the expression level of OTA1 protein.

[0031] Fig.14 (A) and Fig.14 (B) is a graph showing the expression levels of kidney-related transporters among cell membrane proteins. Fig.14 (C) is a graph showing the temporal changes in the expression level of kidney-related transporter proteins in total proteins.

[0032] Fig.15 (A) is a graph showing the results of membrane transport of Adefovir. Fig.15 (B) is a diagram showing the results of membrane transport of metformin. DETAILED DESCRIPTION

[0033] Hereinafter, the present invention will be described based on a preferred embodiment and with reference to the accompanying drawings. The embodiment does not limit the present invention but is merely an example, and all the features and combinations thereof described in the embodiment are not necessarily the essential contents of the invention. The same or equivalent constituent elements, components, and processes shown in the drawings are marked with the same figure numerals, and repeated descriptions are appropriately omitted. In addition, for ease of description, the scales and shapes of the various parts shown in the drawings are conveniently set, and are not to be interpreted in a limiting sense unless otherwise specifically mentioned. In addition, when the terms "first", "second", etc. are used in this specification or claims, unless otherwise specifically mentioned, the terms do not indicate any order or importance, but are used to distinguish a certain structure from other structures. In addition, in each of the drawings, a part of the components that are not important on the basis of describing the embodiment is omitted and displayed.

[0034] The inventors of the present invention have studied the drug evaluation analysis using cells and obtained the following understanding. That is, kidney cells such as proximal tubular epithelial cells isolated from the kidney (primary culture cells) dedifferentiate and their functions gradually decrease or disappear due to the disappearance of the in vivo environment and the culture environment such as two-dimensional culture on the culture dish. Therefore, by simply culturing kidney cells, only cells with insufficient physiological functions of the kidney increase. Cells with insufficient physiological functions may not show normal transport of drugs or normal reactions to drug toxicity. Therefore, the use of kidney cells in new drug development research has stagnated.

[0035] The applicant has discovered a technology that generates cultured cells with greatly improved physiological functions by culturing kidney cells with lost physiological functions in the state of aggregates (International Application No. PCT / JP2018 / 011392). By assembling the cells obtained using this technology in drug evaluation analysis, it is expected that drug evaluation can be implemented with high precision.

[0036] In drug evaluation analysis, especially drug dynamic evaluation (transporter activity assay) related to the reabsorption and excretion of drugs in the kidney, membrane transport evaluation using migration assay (Transwell) is common. If the aggregates of cultured cells are used directly, a uniform monolayer of cells cannot be formed on the cell culture plug (hereinafter appropriately referred to as the plug) of the migration assay (Transwell). If the monolayer of cultured cells cannot be obtained, it is difficult to implement the drug dynamic evaluation using migration assay (Transwell) with high precision. Therefore, it is necessary to treat the aggregates with protease to separate them into individual cultured cells, and inoculate the separated cultured cells on the plug.

[0037] However, if the separated cultured cells are cultured in two dimensions on the plug-in, the physiological function that has been increased by agglomeration culture (three-dimensional culture) may be reduced again. In addition, when the cultured cells are inoculated on the plug-in at a low density, a long period is required to form a monolayer membrane without gaps. Therefore, the physiological function is more likely to be reduced. Therefore, in order to shorten the time from the inoculation of cultured cells to the formation of a monolayer membrane, it is considered to inoculate the cultured cells without gaps on the plug-in. However, the inventors have confirmed that even if the cultured cells are inoculated at a high density like this, a monolayer membrane with substantially no drug leakage suitable for drug dynamic evaluation cannot be obtained. Therefore, in the development of new drugs, it is urgently desired to develop a drug evaluation analysis using a monolayer membrane of cells with good physiological functions.

[0038] On the other hand, the present inventors have found a technique for suppressing the decrease in physiological functions caused by aggregation culture and forming a monolayer membrane of cultured cells on a substrate. The present embodiment is proposed based on such considerations. Figure 1 1 is a diagram schematically showing a cell-supporting complex 10 according to an embodiment. The cell-supporting complex 10 includes a substrate 12 and a single layer of cultured cells 16. The cell-supporting complex 10 according to the present embodiment further includes a coating agent layer 14.

[0039] (Substrate 12)

[0040] The substrate 12 is made of, for example, an artificial material. The substrate 12 is permeable to water and various ions. In addition, the substrate 12 is also permeable to sugars, low-molecular proteins, and the like. The drug 50 present on the cell apical membrane side moves to the cell basal membrane side through the cell-supporting complex 10 via the first transporter 18 on the cell apical membrane side and the second transporter 20 on the cell basal membrane side of the cultured cells 16 and the substrate 12. In addition, the transport of the drug 50 by the cultured cells 16 includes not only the case where the drug 50 present on the cell apical membrane side is moved to the cell basal membrane side, but also the case where the drug 50 present on the cell basal membrane side is moved to the cell apical membrane side.

[0041] In order to have permeability to various substances, for example, holes are provided in the substrate 12. The average pore diameter of the holes provided in the substrate 12 is preferably 5 μm or less. By setting the average pore diameter to 5 μm or less, the possibility of the cultured cells 16 passing through the substrate 12 can be reduced. As such a substrate 12, for example, a migration assay (Transwell) insert (Corning: average pore diameter 0.4 μm or 3.0 μm) can be used.

[0042] In addition, a substrate 12 that is not permeable to various substances may be used. In this case, the drug 50 present on the cell apical membrane side is taken into the interior of the cultured cell 16 via the first transporter 18 of the cultured cell 16. In a short period of time from the start of use, the movement of the drug 50 to the cell basal membrane side via the second transporter 20 of the cultured cell 16 is small. Therefore, the layer of the cultured cell 16 is not deformed. Over a long period of time, although the amount of movement of the drug 50 via the second transporter increases, the drug 50 cannot pass through the substrate 12. This is because the layer of the cultured cell 16 floats and forms a dome.

[0043] The material constituting the substrate 12 is not particularly limited, and examples thereof include polystyrene, polycarbonate (PC), polyester (PET), polyester polymer alloy (PEPA), ethylene-vinyl alcohol copolymer (EVOH), polyethylene, polysulfone (PSf), polyethersulfone (PES), and the like.

[0044] (Cultured cells)

[0045] The cultured cells 16 are attached to the substrate 12. The cultured cells 16 of the present embodiment are fixed to the substrate 12 via the coating layer 14. In addition, when the coating layer 14 is omitted, the cultured cells 16 are directly attached to the substrate 12. As described above, the cultured cells 16 have the first transporter 18 located on the cell apical membrane side and the second transporter 20 located on the cell basal membrane side.

[0046] Cultured cells 16 are produced by culturing kidney cells in a culture container in a non-adhesive state. When cultured cells are used for drug evaluation, cultured cells 16 need to maintain physiological functions. On the other hand, if kidney cells are cultured in an environment different from the in vivo environment, they dedifferentiate and their physiological functions decrease. In contrast, by culturing kidney cells in a culture container in a non-adhesive state, the reduced physiological functions of kidney cells can be restored. Specifically, if kidney cells are cultured in a culture container in a non-adhesive state, kidney cells form aggregates during the culture period. For example, aggregates are formed on the first day of culture (i.e., within 24 hours). Then, by culturing kidney cells in the state of aggregates for at least a portion of the period, the physiological functions of kidney cells can be restored. The cell culture method will be described in detail later. In addition, aggregates also include organoids.

[0047] The kidney cells that form the basis of the cultured cells 16 include kidney cells from tissues. In addition, kidney cells derived from stem cells such as iPS cells, ES cells or Muse cells are included. In addition, kidney cells include at least one of the epithelial cells of the proximal tubule system, the distal tubule system and the collecting duct system. More specifically, as kidney cells, examples can be given such as human proximal tubule epithelial cells, human distal tubule epithelial cells and human collecting duct epithelial cells collected and separated from the kidney. In addition, proximal tubule epithelial cells, distal tubule epithelial cells and collecting duct epithelial cells induced by differentiation of human iPS cells, human ES cells or human Muse cells can be exemplified. Preferably, kidney cells are proximal tubule epithelial cells. In addition, kidney cells include immortalized cells, strain cells (HK-2 cells, etc.) of the above-mentioned kidney cells, transformed cells and renal precursor cells into which genes are introduced in kidney cells in order to express proteins such as specific transporters. Furthermore, as kidney cells, cells derived from other animal species (MDCK cells, LLC-PK1 cells, JTC-12 cells, etc.) may be used instead of human-derived kidney cells.

[0048] The cultured cells 16 are not substantially multilayered on the substrate 12 but form a confluent monolayer. The above-mentioned "single layer" is preferably a layer in which the cultured cells 16 are not multilayered at all. However, the above-mentioned "single layer" may also include a structure (substantially a single layer) in which a portion of the layers are multilayered to the extent that the reduction in the efficiency of the movement of the substance caused by the multilayering does not become a problem. In addition, the above-mentioned "convergence" is preferably a state in which the area of ​​the occupied area of ​​the cultured cells 16 in the observation image is 100% relative to the total area of ​​the observation image. For example, the above-mentioned "convergence" refers to a state in which the ratio of the area occupied by the cells to the entire culture surface of the substrate 12 is 100%, that is, the cells proliferate completely without gaps on the culture surface. However, the above-mentioned "convergence" may also include a state in which a portion of the cells have gaps to the extent that the generation of concentration-dependent material movement in the gaps between adjacent cells does not become a problem (substantially converging). It is easy for a person skilled in the art to judge whether it is a monolayer and whether it is confluent.

[0049] (Coating agent layer)

[0050] The coating agent layer 14 is composed of a coating agent. The coating agent layer 14 covers at least a portion of the substrate 12. The coating agent layer 14 adheres to the surface of the substrate 12 and is fixed to the substrate 12. The coating agent includes one or more adhesion molecules selected from the group consisting of laminin molecules, fragments of laminin molecules, basement membrane matrix mixtures, fragments of basement membrane matrix mixtures, collagen molecules, and fragments of collagen molecules. Laminin molecules, basement membrane matrix mixtures, collagen molecules, and fragments thereof may also be used separately. By containing these adhesion molecules in the coating agent layer 14, a monolayer membrane of the cultured cells 16 can be formed more easily. In addition, in addition to or in place of the above-mentioned adhesion molecules, the coating agent may also include other adhesion proteins such as gelatin, fibronectin, and fragments thereof.

[0051] (Laminin molecule)

[0052] The laminin molecule adopts a heterotrimeric structure having one α chain, one β chain, and one γ chain respectively. Currently, five α chains, three β chains, and three γ chains have been identified. It is known that the laminin molecule forms at least 12 isoforms through these combinations. For example, the laminin molecule selects one or more of laminin 111, laminin 211, laminin 221, laminin 311, laminin 332, laminin 421, laminin 511, and laminin 521.

[0053] The laminin molecule also includes a variant of laminin in which a prescribed modification group is added to one or more of the above isotypes (variant laminin). Variants also include genetic recombinants, i.e., proteins obtained from recombinant genes with mutations introduced into proteins or proteins having partial proteins of genetic recombinants or peptides from genetic recombinants. The modification group of the variant laminin is, for example, a growth factor binding molecule or a cell adhesion molecule.

[0054] The concentration of laminin molecules in the coating agent and the amount of laminin molecules adhered to the substrate 12 are appropriately adjusted so that the cell-supporting complex 10 can maintain its performance during use. The amount of laminin molecules adhered to the substrate 12 can be controlled by adjusting the concentration of laminin molecules in the coating agent. The amount of laminin molecules adhered to the substrate 12 can be measured using a method known to those skilled in the art.

[0055] As a fragment of the laminin molecule, a modified body (expressed as laminin***-E8) of the E8 region of the cell adhesion site (integrin binding site) including domain I in the laminin molecule (full-length laminin) can be exemplified. As such a variant, laminin 111-E8, laminin 211-E8, laminin 421-E8, laminin 511-E8, and laminin 521-E8 can be exemplified. As a fragment of the laminin molecule, not only a variant of the E8 region, but also a laminin peptide with cell adhesion activity or a substance obtained by peptide synthesis of only the cell active site can be used. As such a laminin peptide, a YIGSR peptide, a PDSGR peptide, a RYVVLPR peptide, an RGD peptide, a KAFDITYVRLKF peptide, an IKVAV peptide, and a LRE peptide can be exemplified. In addition, the size of the fragment of the laminin molecule is not particularly limited.

[0056] Laminin molecules and fragments may be mixed and used in multiple isotypes, respectively. Alternatively, laminin molecules and fragments may be mixed and used. Alternatively, multiple coating agents containing different types of laminin molecules and / or fragments may be applied to the substrate 12, and multiple coating agent layers 14 containing different types of laminin may be stacked.

[0057] (Basement membrane matrix mixture)

[0058] Basement membrane matrix mixture is a mixture of extracellular matrix proteins extracted from mouse sarcoma. Basement membrane matrix mixture contains laminin, collagen IV and entactin as main components. Matrigel (matrigel) (registered trademark: Corning company) can be exemplified as basement membrane matrix mixture. Matrigel refers to a soluble basement membrane matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma richly containing extracellular matrix proteins. In addition to the usual Matrigel containing growth factors, Matrigel also includes Matrigel (Growth FactorReduced Matrigel Matrix: Growth Factor Reduced Matrigel Matrix) with reduced growth factors compared to the Matrigel.

[0059] The concentration of the basement membrane matrix mixture in the coating agent and the amount of the basement membrane matrix mixture adhered to the substrate 12 are appropriately adjusted so that the cell-supporting complex 10 can maintain its performance during use. The amount of the basement membrane matrix mixture adhered to the substrate 12 can be controlled by adjusting the concentration of the basement membrane matrix mixture in the coating agent. The amount of the basement membrane matrix mixture adhered to the substrate 12 can be measured using a method known to those skilled in the art.

[0060] The fragment of the basement membrane matrix mixture is mixed with at least one of a fragment of laminin, a fragment of collagen IV, and a fragment of entactin. In addition, the basement membrane matrix mixture and the fragments may be mixed and used in multiple forms. In addition, the basement membrane matrix mixture and the fragments may be mixed and used. In addition, a plurality of coating agents containing different types of basement membrane matrix mixtures and / or fragments may be applied to the substrate 12, so that a plurality of coating agent layers 14 containing different types of mixtures are stacked.

[0061] (Collagen molecule)

[0062] As collagen molecules, collagen I and collagen IV can be exemplified. These can be purchased from Nitta Collagen Co., Ltd., for example. In order to maintain the performance of the cell-supporting complex 10 during use, the concentration of the collagen molecules in the coating agent and the amount of adhesion of the collagen molecules to the substrate 12 are appropriately adjusted. The amount of adhesion of the collagen molecules can be controlled by adjusting the concentration of the collagen molecules in the coating agent. The amount of adhesion of the collagen molecules to the substrate 12 can be measured using methods known to those skilled in the art.

[0063] Collagen molecules and fragments may also be mixed and used in multiple ways. In addition, collagen molecules and fragments may also be mixed and used. In addition, multiple coating agents containing different types of collagen molecules and / or fragments may be applied to the substrate 12, so that multiple coating agent layers 14 containing different types of collagen are stacked.

[0064] (Method for producing a cell-supporting complex and method for producing a drug evaluation device)

[0065] Figure 2 (A)~ Figure 2 (C) Figure 3 (A)~ Figure 3 (C) is a process diagram of a method for manufacturing the cell-supporting complex 10 and a method for manufacturing the drug evaluation device 42 (drug evaluation module) according to the embodiment. Figure 3 (A) and Figure 3 (B) shows only a portion of the substrate 12 .

[0066] The method for producing the cell-supporting complex 10 of the present embodiment includes the steps of culturing kidney cells 24 in the form of aggregates 30, separating the aggregates 30 into individual cultured cells 16, seeding the cultured cells 16 on the substrate 12 at a predetermined seeding density, and culturing the cultured cells 16 on the substrate 12 for a predetermined period of time to form a monolayer film of the cultured cells 16. In addition, the method for producing the drug evaluation device 42 of the present embodiment includes the steps of placing the cell-supporting complex 10 produced by the method for producing the cell-supporting complex 10 of the present embodiment on the support portion 32, or placing the substrate 12 on the support portion 32 and seeding the cultured cells 16 on the substrate 12 by the method for producing the cell-supporting complex 10 of the present embodiment to produce the cell-supporting complex 10, thereby forming a first chamber 34 and a second chamber 36, the first chamber 34 and the second chamber 36 being arranged with the cell-supporting complex 10 interposed therebetween, and at least one of the first chamber 34 and the second chamber 36 containing the drug 50 to be evaluated.

[0067] As an example, Figure 2 As shown in (A), kidney cells 24 are seeded in a culture container 26. In addition, a culture solution 28 is added to the culture container 26. As an example of the culture container 26, a 96-well plate with a U bottom or a V bottom, a 384-well plate with a U bottom, a microplate with a larger number of wells, a spinner flask, a culture dish, a hollow fiber, a culture bottle, a culture bag, a microchannel, a culture tank, etc. can be exemplified.

[0068] The culture container 26 can also be a high-density spheroid culture plate. Thus, the aggregates 30 can be produced in large quantities. As high-density spheroid culture plates, ELPLASIA (registered trademark) plates (Corning Company), EZSPHERE (registered trademark) plates (AGCTECHNO GLASS Company) and the like can be exemplified. For example, ELPLASIA plates have models such as 6-well plates, 24-well plates, 96-well plates, and 384-well plates. The bottom area of ​​the hole varies with the number of holes. Depending on the size of the bottom area, the number of aggregates 30 produced changes. The aggregates produced using the high-density spheroid culture plate can then also be subjected to oscillation floating culture. In an example of oscillation floating culture, the aggregates are accommodated in a dish or plate to which a cell non-adhesion treatment has been implemented, and the dish or the like is placed on an oscillator to culture the aggregates. As an oscillator, a reciprocating oscillator or a rotary oscillator can be used.

[0069] The culture medium 28 is not particularly limited as long as it can culture the kidney cells 24, and can be appropriately selected from known culture media according to the type of cells to be cultured. For example, in the case of culturing proximal tubular cells, REGM (Lonza), EpiCM (ScienCell), KeratinocyteSFM (Life Technologies), etc. can be used. In addition, commercially available tubular cell culture media, epithelial cell culture media, vascular endothelial cell culture media, etc. can also be used.

[0070] Next, the kidney cells 24 are cultured. The kidney cells 24 are cultured in a state where they are not adhered to the surface of the culture container 26. Figure 2 As shown in (B), during the culture period, aggregates 30 of kidney cells 24 are formed. By culturing kidney cells 24 in the state of aggregates 30, cultured cells 16 in a state with better physiological functions can be obtained. The size of the aggregates 30 is, for example, about 100 μm or more and about 350 μm or less. The size of the aggregates 30 is defined as the maximum width of the aggregates 30. That is, the size of the aggregates 30 is the length of the longest straight line among the straight lines connecting two points on the outer edge of the aggregates 30.

[0071] The size of the aggregate 30 can be controlled by adjusting the number of cells seeded in the culture container 26. For example, when a well plate is used as the culture container 26, the number of kidney cells 24 seeded per well is preferably 500 to 5000. In the culture using the well plate, one aggregate 30 is formed in one well. Therefore, when the number of kidney cells 24 seeded per well is 500 to 5000, the number of kidney cells constituting the aggregate 30 is 500 to 5000. When the number of constituent cells is 500 to 5000, the size of the aggregate 30 becomes 100 μm to 350 μm. By setting the number of constituent cells of the aggregate 30 to 500 or more or the size to 100 μm or more, it is easy to avoid the situation where the aggregate 30 is sucked together with the old culture solution when the culture medium is replaced. Furthermore, by setting the number of constituent cells of the aggregate 30 to 5,000 or less or the size to 350 μm or more, it is possible to obtain cultured cells 16 having better physiological functions.

[0072] When it is desired to form more aggregates 30, it is preferable to use a container such as a culture dish or a spinner flask for culturing. In this case, the size of the aggregates 30, in other words, the number of kidney cells 24 constituting one aggregate 30, can be controlled by adjusting the cell density in the container. For example, when an aggregate 30 having a size of about 100 μm to about 350 μm or a constituting cell number of 500 to 5000 is formed on a 60 mm culture dish (Sumitomo Bakelite Co., Ltd.), the cell density in the culture dish is adjusted to 1500 cells / cm 2 Above 15000 pcs / cm 2 Next, by subjecting the kidney cells 24 to static culture, shaking culture, or stirring culture, a plurality of aggregates 30 having a desired size or number of constituent cells can be formed at once.

[0073] Collagen I may also be added to the culture medium 28. Collagen I has the function of adhering kidney cells 24 to each other. Therefore, by culturing kidney cells 24 with a culture medium 28 containing collagen I, aggregates 30 can be formed more easily. The added collagen I is preferably full-length collagen I, but may also be collagen peptides obtained by fragmenting the α1 chain or α2 chain constituting collagen I, as well as fragments of each chain. In addition, the animal species of collagen I is not particularly limited, and not only collagen I from humans but also collagen I from other animals can be used.

[0074] The culture container 26 is preferably subjected to a cell non-adhesion treatment or is composed of a cell non-adhesion material. As a result, the aggregate 30 can be formed more easily. Examples of cell non-adhesion treatments include cell non-adhesion hydrogel coating treatment on the container surface, MPC (2-methacryloyloxyethyl phosphorylcholine) coating treatment, Proteosave (registered trademark) SS coating coarse particles, mirror polishing treatment, etc. Examples of cell non-adhesion materials include glass, etc. In addition, examples of cell non-adhesion materials include low-density polyethylene, medium-density polyethylene, polyvinyl chloride, polyethylene-vinyl acetate copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, poly(ethylene-vinyl acetate) copolymer, and mixtures of two or more of these polymers.

[0075] The culture period of kidney cells 24 is preferably more than 5 days. In addition, the culture period is preferably less than 90 days (i.e. less than 2160 hours), and more preferably less than 14 days (i.e. less than 336 hours). By setting the culture period to more than 5 days, it is easy to obtain cultured cells 16 in a state of expression of higher physiological functions. In addition, by setting the culture period to less than 90 days, it is easy to obtain cultured cells 16 in a state of expression of higher physiological functions. In addition, by setting the culture period to less than 14 days, it is easy to separate cultured cells from aggregates. The culture conditions are, for example, 37°C, 5% CO 2 During the culture period, the culture solution 28 is preferably replaced regularly. For example, the culture solution 28 is replaced every 2 days.

[0076] Next, if Figure 2 As shown in (C), the aggregate 30 is separated into individual cultured cells 16. Separation of the cultured cells 16 from the aggregate 30 can be performed by enzymatically treating the aggregate 30 using Accumax, trypsin / EDTA, Accutase, collagenase, EDTA, TrypLESelect, etc. In one example of enzyme dispersion treatment, culture is performed at 37°C for more than 10 minutes and pipetting is performed using a micropipette. Thus, a cell suspension in which a single cultured cell 16 is dispersed is obtained. The concentration of the enzyme used for the treatment can be appropriately set according to the type of cultured cell 16. However, when the enzyme concentration is high, the separation of the cultured cells 16 becomes easy, but on the other hand, the possibility of damage to the cell surface protein of the cultured cells 16 increases. Therefore, the enzyme concentration is preferably as low as possible.

[0077] In addition, if Figure 3 As shown in (A), a coating agent is applied to at least a portion of a substrate 12. Thus, a coating agent layer 14 is formed on the surface of the substrate 12. In addition, the preparation and separation of the cultured cells 16 and the application of the coating agent can be performed independently of each other. That is, the two steps can be performed either one first or in parallel.

[0078] Next, if Figure 3 As shown in (B), cultured cells 16 are seeded on the substrate 12. For example, by applying a cell suspension obtained by enzyme dispersion treatment on the substrate 12, the cultured cells 16 can be seeded on the substrate 12. Then, the cultured cells 16 are cultured on the substrate 12 to form a monolayer film of the cultured cells 16.

[0079] Culture cells at 16 × 10 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2 The following seeding densities are seeded on the substrate 12. For example, when the seeding area is 0.336 cm 2In the case of a 24-well plate with a cell culture insert used as the substrate, 16 cells were cultured at 2 × 10 4 pcs / hole or more 6×10 4 The inoculation density was less than 1 / well and the inoculation area was 1.131 cm 2 A cell culture insert for a 12-well plate or the like can also be used as the substrate 12 .

[0080] The cultured cells 16 are cultured on the substrate 12 for a period of 12 hours to 48 hours. The cultured cells 16 are cultured using, for example, REGM (Lonza) as a culture medium at 37°C and 5% CO. 2 The cultured cells 16 proliferate on the substrate 12 until they become confluent and then maintain the confluent state. Through the above steps, the cell-supporting complex 10 is obtained.

[0081] By setting the seeding density to 5 × 10 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2 When the culture time is set to 12 hours or more and 48 hours or less, a monolayer membrane composed of cultured cells 16 with good physiological functions and substantially no drug leakage can be obtained. More preferably, the cultured cells 16 are cultured at 5×10 4 Pieces / cm 2 Above 12×10 4 Pieces / cm 2 The following seeding density was inoculated on substrate 12. In the inoculation area of ​​0.336 cm 2 In the case of cell culture inserts, culture cells at 16 × 10 4 pcs / hole or more 4×10 4 The cells are seeded at a seeding density of less than 100 cells / well on the substrate 12. This makes it easy to extend the period during which the monolayer membrane of the cultured cells 16 is well maintained, in other words, the period during which the cell-supporting complex 10 can be used for drug evaluation.

[0082] Next, if Figure 3 As shown in (C), the cell support complex 10 is set on the support part 32. The drug evaluation device 42 of this embodiment adopts the form of migration assay (Transwell). Therefore, the substrate 12 is a cell culture plug-in, and the support part 32 is a hole of a well plate. The plug-in as the substrate 12 is accommodated in the hole as the support part 32. As a result, the space in the hole is divided into two by the cell support complex 10, forming a first chamber 34 and a second chamber 36 arranged across the cell support complex 10.

[0083] The first chamber 34 is arranged above the cell-supporting complex 10, that is, on the cell monolayer membrane side, and the second chamber 36 is arranged below the cell-supporting complex 10, that is, on the substrate 12 side. Through the above steps, a drug evaluation device 42 including the cell-supporting complex 10, the support portion 32, and the first chamber 34 and the second chamber 36 is obtained. In addition, in the above description, before the substrate 12 is set on the support portion 32, the cultured cells 16 are seeded on the substrate 12 to produce the cell-supporting complex 10, but the cell-supporting complex 10 may also be produced by seeding the cultured cells 16 on the substrate 12 after the substrate 12 is set on the support portion 32.

[0084] At least one of the first chamber 34 and the second chamber 36 contains the drug 50 to be evaluated. As an example, a liquid obtained by adding the drug 50 to a balanced salt solution such as HBSS buffer is contained in at least one chamber. Figure 3 (C) shows a drug evaluation device 42 in which a first liquid 38 is contained in a first chamber 34 and a second liquid 40 is contained in a second chamber 36. A drug 50 is added to at least one of the first liquid 38 and the second liquid 40. The drug 50 can contact the cultured cells 16 in the state of being contained in the first chamber 34 and in the state of being contained in the second chamber 36.

[0085] (Drug Evaluation Method)

[0086] The drug evaluation method of this embodiment includes the following method: using the drug evaluation device 42 obtained by the above-mentioned manufacturing method, the transport of the drug 50 by the cultured cells 16 provided in the cell-supporting complex 10 is evaluated. In other words, according to the drug evaluation device 42, the intake and excretion of the drug 50 by the first transporter 18 or the second transporter 20 can be evaluated. In addition, based on this evaluation, the effect of the drug 50 on the kidney can be predicted.

[0087] For example, by adding the drug 50 to the first liquid 38 located on the cell apical membrane side of the cultured cells 16 and measuring the amount of the drug 50 contained in the cultured cells 16 after a predetermined time, the uptake capacity of the first transporter 18 for the drug 50 can be evaluated. In addition, by adding the drug 50 to the second liquid 40 located on the cell basal membrane side of the cultured cells 16 and measuring the amount of the drug 50 contained in the cultured cells 16 after a predetermined time, the uptake capacity of the second transporter 20 for the drug 50 can be evaluated. In addition, by adding the drug 50 only to the first liquid 38 of the first liquid 38 and the second liquid 40 and measuring the amount of the drug 50 contained in the second liquid 40 after a predetermined time, the absorption capacity of the cultured cells 16 from the cell apical membrane side to the cell basal membrane side can be evaluated. In addition, by adding the drug 50 only to the second liquid 40 of the first liquid 38 and the second liquid 40 and measuring the amount of the drug 50 contained in the first liquid 38 after a predetermined period of time, the ability of the cultured cells 16 to excrete the drug 50 from the cell basal membrane side to the cell apical membrane side can be evaluated.

[0088] As described above, by setting the culture period of the cultured cells 16 on the substrate 12 to be 12 hours or more and 48 hours or less after inoculation, a monolayer of cells can be obtained in which the physiological function of the kidney is maintained in a good state and there is substantially no leakage of the drug. Therefore, the drug evaluation is preferably performed within a period of 12 hours or more and 48 hours from the inoculation of the cultured cells 16 on the substrate 12 and the start of the culture.

[0089] (Other devices using cell-supporting complexes)

[0090] The cell-supporting complex 10 can also be used in devices other than the above-mentioned migration assay (Transwell). Figure 4 (A)~ Figure 4 (E) is a diagram schematically showing an example of application of the cell-supporting complex 10. In addition, each figure shows a part of the structure in which the cell-supporting complex 10 is incorporated.

[0091] Figure 4 (A) shows a device in which a cell-supporting complex 10 using a hollow fiber membrane as a substrate 12 is assembled. In this device, a monolayer membrane of cultured cells 16 is formed in the lumen of the hollow fiber membrane. By flowing a liquid in the lumen of the hollow fiber membrane, the device can take a predetermined substance contained in the liquid into the cultured cells 16 and move it to the outside of the lumen of the hollow fiber membrane. This device can be used, for example, as a bioartificial kidney module that recovers useful substances from plasma components filtered by a blood filter.

[0092] Figure 4(B) shows a situation where a cell-supporting complex 10 is assembled on a microchannel chip. In the microchannel chip, a substrate 12 constitutes a microchannel. Then, a monolayer membrane of cultured cells 16 is formed on the inner wall of the microchannel. In the microchannel chip, a trace amount of liquid flows in the channel, that is, on the side where the cultured cells 16 are arranged. Then, a predetermined substance in the liquid is taken into the cultured cells 16. The microchannel chip can be used as a drug evaluation module for investigating the function of cells and the intake / excretion of drugs with a trace amount of liquid. The microchannel chip can be housed in a box and used.

[0093] Figure 4 (C) shows a case where the cell-supporting complex 10 constitutes a hollow microcarrier. Figure 4 (D) shows the situation that the cell support complex 10 constitutes a solid microcarrier. In the hollow microcarrier and the solid microcarrier, the substrate 12 constitutes the carrier body. Then, a monolayer membrane of the cultured cells 16 is formed on the outer surface of the substrate 12. In the hollow microcarrier and the solid microcarrier, a trace amount of liquid flows through the side where the cultured cells 16 are configured. Then, the specified substance in the liquid is taken into the cultured cells 16. Each microcarrier can be used as a drug evaluation module for investigating the function of cells and the intake / discharge of drugs with a trace amount of liquid. Each microcarrier can be accommodated in a box and used.

[0094] Figure 4 (E) shows a situation where a cell-supporting complex 10 is assembled on the bottom surface of a well of a well plate. When the cell-supporting complex 10 is arranged on the bottom surface of the well, the cultured cells 16 face the upper side of the well. In the well plate, a small amount of liquid 49 is injected into the well. Then, a predetermined substance in the liquid 49 is taken into the cultured cells 16. The well plate can be used, for example, as a drug evaluation module for investigating the function of cells and the intake / excretion of drugs with a small amount of liquid. In addition, the cell-supporting complex 10 can also be assembled in a culture dish (culture dish, etc.) instead of the well plate.

[0095] As described above, the method for producing the cell-supporting complex 10 of the present embodiment includes the steps of culturing kidney cells 24 in the form of aggregates 30 to produce cultured cells 16, and 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2 The following seeding density is set on the substrate 12, and the cultured cells 16 are cultured on the substrate 12 for a period of 12 hours to 48 hours to form a monolayer of the cultured cells 16. Thus, the monolayer of the cultured cells 16 with improved kidney physiological function and substantially no drug leakage can be used to evaluate the drug. Therefore, the accuracy of drug evaluation analysis can be improved. In addition, by setting the seeding density of the cultured cells 16 to 5×10 4 Pieces / cm2 Above 18×10 4 Pieces / cm 2 As a result, the period during which the cell-supporting complex 10 can be used for drug evaluation can be easily extended.

[0096] In addition, the manufacturing method of the drug evaluation device 42 of the present embodiment includes the following steps: by placing the cell-supporting complex 10 of the present embodiment on the supporting portion 32, or manufacturing the cell-supporting complex 10 in a state where the substrate 12 is placed on the supporting portion 32, a first chamber 34 and a second chamber 36 are formed, the first chamber 34 and the second chamber 36 are arranged across the cell-supporting complex 10, and at least one of the first chamber 34 and the second chamber 36 contains the drug 50 to be evaluated. Thus, the drug evaluation device 42 including the cell-supporting complex 10, the supporting portion 32, and at least one of the first chamber 34 and the second chamber 36 containing the drug 50 is obtained. In addition, the drug evaluation method of the present embodiment includes the following steps: using the drug evaluation device 42 to evaluate the transport of the drug 50 by the cultured cells 16. As described above, according to the present embodiment, the membrane transport evaluation using the migration assay (Transwell), which is a general method for dynamic evaluation of drugs, can be performed with higher accuracy.

[0097] The above describes the embodiments of the present invention in detail. The above embodiments only show specific examples when the present invention is implemented. The contents of the embodiments do not limit the technical scope of the present invention. Various design changes such as changes, additions, and deletions of constituent elements can be made without departing from the scope of the idea of ​​the invention specified in the claims. The new embodiment with the design changes has the effects of the combined embodiments and the deformations. In the above embodiments, the contents that can be subjected to such design changes are marked with marks such as "in this embodiment" and "in this embodiment" to emphasize, but the contents without such marks also allow design changes. Any combination of the above constituent elements is also effective as a mode of the present invention.

[0098] The embodiment can also be determined by the items described below.

[0099] (Item 1)

[0100] A method for manufacturing a cell-supporting complex (10) comprises the following steps:

[0101] Cultivating kidney cells (24) in the form of aggregates (30),

[0102] separating the aggregate (30) into individual cultured cells (16),

[0103] 5×10 4 Pieces / cm 2 Above 18×10 4 Pieces / cm2 The cultured cells (16) are inoculated on the substrate (12) at the following inoculation density, and

[0104] The cultured cells (16) are cultured on the substrate (12) for a period of 12 hours to 48 hours to form a monolayer membrane of the cultured cells (16).

[0105] (Item 2)

[0106] The method for producing a cell-supporting complex as described in item 1,

[0107] The seeding density was set to 5×10 4 Pieces / cm 2 Above 12×10 4 Pieces / cm 2 The following steps.

[0108] (Item 3)

[0109] A method for manufacturing a drug evaluation device (42), comprising the following steps:

[0110] The cell-supporting complex (10) is prepared by placing the cell-supporting complex (10) prepared by the method for preparing the cell-supporting complex (10) as described in item 1 or item 2 on a supporting portion (32), or a substrate (12) is provided on the supporting portion (32) and cultured cells (16) are inoculated on the substrate (12) by the method for preparing the cell-supporting complex (10) as described in item 1 or item 2, thereby preparing the cell-supporting complex (10); and

[0111] A first chamber (34) and a second chamber (36) are formed, the first chamber (34) and the second chamber (36) are arranged with the cell-supporting complex (10) interposed therebetween, and at least one of the first chamber (34) and the second chamber (36) contains a drug (50) to be evaluated.

[0112] (Item 4)

[0113] A method for evaluating a drug comprises the following steps:

[0114] Using the drug evaluation device (42) obtained by the method for producing the drug evaluation device (42) as described in Item 3, the transport of a drug (50) by cultured cells (16) included in a cell-supporting complex (10) is evaluated.

[0115] (Item 5)

[0116] A drug evaluation device (42) comprising:

[0117] A cell-supporting complex (10) produced by the method for producing a cell-supporting complex (10) as described in item 1 or item 2,

[0118] a support portion (32) supporting the cell-supporting complex (10), and

[0119] The first chamber (34) and the second chamber (36) are arranged with the cell-supporting complex (10) interposed therebetween, and at least one of the first chamber (34) and the second chamber (36) contains a drug (50) to be evaluated.

[0120] Example

[0121] Hereinafter, although an Example of the present invention is described, it is only an illustration for explaining the present invention appropriately, and it does not limit the present invention.

[0122] (Analysis of temporal changes in gene expression levels in cells constituting aggregates: Experiment 1)

[0123] First, 100,000 human proximal tubule epithelial cells (model CC-2553, Lonza) as an example of kidney cells were seeded on a 60 mm culture dish (Corning). The culture dish was an adhesion-treated culture dish coated with a gelatin solution (Sigma). Then, REGM (Lonza) was used as a culture medium and the cells were incubated at 37°C and 5% CO. 2 The cells were cultured under the conditions of . Since the culture dish was subjected to an adhesion treatment, the cells did not aggregate during the culture. By using the two-dimensional culture in the culture dish, proximal tubule epithelial cells with dedifferentiation and reduced physiological function were obtained.

[0124] Prepare a suspension of dedifferentiated cells. The cell concentration in the suspension is 10,000 cells / ml. Inoculate the cells by dropping 100 μl of the cell suspension on a 96-well U-bottom plate (Sumitomo Bakelite) treated for cell non-adhesion. Therefore, the inoculation density is 1,000 cells / well. Then, use REGM (model CC-3190, Lonza) as the culture medium and incubate at 37°C, 5% CO 2 The cells were cultured under the conditions of . The culture medium was replaced every 2 days. Through this culture, the cells formed aggregates.

[0125] Using RNeasy Mini Kit (RNeasy Mini Kit) (QIAGEN), extract mRNA and purify from cells just after inoculation (i.e. 0 hour) and cells constituting agglomerates cultured for 3, 4, 5, 6, 7, 8, 10, 12, 14 days (i.e. 72, 96, 120, 144, 168, 192, 240, 288, 336 hours). Then, using QuantiTect Reverse Transcription Kit (QuantiTect Reverse Transcription Kit) (QIAGEN), cDNA is synthesized from the purified mRNA. Using these cDNAs as templates, using Thermal Cycler Dice Real Time System I (thermal cycler dilution real-time system I) (TakaraBio), the expression level of each gene of APP1, SGLT2 and OAT1 is measured by real-time PCR.

[0126] These genes are genes related to the physiological functions of the kidney. Specifically, the AQP1 (aquaporin 1) gene is a gene encoding the AQP1 protein involved in water transport. The SGLT2 (sodium glucose cotransporter 2) gene is a gene encoding the SGLT2 protein involved in the transport of sodium and glucose. The OAT1 (organic anion transporter 1) gene is a gene encoding the OAT1 protein involved in the transport of drugs, especially the transport of relatively low molecular weight compounds.

[0127] For each gene, the ratio of the expression level after M days of culture (M = 3, 4, 5, 6, 7, 8, 10, 12, 14) to the expression level immediately after inoculation was calculated (day M / day 0). The results are shown in Figure 5 . Figure 5 Graph showing the temporal changes in gene expression levels in cells constituting aggregates. Figure 5 As shown, it was confirmed that the expression level of each gene increased by culturing human proximal tubule epithelial cells in the state of aggregates. In other words, it was confirmed that the physiological function of the cells was restored by aggregate culture. In particular, it was confirmed that the expression level of the OAT1 gene increased significantly in culture for more than 5 days.

[0128] In addition, the aggregates were cultured for a longer period of time, and the expression levels of kidney-related genes in the cells constituting the aggregates were analyzed over time. First, 1000 dedifferentiated proximal tubule epithelial cells (model CC-2553, Lonza) were added to a 96-well plate treated with cell non-adhesion, and 100 μl of REGM (model CC-3190, Lonza) was used to culture the plates at 37°C and 5% CO. 2 The cells were cultured under the conditions of . The culture medium was replaced every 2 days. Through this culture, the cells formed aggregates.

[0129] Using RNeasy Mini Kit (QIAGEN), mRNA was extracted and purified from cells cultured for 0, 4, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, 50, 60, 90 days (i.e., 0, 96, 192, 240, 288, 336, 384, 480, 600, 720, 840, 960, 1200, 1440, 2160 hours). Next, cDNA was synthesized from the purified mRNA using QuantiTect Reverse Transcription Kit (QIAGEN).

[0130] Using these cDNAs as templates, the expression levels of each gene of OAT1, OAT3 (organic anion transporter 3), OCT2 (organic cation transporter 2), MDR1 (multiple drug resistance 1), MATE1 (multidrug and toxin extrusion 1), MATE2 (multidrug and toxin extrusion 2), URAT1 (urate transporter 1), PEPT2 (peptide transporter 2) and SGLT2 were measured by real-time PCR using Thermal Cycler Dice Real Time System I (Takara Bio). In addition, as a positive control, renal cortex mRNA directly collected from human kidney was used.

[0131] For each gene, the ratio of the expression level after culture for M days (M = 4, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, 50, 60, 90) to the expression level immediately after inoculation (day M / day 0) was calculated. The results are shown in Figure 6 (A) Figure 6 (B) Figure 6 (C) Figure 7 (A) Figure 7 (B) Figure 7 (C) Figure 8 (A) Figure 8 (B) and Figure 8 (C). Figure 6 (A)~ Figure 8 (C) is a graph showing the temporal changes in gene expression levels in cells constituting aggregates.

[0132] like Figure 6 (A)~ Figure 8 As shown in (C), it was confirmed that the expression level of each gene of OTA1, OTA3, OCT2, MDR1, MATE1, and MATE2, which encode drug transporters related to drug excretion from the vascular side to the renal tubular side, increased by agglutination culture. In addition, it was confirmed that the state of increased expression level could be maintained until 90 days of culture. For each gene of URAT1, PEPT2, and SGLT2, which encodes endogenous transporters related to the reabsorption of substances from the renal tubular side to the vascular side, it was also confirmed that the expression level increased by agglutination culture, and the state of increased expression level could be maintained until 90 days of culture.

[0133] (Separation and reseeding of cells from aggregates: Experiment 2)

[0134] First, human proximal tubule epithelial cells (model CC-2553, Lonza) were revived and seeded on an adhesion-treated culture dish. REGM (model CC-3190, Lonza) was used to culture the dish at 37°C and 5% CO. 2 Two-dimensional culture was performed under the conditions of . The culture medium was replaced every 2 days. Before the cells converged, Accutase (Innovative Cell Technologies) was added to the culture dish and reacted at room temperature for 3 minutes to recover the dedifferentiated cells. Then, the cells were inoculated by dropping the cell suspension on a 96-well V-bottom plate (PrimeSurface plate 96V, Sumitomo Bakerite) that had been treated for cell non-adhesion. The inoculation density was 1000 cells / well. Then, REGM was used to culture at 37°C and 5% CO 2 The cells were cultured for 10 days under the conditions of . The culture medium was replaced every 2 days. Through this culture, the cells formed aggregates.

[0135] After 10 days of culture, the aggregates were collected and washed with PBS. Accumax (Innovative Cell Technologies) was then added to the aggregates, reacted at 37°C for 10 minutes, and separated into individual cells by pipetting. The reaction was further continued for 3 to 5 minutes to obtain a cell suspension in which almost all cells were separated.

[0136] Prepare a migration assay (Transwell) with a cell culture insert (model 662-641, Greiner) in each well of a 24-well plate (model 662-160, Greiner). For the insert, use an insert that has been dripped with a coating solution mixed with 3.3 μL / well of laminin (iMatrix-511silk, Nippi) and 100 μL / well of PBS and left to stand overnight in the refrigerator the day before use. Remove the coating solution dripped onto the insert before cell inoculation, and re-inoculate the cells by dripping a cell suspension onto the insert. The amount of cell suspension dripped is 150 μL / well. The inoculation density is 4×10 4 pcs / hole (12×10 4 Pieces / cm 2 ).

[0137] 600 μL / well of REGM (Lonza) was dripped into the bottom of the well (chamber 36) of the migration assay (Transwell) and the cells were cultured. The culture medium was replaced every day. The optical microscope image of the cells on the day after reseeding (21 hours after reseeding) is shown in Fig. 9 . Fig. 9 This is an optical microscope image of cells separated from the aggregates on the day after reseeding. Fig. 9 It was confirmed that the cells can be homogenized by subjecting the aggregates to enzyme treatment, and the homogenized cells can be uniformly dispersed on the insert and the cells can be grown to a confluent state. In other words, it was confirmed that a cell-supporting complex can be formed using cells separated from the aggregates.

[0138] (Analysis of the temporal change of the single-layer film: Test 3)

[0139] First, the formation of aggregates, preparation of cell suspension, and reseeding of cells were carried out in the same manner as in Experiment 2. The seeding density was 4×10 4 pcs / hole (12×10 4 Pieces / cm 2). Add REGM (Lonza) to the bottom of the wells of the migration assay (Transwell) to culture the cells. Change the culture medium every day. After re-seeding the cells, use the Millicell ERS-2 resistance measurement system (Millipore) to measure the TEER (transepithelial electrical resistance) of the cells on the plug over time. Specifically, on the day of re-seeding, the TEER is measured from re-seeding to 13 hours later, and after the day after re-seeding, the TEER is measured every 24 hours until 72 hours. TEER is a resistance value that serves as an indicator of the formation of a monolayer membrane of cells, and TEER is 100Ω·cm 2 When the above values ​​are obtained, it means that a uniform single-layer film with substantially no drug leakage is formed. The results are shown in Fig.10 .

[0140] Fig.10 is a graph showing TEER at various elapsed times from re-inoculation. Fig.10 As shown, TEER was 100 Ω·cm from 12 to 48 hours after reseeding. 2 Therefore, it was confirmed that a cell monolayer membrane with substantially no drug leakage can be obtained by setting the culture period of the cultured cells 16 on the substrate 12 to 12 hours or more and 48 hours or less.

[0141] In addition, Fig.10 The TEER of the case where the cells cultured in two dimensions were reseeded on the insert as a reference (24 hours after reseeding) is also shown. When the cells cultured in two dimensions were reseeded, the appearance was also a confluent monolayer. However, the TEER did not rise to 100 Ω·cm 2 This is believed to be because the physiological function of the cells was not restored in the two-dimensional culture, and the expression of the binding protein that contributes to the binding of the cells did not increase. On the other hand, it is believed that the expression of the binding protein in the cells that have restored their physiological function through agglutination culture also increased, so a uniform monolayer was formed, and the TEER increased to 100Ω·cm 2 above.

[0142] (Analysis of the relationship between seeding density and monolayer membrane: Experiment 4)

[0143] First, the formation of aggregates, preparation of cell suspension, and reseeding of cells were carried out in the same manner as in Experiment 2. The seeding density was 2×10 4 pcs / hole (5×10 4 Pieces / cm 2 ), 4×10 4 pcs / hole (12×10 4 Pieces / cm 2), 6×10 4 pcs / hole(18×10 4 Pieces / cm 2 ), 6.5×10 4 pcs / hole(19×10 4 Pieces / cm 2 ), 8×10 4 pcs / hole(24×10 4 Pieces / cm 2 ). REGM (Lonza) was added to the bottom of the wells of the migration assay (Transwell) to culture the cells. The culture medium was changed every day. After the cells were reseeded, the TEER of the cells on the insert was measured over time using the Millicell ERS-2 resistance value measurement system (Millipore). The results are shown in Fig.11 (A) and Fig.11 (B).

[0144] Fig.11 (A) and Fig.11 (B) is a graph showing the temporal changes in TEER at various seeding densities. Fig.11 (A) and Fig.11 As shown in (B), at a seeding density of 6.5×10 4 pcs / hole(19×10 4 Pieces / cm 2 ) or above, TEER did not rise to 100Ω·cm 2 On the other hand, at a seeding density of 2×10 4 pcs / hole~6×10 4 pcs / hole (5×10 4 Pieces / cm 2 ~18×10 4 Pieces / cm 2 ) case, TEER rises to 100Ω·cm 2 Therefore, it was confirmed that by setting the seeding density of the cultured cells 16 on the substrate 12 to 5×10 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2 In the following manner, a cell monolayer membrane with substantially no drug leakage can be obtained.

[0145] In addition, at a seeding density of 2×10 4 pcs / hole~4×10 4 pcs / hole (5×10 4 Pieces / cm 2 ~12×10 4 Pieces / cm 2 ) and the inoculation density was 6×104 pcs / hole(18×10 4 Pieces / cm 2 ) can maintain TEER at 100Ω·cm for a longer period of time. 2 Therefore, it was confirmed that by setting the seeding density to 5×10 4 Pieces / cm 2 Above 12×10 4 Pieces / cm 2 As a result, the usable period of the cell-supporting complex 10 or the drug evaluation device 42 can be easily extended.

[0146] In addition, Fig.11 (A) and Fig.11 (B) The seeding density was 4×10 4 The TEER of each well may not be consistent. Fig.11 In (A), TEER becomes 100 Ω·cm 21 hours after reseeding. 2 In addition, Fig.11 In (B), TEER becomes 100 Ω·cm 19 hours after reseeding 2 This is presumably because the start time of TEER measurement is different. Fig.11 (A) and Fig.11 (B) shows that the maximum TEER value is different. This is presumably because the TEER value is easily changed depending on the measurement position in the well, the amount of culture medium in the well, the state of migration measurement, etc. However, the present inventors have confirmed with high reproducibility that the maximum TEER value is 5×10 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2 When the seeding density is less than 100 Ω·cm and the culture period is 12 hours to 48 hours, the TEER becomes 100 Ω·cm 2 above.

[0147] (Analysis of temporal changes in gene expression levels in cells after reseeding: Experiment 5)

[0148] First, the formation of aggregates, preparation of cell suspension, and reseeding of cells were carried out in the same manner as in Experiment 2. The seeding density was 4×10 4 pcs / hole (12×10 4 Pieces / cm 2). Add REGM (Lonza company) to culture cells at the bottom of the well of migration assay (Transwell). Change the culture medium every day. Recover the cells on the plug after 0, 4, 24, 48, and 72 hours from re-seeding. The recovery of cells after 0 hour from re-seeding is synonymous with the recovery of cells from the aggregate. Use RNeasyMini Kit (RNeasy mini kit) (QIAGEN company), extract mRNA from the recovered cells and purify. Then, use QuantiTectWhole Transcriptome Kit (QuantiTect whole transcriptome kit) (QIAGEN company) to synthesize cDNA from the purified mRNA. Using these cDNA as templates, use Thermal Cycler Dice Real Time SystemI (thermal cycler dilution real-time system I) (Takara Bio company), measure the expression of OAT1 gene by real-time PCR method. Then, calculate the relative value of the expression of OAT1 gene relative to the expression of GAPDH gene as housekeeping gene. In addition, as a control, the expression level of the OAT1 gene in human renal cortex was also measured, and the relative value to the expression level of the GAPDH gene was calculated. The results are shown in Fig.12 .

[0149] Fig.12 : is a graph showing the temporal changes in the expression level of the OAT1 gene. Fig.12 As shown in Figure 1, the expression level of the OAT1 gene decreases over time after re-inoculation. At 48 hours after re-inoculation, the expression level of the OAT1 gene decreases to about 1 / 100 of the expression level of the OAT1 gene in the aggregate and human renal cortex. In other words, from the perspective of gene expression, it was confirmed that the physiological function required for drug evaluation was significantly reduced 48 hours after re-inoculation.

[0150] (Analysis 1 of the temporal changes in protein expression levels in cells after reseeding: Experiment 6)

[0151] Through test 5, it was confirmed that the gene related to the physiological function of the kidney disappears to a degree that is not suitable for drug evaluation 48 hours after re-inoculation. However, even if the gene related to the physiological function of the kidney disappears, if the expression of the protein encoded by the gene is still maintained, it can be said that the cell maintains a state suitable for drug evaluation. And, in general, the expression level of the protein is delayed from the decrease in the expression level of the gene. Therefore, the inventors further observed the expression of the OAT1 protein in the cells after re-inoculation by immunocytostaining.

[0152] First, the formation of aggregates, preparation of cell suspension, and reseeding of cells were carried out in the same manner as in Experiment 2. The seeding density was 4×104 pcs / hole (12×10 4 Pieces / cm 2 ). REGM (Lonza) was added to the bottom of the wells of the migration assay (Transwell) to culture the cells. The culture medium was changed every day. In addition, a primary antibody solution in which OAT1 antibody (model KE038, from rabbit, Transgenic) was diluted to 50 times with a blocking solution and a secondary antibody solution in which Alexa Fluor-labeled anti-rabbit antibody was diluted to 500 times with a blocking solution was prepared.

[0153] The cells were washed with PBS 2, 6, 24, 48, and 120 hours after reseeding, and fixed with paraformaldehyde phosphate buffer (PFA). After fixation for more than 30 minutes, the cells were washed with PBS. The PBS was replaced with a blocking solution and treated at room temperature for 1 hour. The blocking solution is a solution in which donkey serum is mixed in 0.1% PBT in a manner that the concentration is 5%. 0.1% PBT is a solution in which Triton X is added in a manner that the concentration is 0.1% in PBS. Next, the blocking solution was replaced with a primary antibody solution and treated overnight under refrigeration. Thereafter, the cells were washed three times with 0.1% PBT. Then, PBT was replaced with a secondary antibody solution and treated at room temperature for 1 hour. In addition, the cell nucleus was stained with Hoechst33342 to confirm the cells. Thereafter, the cells were washed three times with 0.1% PBT. PBT was replaced with PBS, and the cells were observed using a fluorescence microscope (Keyence). The results are shown in Fig.13 (A)~ Fig.13 (F).

[0154] Fig.13 (A)~ Fig.13 (F) is a graph showing the temporal changes in the expression level of the OAT1 protein. Fig.13 (A) is a fluorescence microscope image of cells cultured in two dimensions when they were replated as a reference (2, 6, 24, 48, and 120 hours after replated). Fig.13 (B)~ Fig.13 (F) is a fluorescence microscope image of cells cultured after reseeding (24 hours after reseeding). Fig.13 As shown in (A), the expression of OAT1 protein could not be observed in cells cultured in two dimensions. Fig.13 (B)~ Fig.13 As shown in (F), in the cells cultured in agglomeration, the expression of OAT1 protein can be observed until 48 hours after reseeding. In addition, OAT1 protein is uniformly expressed on the insert. Therefore, it is confirmed that the cultured cells 16 also maintain the physiological functions required for drug evaluation 48 hours after reseeding.

[0155] (Analysis 2 of the temporal changes in protein expression levels in cells after reseeding: Experiment 6)

[0156] First, the formation of aggregates, preparation of cell suspension, and reseeding of cells were carried out in the same manner as in Experiment 2. The seeding density was 4×10 4 pcs / hole (12×10 4 Pieces / cm 2 ). The cells on the plug were recovered after 0, 4, 24, 28, and 48 hours from the time of re-seeding. Proteins were extracted from the recovered cells, and the extracted proteins were used to perform known proteomic analysis. In the proteomic analysis, the total protein (Total Lysate) and cell membrane protein (Plasma membrane) of the recovered cells were comprehensively analyzed for expression. Then, based on the results of the proteomic analysis, the expression level of the main transporter in the kidney was calculated. The results are shown in Fig.14 (A) Fig.14 (B) and Fig.14 (C).

[0157] Fig.14 (A) and Fig.14 (B) is a graph showing the expression levels of kidney-related transporters among cell membrane proteins. Fig.14 (C) is a graph showing the temporal changes in the expression level of kidney-related transporter proteins in total protein. Fig.14 (A)~ Fig.14 (C) shows the relative value of the expression level of the transporter protein after 0 hours from re-inoculation. Fig.14 (A) and Fig.14 (B) shows the expression level 24 hours after reinoculation. Fig.14 (C) shows the expression levels up to 48 hours after reinoculation.

[0158] In addition, Fig.14(A) shows the expression levels of the following proteins: SLC transporter (solute carrier transporter) proteins as uptake system, GLUT2 (glucose transporter 2), GLUT9 (glucose transporter 9), OCTN1 (organic cation / carnitine transporter 1), OCTN2 (organic cation / carnitine transporter 1), PEPT1 (peptide transporter 1), OAT1, OCT2, MATE1, MATE2-K (multidrug and toxin extrusion transporter 2-K), and OATP4C1 (organic anion-transporting polypeptide 4C1). Fig.14 (B) shows the expression levels of the following proteins: ABC transporter (ATP Binding Cassette transporter) proteins, ABCA3, ABCB1 (MDR1), ABCC1, ABCC2, ABCC3, and ABCC4, which are ABC transporters of the excretion system. Fig.14 (C) shows the expression levels of each protein of OAT1, OCT2, MATE1, MATE2, and ABCB1.

[0159] like Fig.14 As shown in (A), the expression level of each SLC transporter was maintained at 40% or more in cells 24 hours after reseeding. Fig.14 As shown in (B), the expression level of each ABC transporter was maintained at more than 60% in cells 24 hours after reseeding. Fig.14 As shown in (C), the expression level of each transporter protein was maintained at more than 25% from the time of reseeding to 48 hours. If the expression level of the transporter protein remains more than 20% relative to the expression level in the aggregate, it can be said that the cell maintains the physiological function required for drug evaluation. Therefore, it was confirmed that the cultured cells 16 also maintained the physiological function required for drug evaluation 48 hours after reseeding.

[0160] (Membrane transport evaluation using migration assay (Transwell): Test 7)

[0161] First, aggregates were formed by the same method as in Experiment 2, and a cell suspension was prepared from the aggregates, and the cells were seeded on the insert. The seeding density was 4×10 4 pcs / hole (12×10 4 Pieces / cm 2 ). Then, on the day after reinoculation (within 18 to 24 hours from reinoculation), membrane transport evaluation was performed using a migration assay (Transwell). In the membrane transport evaluation, radioactive isotope-labeled Adefovir and radioactive isotope-labeled Metformin were used. Adefovir is a drug that becomes a substrate for OAT1, that is, a drug that is transported by OAT1. Metformin is a drug that becomes a substrate for OCT2, that is, a drug that is transported by OCT2.

[0162] The analysis buffer to which each matrix is ​​added in the HBSS buffer is added to the plug-in of the migration assay (Transwell) and processed for a certain time. In the test of Adefovir (Adefovir), the processing time is set to 2 minutes. In the test of Metformin (Metformin), the processing time is set to 10 minutes. Afterwards, the cells are recovered and a cell suspension is prepared, and the radioactive isotope in the cell suspension is quantified. Thus, the intake of the matrix from the cell top membrane side to the cell is evaluated. In addition, for the situation of adding the inhibitor Probenecid (probenecid) of OAT1 in the analysis buffer and the situation of adding the inhibitor Cimetidine (cimetidine) of OCT2, the intake of the matrix is ​​evaluated in the same manner respectively.

[0163] In addition, the analysis buffer is added to the wells of the migration assay (Transwell) and treated for a certain period of time. In the test of Adefovir, the treatment time is set to 2 minutes. In the test of Metformin, the treatment time is set to 10 minutes. Afterwards, the cells are recovered and a cell suspension is prepared, and the radioactive isotope in the cell suspension is quantified. Thus, the uptake of the matrix from the cell basement membrane side to the cell is evaluated. In addition, the uptake of the matrix is ​​evaluated in the same manner for the case where the inhibitor Probenecid is added to the analysis buffer and the case where the inhibitor Cimetidine is added. The results are shown in Fig.15 (A) and Fig.15 (B).

[0164] Fig.15 (A) is a graph showing the results of membrane transport of Adefovir. Fig.15 (B) is a graph showing the results of membrane transport of metformin. Fig.15 (A) and Fig.15 In (B), "A to C" indicates the amount of matrix taken up into the cell from the apical membrane side, and "B to C" indicates the amount of matrix taken up into the cell from the basal membrane side.

[0165] like Fig.15 (A) and Fig.15 As shown in (B), it was confirmed that both Adefovir (Adefovir) and Metformin (Metformin) were taken into the cell. In addition, OAT1 and OCT2 are proteins expressed in large quantities on the cell basement membrane side. Therefore, for any one of Adefovir (Adefovir) and Metformin (Metformin), the amount of intake from the cell basement membrane side to the cell is more than from the cell apical membrane side. In addition, on the substrate, the apical membrane side and the base end membrane side are sometimes in opposite directions for some cells. Therefore, the intake of the substrate when adding the substrate to the plug-in ("A to C" in the figure) was also observed.

[0166] In addition, due to the addition of each inhibitor, the intake of the substrate was significantly reduced compared to the case where no inhibitor was added. It can be confirmed that the intake of Adefovir is based on OAT1, and the intake of Metformin is based on OCT2. As described above, it was confirmed that the cell-supporting complex 10 and the drug evaluation device 42 of this embodiment can be used to implement high-precision drug dynamic evaluation.

[0167] [Industrial Applicability]

[0168] The present invention can be used for a method for producing a cell-supporting complex, a method for producing a drug evaluation device, a drug evaluation method, and a drug evaluation device.

[0169] [Explanation of Reference Numerals]

[0170] 10 cell-supporting complex, 12 substrate, 16 cultured cells, 24 kidney cells, 30 aggregates, 32 supporting portion, 34 first chamber, 36 second chamber, 42 drug evaluation device, 50 drug.

Claims

1. A method for producing a cell-supporting complex, comprising the following steps: Cultivating kidney cells in the form of aggregates, separating the aggregates into individual cultured cells, 5×10 4 Pieces / cm 2 Above 18×10 4 Pieces / cm 2 The cultured cells are seeded on the substrate at the following seeding density, and The cultured cells are cultured on the substrate for a period of 12 hours to 48 hours to form a monolayer film of the cultured cells.

2. The method for producing a cell-supporting complex according to claim 1, Including setting the seeding density to 5×10 4 Pieces / cm 2 Above 12×10 4 Pieces / cm 2 The following steps.

3. A method for manufacturing a drug evaluation device, comprising the following steps: The cell-supporting complex produced by the method for producing a cell-supporting complex according to claim 1 or 2 is placed on a support, or a substrate is provided on a support and cultured cells are seeded on the substrate by the method for producing a cell-supporting complex according to claim 1 or 2 to produce a cell-supporting complex, and A first chamber and a second chamber are formed, the first chamber and the second chamber are arranged with the cell-supporting complex interposed therebetween, and at least one of the first chamber and the second chamber contains a drug to be evaluated.

4. A method for evaluating a drug, comprising the following steps: Using the drug evaluation device obtained by the method for manufacturing a drug evaluation device according to claim 3, the transport of a drug by cultured cells included in the cell-supporting complex is evaluated.

5. A drug evaluation device comprising: A cell-supporting complex produced by the method for producing a cell-supporting complex according to claim 1 or 2, a supporting portion that supports the cell-supporting complex, and A first chamber and a second chamber are arranged with the cell-supporting complex interposed therebetween, and at least one of the first chamber and the second chamber contains a drug to be evaluated.

Images