Proximal tubule biomimetic system

Abstract

To provide a biomimetic system that mimics the human proximal tubule, which is a high-performance in vitro model that can reproduce the physiological functions of the human proximal tubule in vitro and can be used as an evaluation tool in drug discovery.SOLUTION: The present invention provides a proximal tubule biomimetic system comprising a microfluidic device and LTL-positive cells derived from pluripotent stem cells contained within the microfluidic device. The microfluidic device has (A) a device body, (B) a first chamber provided in the device body, (C) a second chamber provided in the device body, and (D) a porous membrane positioned between the first chamber and the second chamber, separating the first chamber from the second chamber. The LTL-positive cells are contained within the first chamber and adhere to a first surface of the porous membrane facing the first chamber. The first surface of the porous membrane is coated with an extracellular matrix.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a proximal tubule biomimetic system, a kit for producing a proximal tubule biomimetic system, a microfluidic device for a proximal tubule biomimetic system, and a method for producing a proximal tubule biomimetic system. [Background technology]

[0002] A micro-physiological system (MPS) is an in vitro culture system that recreates a culture environment similar to that of a living organism (in vivo) in a microspace created using MEMS (microelectromechanical systems) technology. This biomimetic system can reproduce the physiological functions of living tissue more accurately than conventional cell-based assays and has the potential to replace time-consuming and expensive animal testing. Therefore, it is expected to replace cell or animal experiments in drug discovery, such as screening of candidate drugs and pharmacokinetic or toxicity experiments.

[0003] Renal tubules are winding tubes measuring 20-30 μm in diameter and 4-7 cm in length that extend from Bowman's capsule, which surrounds the glomerulus in the kidney. They divide into the proximal tubule, the loop of Henle, and the distal tubule. Finally, multiple distal tubules join together to form a collecting duct, which opens into the renal papilla. Renal tubules play an important role in reabsorbing necessary substances from the blood (enzymes, sugars, amino acids, etc.) while simultaneously excreting drugs and toxins into the urine. The proximal tubule is responsible for up to 80% of nutrient reabsorption from primary urine, making it a site where drugs in the circulating blood tend to accumulate.

[0004] In recent years, many biomimetic systems (also known as proximal tubule-on-a-chip) have been developed that mimic the physiological functions of the proximal tubule, such as its filtration function, in vitro. For example, Patent Document 1 (US 2020 / 0269234 A1) discloses a microfluidic device including a membrane, proximal tubule cells on a first surface of the membrane, and glomerular microvascular endothelial cells on a second surface of the membrane. Patent Document 2 (WO 2020 / 172670 A1) discloses microfluidic kidney-on-a-chip microfluidic devices, such as a human proximal tubule-kidney-chip, a glomerulus (kidney)-chip, and a collecting duct (kidney)-chip. Furthermore, Patent Document 3 (WO2016 / 057571A1) discloses a three-dimensional (3D) renal tubule model comprising a renal interstitial layer containing renal fibroblasts and endothelial cells, and a renal epithelial tissue layer containing renal tubular epithelial cells, with the renal epithelial tissue in contact with the renal interstitial tissue layer.

[0005] Furthermore, Non-Patent Document 1 (Jang, KJ et al., Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integr Biol (Camb) 5, 1119-1129, doi:10.1039 / c3ib40049b (2013)) discloses a human kidney proximal tubule-on-a-chip in which epithelial cell polarization and primary cilium formation are improved compared to conventional Transwell (Transwell®) culture systems. Non-Patent Document 2 (Vedula EM et al., A microfluidic renal proximal tubule with active reabsorptive function. PLoS ONE 12(10):e0184330. https: / / doi.org / 10.1371 / journal.pone.0184330 (2017)) discloses a microfluidic renal proximal tubule with active reabsorptive function, which is formed by co-culturing primary cultured human proximal tubule epithelial cells (hRPTEC) and human vascular endothelial cells (hMVEC). Furthermore, Non-Patent Document 3 (Lin, NYC et al., Renal reabsorption in 3D vascularized proximal tubule models. Proceedings of the National Academy of Sciences of the United States of America 116, 5399-5404, doi:10.1073 / pnas.1815208116 (2019)) discloses a proximal tubule model in which 3D blood vessels are formed by co-culturing immortalized proximal tubule epithelial cells and glomerular endothelial cells.

[0006] However, although these conventional proximal tubule models can mimic the function of human proximal tubules to some extent, they do not accurately reflect the in vivo situation in which multiple transporters are involved in a complex manner. [Prior art documents] [Patent documents]

[0007] [License 1] US2020 / 0269234A1 [License 2] International Open 2020 / 172670A1 [License 3] International Open 2016 / 057571A1 [Non-licensed literature]

[0008] [Non-licensed Document 1] Jang, KJ et al., Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integr Biol (Camb) 5, 1119-1129, doi:10.1039 / c3ib40049b (2013) [Non-licensed Document 2] Vedula EM et al., A microfluidic renal proximal tubule with active reabsorptive function. PLoS ONE 12(10):e0184330. https: / / doi.org / 10.1371 / journal.pone.0184330 (2017) [Non-licensed Document 3] Lin, NYC et al., Renal reabsorption in 3D vascularized proximal tubule models. Proceedings of the National Academy of Sciences of the United States of America 116, 5399-5404, doi:10.1073 / pnas.1815208116 (2019) [Non-licensed Document 4] Ramin Banan Sadeghian et al., A bioinspired human proximal tubule-on-a-chip with both Epithelial and endothelial tissue layers for online Monitoring of nephrotoxicity and filtration properties, p. 1631-1634, 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, November 11-15, 2018, Kaohsiung, Taiwan Summary of the Invention [Problem to be solved by the invention]

[0009] Under the circumstances described above, the present invention aims to provide a high-performance in vitro proximal tubule model that is a biomimetic system that mimics the human proximal tubule, can reproduce the physiological functions of the human proximal tubule in vitro, and can be used as an evaluation tool in drug discovery. [Means for solving the problem]

[0010] The present inventors previously developed a biomimetic system with proximal tubule epithelial cells and vascular endothelial cells on both sides of a porous membrane (Non-Patent Document 4). The present inventors further improved and investigated this biomimetic system, and by using LTL-positive cells derived from iPS cells instead of proximal tubule epithelial cells, they were able to obtain a human proximal tubule model that expresses the organic anion transporters SLC22A6 (OAT1) and SLC22A8 (OAT3) and can reproduce the intracellular uptake and transport of substrates as in vivo. Specifically, the present invention is as follows:

[0011] [1] A proximal tubule biomimetic system, comprising: A method for producing a pluripotent stem cell-derived LTL-positive cell culture medium comprising: a microfluidic device; and LTL-positive cells derived from pluripotent stem cells contained in the microfluidic device. The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber and separating the first chamber from the second chamber; and the LTL-positive cells are contained in the first chamber and adhere to a first surface of the porous membrane facing the first chamber; The first surface of the porous membrane is coated with an extracellular matrix. A proximal tubule biomimetic system characterized by: [2] The proximal tubule biomimetic system described in [1], wherein the pluripotent stem cells are iPS cells or ES cells. [3] The proximal tubule biomimetic system described in [2], wherein the LTL-positive cells are LTL-positive cells derived from kidney organoids produced from iPS cells or ES cells. [4] The proximal tubule biomimetic system described in [3], wherein the LTL-positive cells adhering to the first surface of the porous membrane facing the first chamber express OAT1 and OAT3. [5] A kit for producing a proximal tubule biomimetic system, comprising: A microfluidic device and LTL-positive cells derived from pluripotent stem cells, The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber and separating the first chamber from the second chamber; and A first surface of the porous membrane facing the first chamber is coated with an extracellular matrix. A kit for producing a proximal tubule biomimetic system, comprising: [6] The kit for producing a proximal tubule biomimetic system described in [5], wherein the pluripotent stem cells are iPS cells or ES cells. [7] The kit for producing a proximal tubule biomimetic system described in [6], wherein the LTL-positive cells are LTL-positive cells derived from kidney organoids produced from iPS cells or ES cells. [8] A microfluidic device for a proximal tubule biomimetic system, comprising: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber and separating the first chamber from the second chamber; and A microfluidic device for a proximal tubule biomimetic system, wherein a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix. [9] A method for producing a proximal tubule biomimetic system, comprising: (1) providing a microfluidic device; (2) preparing LTL-positive cells derived from pluripotent stem cells; and (3) Culturing LTL-positive cells derived from pluripotent stem cells in a microfluidic device Including, The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber and separating the first chamber from the second chamber; and a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix; In the step (3), LTL-positive cells derived from pluripotent stem cells are seeded in the first chamber and cultured, and the LTL-positive cells are allowed to adhere to the first surface of the porous membrane. A method for producing a proximal tubule biomimetic system, comprising:

[10] A method for producing a proximal tubule biomimetic system as described in claim 9, wherein the pluripotent stem cells are iPS cells or ES cells.

[11] The method for producing a proximal tubule biomimetic system according to

[10] , wherein the LTL-positive cells are LTL-positive cells derived from kidney organoids produced from iPS cells or ES cells. [Effects of the Invention]

[0012] The biomimetic system of the present invention enables in vitro evaluation of human proximal tubules that is closer to that of human proximal tubules than conventional biomimetic systems. This not only makes it possible to easily evaluate the efficacy, nephrotoxicity, and pharmacokinetics of candidate drugs in preclinical trials in drug discovery, but also serves as an alternative to animal experiments and cultured cell experiments, thereby saving resources such as experimental animals and leading to a reduction in the dropout of candidate drugs in clinical trials and a reduction in drug discovery costs. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram of a proximal tubule biomimetic system of the present invention. [Figure 2] 1 is an exploded perspective view of a microfluidic device according to one embodiment, and a cross-sectional view of a biomimetic system. [Figure 3] FIG. 1 is a diagram showing a schematic diagram of a method for producing a proximal tubule biomimetic system (iPSC-PToC) of the present invention. [Figure 4] FIG. 1 shows the results of culturing LTL-positive cells derived from kidney organoids induced from iPS cells on a porous membrane. [Figure 5]FIG. 1 shows the results of evaluating the barrier function of epithelial tissue formed on a porous membrane of a proximal tubule biomimetic system (iPSC-PToC). [Figure 6] FIG. 1 shows the results of evaluating the barrier function of epithelial tissue formed on a porous membrane of a proximal tubule biomimetic system (iPSC-PToC). [Figure 7] FIG. 1 shows transporter (OAT1, OAT3) expression in epithelial tissue formed on a porous membrane of a proximal tubule biomimetic system (iPSC-PToC). [Figure 8] FIG. 1 shows cross-sectional fluorescence images of epithelial tissue formed on a porous membrane in a proximal tubule biomimetic system (iPSC-PToC). [Figure 9] FIG. 1 shows the results of measuring the distance (Δd) between the position of OAT1 expression and the position of the nucleus (DAPI) in epithelial tissue formed on the porous membrane of a proximal tubule biomimetic system (iPSC-PToC). [Figure 10] FIG. 1 shows the results of measuring the distance (Δd) between the position of OAT1 expression and the position of the nucleus (DAPI) in epithelial tissue formed on the porous membrane of a proximal tubule biomimetic system (iPSC-PToC). [Figure 11] FIG. 1 is a schematic diagram showing the method for testing fluorescein uptake and transport using the proximal tubule biomimetic system (iPSC-PToC). [Figure 12] FIG. 1 is a diagram showing a schematic diagram of fluorescein uptake and transport from the vascular side (B: basal) to the primary urine side (A: apical) and the effect of inhibitors. [Figure 13] FIG. 1 shows the results of a fluorescein uptake test using a proximal tubule biomimetic system (iPSC-PToC) (fluorescence micrographs). [Figure 14] FIG. 1 shows the results of fluorescein uptake tests under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 15] FIG. 1 shows the results of fluorescein uptake tests under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 16]FIG. 1 shows the results of a B to A transport test of fluorescein under various conditions using a renal proximal tubule biomimetic system (iPSC-PToC). [Figure 17] FIG. 1 shows the results of comparing MPR4 gene expression in epithelial tissues derived from different kidney organoids formed on the porous membrane of a proximal tubule biomimetic system (iPSC-PToC). [Figure 18] FIG. 1 shows the results of fluorescein uptake tests under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 19] FIG. 1 shows the results of a B to A transport test of fluorescein under various conditions using a renal proximal tubule biomimetic system (iPSC-PToC). [Figure 20] FIG. 1 shows the results of a transport test of Adefovir under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 21] FIG. 1 shows the results of PAH transport tests under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 22] FIG. 1 shows the results of a transport test of Rosuvastatin under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 23] FIG. 1 shows the results of substrate transport studies under various conditions using a proximal tubule biomimetic system (iPSC-PToC) (inhibitory effect of MK571). [Figure 24] FIG. 1 shows the results of metformin transport tests under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 25] FIG. 1 shows the results of metformin transport tests under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 26] FIG. 1 shows the results of a quinidine transport test under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 27]FIG. 1 shows the results of a quinidine transport test under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 28] FIG. 1 shows the results of cell damage caused by aristolochic acid under various conditions using a renal proximal tubule biomimetic system (iPSC-PToC) (LDH assay). [Figure 29] FIG. 1 shows the results of cell damage caused by cisplatin under various conditions using a proximal tubule biomimetic system (iPSC-PToC). [Figure 30] This shows the percentage of dead cells induced by cisplatin under various conditions using a proximal tubule biomimetic system (iPSC-PToC) (quantification of cell death from fluorescent images). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below.

[0015] <Proximal tubule biomimetic system> The proximal tubule biomimetic system of the present invention comprises a microfluidic device and LTL-positive cells derived from pluripotent stem cells contained in the microfluidic device, wherein the microfluidic device: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber, separating the first chamber from the second chamber; The LTL-positive cells are contained in the first chamber and adhere to a first surface of the porous membrane facing the first chamber, and the first surface of the porous membrane is coated with an extracellular matrix. Each component of the proximal tubule biomimetic system of the present invention is described in detail below. In this specification, the proximal tubule biomimetic system is also referred to as a proximal tubule-on-a-chip (PToC). In this specification, the proximal tubule biomimetic system of the present invention is also referred to as an "iPSC-PToC" or "hiPSC-derived kidney organoid-based PToC."

[0016] [Microfluidic Devices] The microfluidic device of the present invention is a microfluidic device for a proximal tubule biomimetic system, comprising: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber and separating the first chamber from the second chamber; and a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix.

[0017] As shown in Fig. 2, a microfluidic device according to one embodiment of the present invention includes a device body 1, a first chamber 2 provided in the device body 1, a second chamber 3 provided in the device body 1, and a porous membrane 4 located between the first chamber 2 and the second chamber 3, separating the first chamber 2 from the second chamber 3. The microfluidic device 1 may further include a first supply unit 5 provided in the device body 1 and communicating with the first chamber 2, and a second supply unit 6 provided in the device body 1 and communicating with the second chamber 3. Note that Fig. 2 shows an exploded perspective view of the microfluidic device 1, and the microfluidic device 1 is in use with the respective parts shown in Fig. 2 overlapping each other. The second supply unit 6 is formed by communication between a through-hole 6a and a recess 6b.

[0018] The microfluidic device 1 is not particularly limited as long as it is made of a material suitable for cell culture. Examples of materials include, but are not limited to, plastic resins such as polydimethylsiloxane (PDMS). The shape of the microfluidic device is not particularly limited, and the size is not particularly limited as long as it is a normal size for a microfluidic device, such as a size of 10 mm to 100 mm x 10 mm to 100 mm x 5 to 20 mm.

[0019] The first chamber 2 is a chamber (space) for accommodating LTL-positive cells derived from pluripotent stem cells and a first culture medium, and for forming and maintaining a cell layer of LTL-positive cells derived from pluripotent stem cells. There are no particular limitations on its shape or size. The height (depth) of the first chamber 2 can be set appropriately, and may be, for example, 50 μm to 2 mm, 50 μm to 1 mm, 50 μm to 500 μm, or 100 μm to 300 μm. The length of the first chamber 2 may be 1 mm to 20 mm, and the width may be 1 mm to 5 mm.

[0020] The second chamber 3 is a chamber (space) for accommodating the second culture medium. There are no particular limitations on its shape and size. The height (depth) of the second chamber 3 can be set appropriately, and may be, for example, 50 μm to 2 mm, 50 μm to 1 mm, 50 μm to 500 μm, or 100 μm to 300 μm. The length of the second chamber 3 may be, for example, 1 to 20 mm, and the width may be, for example, 1 to 5 mm. Furthermore, the second chamber 3 may be the same size as the first chamber 2.

[0021] The porous membrane 4 is a porous membrane that separates the first chamber 2 and the second chamber 3, and the first chamber 2 and the second chamber 3 form independent spaces that do not communicate with each other due to the partition made of this porous membrane 4. The porous membrane 4 has a surface facing the first chamber 2 (first surface 41) and a surface facing the second chamber 3 (second surface 42), and pluripotent stem cell-derived LTL-positive cells are adhered to the first surface 41 of the porous membrane 4.

[0022] The porous membrane 4 preferably has pores of 0.4 μm to 3.0 μm, and more preferably 1 μm to 3.0 μm, which allows the porous membrane 4 to function as a basement membrane for cells.

[0023] The material of the porous membrane 4 is not particularly limited as long as it allows interaction between cells on both sides of the porous membrane 4, and examples thereof include polyethylene terephthalate (PET) and polycarbonate, with PET being preferred. The material of the porous membrane 4 is also preferably a material to which cells can adhere. The size of the porous membrane 4 is determined by the sizes of the first chamber 2 and the second chamber 3, and the thickness of the membrane is not particularly limited, and may be, for example, 5 μm to 50 μm, and preferably 5 μm to 20 μm, or 5 μm to 10 μm.

[0024] The surface (first surface 41) of the porous membrane 4 facing the first chamber 2 is coated with an extracellular matrix such as laminin, fibronectin, collagen, proteoglycan, etc. As such an extracellular matrix, laminin is particularly preferred, and examples thereof include laminin-511, laminin-332, laminin-211, laminin-121, laminin-221, laminin-3A11, laminin-3A21, laminin-411, laminin-421, laminin-521, laminin-213, laminin-423, laminin-523, laminin-212 / 222, laminin-522, and laminin-111, with laminin-511 and laminin-332 being more preferred. By coating the first surface 41 of the porous membrane 4 with the above-mentioned extracellular matrix, when pluripotent stem cell-derived LTL-positive cells are cultured on the porous membrane 4, uniform epithelial tissue with high barrier function can be formed, and a human proximal tubule model can be obtained that expresses organic anion transporters SLC22A6 (OAT1) and SLC22A8 (OAT3) and can reproduce the intracellular uptake and transport of substrates as in vivo.

[0025] Prior to seeding pluripotent stem cell-derived LTL-positive cells, the porous membrane 4 is sterilized by filling the first chamber 2 and the second chamber 3 with 70% ethanol or the like, drying overnight at 60°C, and then irradiating with UV light for approximately one hour. The method for coating the porous membrane 4 with an extracellular matrix is ​​not particularly limited; for example, the first chamber 2 and the second chamber 3 may be filled with a coating solution containing the extracellular matrix and incubated for 10 minutes to 24 hours, preferably 1 hour to 8 hours, more preferably 4 hours to 6 hours, and even more preferably 5 hours. The concentration of the extracellular matrix in the coating solution can be adjusted appropriately depending on the type of extracellular matrix, but is, for example, 0.01 mg / mL to 1.0 g / mL, preferably 0.05 mg / mL to 100 mg / mL, more preferably 0.08 mg / mL to 10 mg / mL, and even more preferably 0.1 mg / mL to 1.0 mg / mL.

[0026] The microfluidic device 1 may further include a first supply unit provided on the device body 1 and communicating with the first chamber 2, and a second supply unit provided on the device body 1 and communicating with the second chamber 3. The first supply unit 5 is a supply unit for supplying a first medium to the first chamber 2 to perform perfusion, and the second supply unit 6 is a supply unit for supplying a second medium to the second chamber 3 to perform perfusion. It is preferable to have two or more first supply units 5 or second supply units 6 to perform unidirectional perfusion. For example, when there are two supply units, one can be a medium input unit and the other can be a medium output unit. The first supply unit 5 and the second supply unit 6 may be connected to a pump to perform perfusion on the microfluidic device 1. The sizes of the first supply unit 5 and the second supply unit 6 can be appropriately determined by those skilled in the art. Perfusion culture is thought to affect the morphology and maturation of brush borders.

[0027] [LTL-positive cells derived from pluripotent stem cells] LTL-positive cells derived from pluripotent stem cells are cells that can be selected by LTL (Lotus Tetragonolobus lectin), which specifically binds to renal tubules. LTL-positive cells are thought to have the functions of proximal tubule cells (PT), i.e., human renal proximal tubule epithelial cells. LTL-positive cells secrete laminin and collagen IV, and are thought to contribute to the improvement of proximal tubule function by increasing mRNA and protein expression levels.

[0028] Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells used in the present invention include embryonic stem cells (ES cells: Embryonic stem cells), induced pluripotent stem cells (iPS cells: induced pluripotent stem cells), and EG cells (Embryonic germ cells). Pluripotent stem cells also include Muse cells (Multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSCs) and GS cells prepared from germ cells (e.g., testes). As pluripotent stem cells in the present invention, ES cells and iPS cells are preferred, with iPS cells being more preferred.

[0029] iPS cells are cells induced to pluripotency by reprogramming somatic cells using known methods. Since Yamanaka et al. established iPS cells in mouse cells in 2006 (Cell, 2006, 126(4), pp. 663-676), various established induced pluripotent stem cell lines have become available. For example, human iPS cell line CRL1502.3 (MCRI, Australia) and human induced pluripotent stem cell lines established at Kyoto University, such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, are available from Kyoto University and iPS Academia Japan, Inc. As established induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University are available from Kyoto University. The iPS cells of the present invention are not particularly limited, and are preferably human iPS cells, or may be any of the above-mentioned iPS cell lines and known iPS cells thereof.

[0030] LTL-positive cells derived from pluripotent stem cells can be obtained by inducing differentiation of pluripotent stem cells. LTL-positive cells are preferably LTL-positive cells derived from kidney organoids prepared from iPS cells. Kidney organoids can be prepared from iPS cells using known differentiation methods. The method for preparing kidney organoids is not particularly limited, but the protocols of Takasato et al. ("Generation of kidney organoids from human pluripotent stem cells," Nat Protoc, vol. 11, pp. 1681-1692, 2016) and Tsujimoto et al. (Cell Reports 31, 10476, April 7, 2020) are preferably used. Briefly, human iPS cells are cultured with a specific differentiation inducer to induce intermediate mesoderm, which is then transferred to a three-dimensional culture environment to form kidney organoids. LTL-positive cells can be enriched and selected from kidney organoids using, for example, magnetic-activated cell sorting (MACS). LTL-positive cells are primarily proximal tubule epithelial cells, but they also contain distal tubule epithelial cells, and are thought to have functions in water reabsorption and ion homeostasis in addition to the functions of proximal tubule epithelial cells.

[0031] In the biomimetic system of the present invention, as shown in the cross-sectional view of the biomimetic system in Figure 2, LTL-positive cells derived from pluripotent stem cells are contained in the first chamber 2 and adhere to the first surface 41 of the porous membrane 4 of the microfluidic device 1. The LTL-positive cells derived from pluripotent stem cells form a cell layer 7 on the first surface 41 of the porous membrane 4, and this cell layer 7 preferably forms epithelial tissue. The LTL-positive cells derived from pluripotent stem cells are seeded in the first chamber 2 and cultured with the first surface 41 of the porous membrane 4 as the bottom surface, thereby causing the LTL-positive cells derived from pluripotent stem cells to adhere to the porous membrane 4.

[0032] The pluripotent stem cell-derived LTL-positive cell culture formed on the porous membrane 4 by adhering to the porous membrane 4 may be partially multilayered, but basically forms a monolayer of proximal tubular epithelial-like tissue (also simply referred to as "epithelial tissue") and has polarity. The side not in contact with the porous membrane 4 is the apical side, the surface of which is covered with microvilli. The side in contact with the porous membrane 4 is the basal side. In this specification, the apical side is referred to as "A" and the basal side as "B." Substrate transport from the basal side to the apical side is sometimes referred to as "B to A," "b to a," "B→A," or "b→a," and substrate transport from the apical side to the basal side is sometimes referred to as "A to B," "a to b," "A→B," or "a→b."

[0033] The LTL-positive cells adhering to the first surface of the porous membrane 4 facing the first chamber express the organic anion transporters (membrane transporters) SLC22A6 (OAT1) and SLC22A8 (OAT3). Furthermore, they also express the excretory transporter MRP4. The LTL-positive cells are also positive for ZO-1, which indicates tight junction formation, as well as P-gp, SGLT2, megalin, OAT2, OAT4, EpCAM, MRP2, and OCT2. The expression of these transporters by the LTL-positive cells adhering to the first surface of the porous membrane 4 facing the first chamber allows the physiological functions of human proximal tubules to be reproduced. Therefore, the biomimetic system of the present invention is a high-performance in vitro model of proximal tubules and can be suitably used as an evaluation tool in drug discovery.

[0034] Human OAT is a protein with a 12-transmembrane consensus structure and is classified by the Human Gene Nomenclature Committee as part of the Solute Carrier Superfamily 22 (SLC22) family, along with organic cation transporters (OCTs) and zwitterion / cation transporters (OCTNs). Human OAT1 and human OAT3 recognize a wide variety of drugs as substrates. Furthermore, human OAT3 generally has higher affinity for various drugs, followed by human OAT1. MRP4 is known as an ATP-dependent transporter for uric acid excretion located in the luminal membrane of the proximal tubule.

[0035] The number of LTL-positive cells derived from pluripotent stem cells contained in the microfluidic device 1 is, for example, 1 × 10 relative to the surface area of ​​the porous membrane 4. 4 pieces / cm 2 ~1×10 6 pieces / cm 2 may be, preferably 6×10 4 pieces / cm 2 ~1×10 5 pieces / cm 2 In particular, 8×10 4 pieces / cm 2 ~1×10 5 pieces / cm 2 The LTL-positive cells in the porous membrane 4 may be preferably 70% or more, 80% or more, 90% or more, 95% or more, or 100% confluent. When the cells are 100% confluent, the epithelial tissue is fused and becomes impermeable.

[0036] The medium for culturing pluripotent stem cell-derived LTL-positive cells in the microfluidic device 1 may be any medium typically used for culturing epithelial cells, such as REGM (Lonza™ Renal Epithelial Growth Medium, CC-3190), RELAR (registered trademark, serum-free medium for culturing human renal proximal tubular epithelial cells, Cell Science Institute, Inc., 2112P05), or DMEM / F12 medium containing hTERT Immortalized RPTEC Growth Kit (ATCC™, ACS-4007). Of these, REGM is particularly preferred.

[0037] The proximal tubule biomimetic system of the present invention can be maintained and used in static culture or perfusion culture. In static culture, for example, the cells can be cultured at 37°C in a 5% CO2 environment while changing the medium at a rate of 200 μL per chamber every day, and static culture can be maintained for approximately 20 days. In perfusion culture, for example, the cells can be cultured at 37°C in a 5% CO2 environment while perfusing the medium at a flow rate of 10 μL / min.

[0038] [Function of the proximal tubule biomimetic system of the present invention] In the human iPSC-based proximal tubule mimetic system (iPSC-PToC) of the present invention, organoid-derived LTL cells are cultured on a porous membrane coated with extracellular matrix, resulting in the formation of uniform epithelial tissue. Conventional proximal tubule mimetic systems express only a limited number of transporters, failing to accurately reflect the in vivo situation in which multiple transporters interact in a complex manner. However, the epithelial tissue in the human iPSC-based proximal tubule mimetic system of the present invention expresses transporters SLC22A6 (OAT1) and SLC22A8 (OAT3), which are not expressed when conventional RPTECs are used. The intracellular uptake of substrates by the expressed OAT1 / 3, B-to-A transport (transport from the basal side (B) to the apical side (A)), inhibition of substrate uptake by inhibitors such as probenecid, B-to-A transport of adeforvir and PAH by OAT1, B-to-A transport of rosuvastatin by OAT3 (transport from the basal side (B) to the apical side (A)), inhibition of uptake by probenecid, inhibition of MRP4 efflux transporter by MK571, and transport of metformin, a substrate of SLC22A2 (OCT2), and quinidine, a substrate of MDR1 (P-gp) were also confirmed. Because the present biomimetic system can reproduce the physiological functions of the human proximal tubule, it is a highly efficient in vitro model of the proximal tubule and can be used as an evaluation tool in drug discovery.

[0039] The proximal tubule biomimetic system of the present invention can reproduce the function of the proximal tubule in vitro and evaluate the reabsorption and filtration efficiency in vitro. It can also be used for screening candidate drugs, confirming pharmacokinetics, or conducting toxicity experiments. By using the biomimetic system of the present invention, screening for substances with high efficacy, favorable pharmacokinetics, and / or low toxicity in the preclinical stage can lead to a higher probability of successful drug discovery and reduce drug discovery costs.

[0040] <Method for manufacturing a proximal tubule biomimetic system> The method for producing the proximal tubule biomimetic system of the present invention includes the steps of: (1) providing a microfluidic device; (2) preparing LTL-positive cells derived from pluripotent stem cells; and (3) Culturing LTL-positive cells derived from pluripotent stem cells in a microfluidic device Including, The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber, separating the first chamber from the second chamber; a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix; In the step (3), LTL-positive cells derived from pluripotent stem cells are seeded in the first chamber and cultured, and the LTL-positive cells are allowed to adhere to the first surface of the porous membrane. It is characterized by:

[0041] [Process (1)] In step (1) of preparing a microfluidic device, the above-described microfluidic device is fabricated. The fabrication method will be described below using the microfluidic device 1 shown in FIG. 2 as an example. The complex internal structure of a microfluidic device makes it difficult to mold it into a single unit. For example, as shown in FIG. 1, a microfluidic device can be fabricated by preparing two layers (substrates) each having a recess corresponding to a first chamber 2 and a second chamber 3, and a porous membrane 4. The substrate (first substrate 1a) having a recess (first recess) for forming the first chamber 2, the porous membrane 4, and the substrate (second substrate 1b) having a recess (second recess) for forming the second chamber 3 are aligned and bonded in this order. A portion of the first recess in the first substrate 1a is blocked by the porous membrane 4 to form the first chamber 2, while the portion of the first recess not forming the first chamber 2 is blocked by the second substrate 1b, forming a first channel for communication with a first supply unit 5 (described below). A portion of the second recess in second substrate 1b is blocked by porous membrane 4 to form second chamber 3, and the portion of the second recess not forming second chamber 3 is blocked by first substrate 1a to form a second channel for communicating with second supply unit 6 described below. The portion of the first recess in first substrate 1a for forming first chamber 2 and the portion of the second recess in second substrate 1b for forming second chamber 3 may have the same shape and may correspond in position. There are preferably two first channels and two second channels each for perfusing cells, and they are preferably located on both sides of the first chamber or the second chamber.

[0042] The first substrate 1a and the second substrate 1b can be fabricated by forming a cured product having a desired shape using a technique capable of forming a fine three-dimensional structure, such as soft lithography. Materials used in soft lithography may be commonly used, such as epoxy resins and silicone resins. For example, a thermosetting resin polymer such as polydimethylsiloxane (PDMS) may also be used. An example of a PDMS prepolymer is one in which a curing agent is mixed into a PDMS base material. The ratio of polymer base material to curing agent is not particularly limited, but may be, for example, 5:1 to 15:1.

[0043] It is preferable that the first substrate 1a further includes a through-hole corresponding to the first supply unit 5 communicating with the first channel. Furthermore, it is preferable that the first substrate 1a and the second substrate 1b further include a through-hole in the first substrate 1a and a recess in the second substrate 1b corresponding to the second supply unit 6 communicating with the second channel. The through-hole 6a formed in the first substrate 1a and the recess 6b formed in the second substrate 1b communicate with each other to form the second supply unit 6. A medium can be supplied from the first substrate 1a side via the first supply unit 5 and the second supply unit 6. It is preferable that there are two first supply units 5 and two second supply units 6 to perfuse the cells in the first chamber 2 and the second chamber 3. The shapes of the first substrate 1a and the second substrate 1b are not particularly limited, but are preferably rectangular plates. In this case, the planes of the first substrate 1a and the second substrate 1b may be rectangular and of the same size, and the thickness of each substrate may be the same, or the first substrate 1a may be thicker than the second substrate 1b, taking into account the desired depth of the first supply section 5 and the second supply section 6.

[0044] The fabricated microfluidic device 1 may be sterilized with 70% ethanol, UV irradiation, or the like before being used for cell culture.

[0045] [Process (2)] Step (2) is a step of preparing LTL-positive cells to be seeded and cultured in the first chamber. The LTL-positive cells are prepared from pluripotent stem cells.

[0046] Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells used in the present invention include embryonic stem cells (ES cells: Embryonic stem cells), induced pluripotent stem cells (iPS cells: induced pluripotent stem cells), and EG cells (Embryonic germ cells). Pluripotent stem cells also include Muse cells (Multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSCs) and GS cells prepared from germ cells (e.g., testes). As pluripotent stem cells in the present invention, ES cells and iPS cells are preferred, with iPS cells being more preferred.

[0047] iPS cells are cells induced to pluripotency by reprogramming somatic cells using known methods. Since Yamanaka et al. established iPS cells in mouse cells in 2006 (Cell, 2006, 126(4), pp. 663-676), various established induced pluripotent stem cell lines have become available. For example, human iPS cell line CRL1502.3 (MCRI, Australia) and human induced pluripotent stem cell lines established at Kyoto University, such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, are available from Kyoto University and iPS Academia Japan, Inc. As established induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University are available from Kyoto University. The iPS cells of the present invention are not particularly limited, and are preferably human iPS cells, or may be any of the above-mentioned iPS cell lines and known iPS cells thereof.

[0048] LTL-positive cells derived from pluripotent stem cells can be obtained by inducing differentiation of pluripotent stem cells. LTL-positive cells are preferably LTL-positive cells derived from kidney organoids prepared from iPS cells. Kidney organoids can be prepared from iPS cells using known differentiation methods. The method for preparing kidney organoids is not particularly limited, but the protocols of Takasato et al. ("Generation of kidney organoids from human pluripotent stem cells," Nat Protoc, vol. 11, pp. 1681-1692, 2016) and Tsujimoto et al. (Cell Reports 31, 10476, April 7, 2020) are preferably used. Briefly, human iPS cells are cultured with a specific differentiation inducer to induce intermediate mesoderm, which is then transferred to a three-dimensional culture environment to form kidney organoids. LTL-positive cells can be enriched and selected from kidney organoids using, for example, magnetic-activated cell sorting (MACS). LTL-positive cells are primarily proximal tubule epithelial cells, but they also contain distal tubule epithelial cells, and are thought to have functions in water reabsorption and ion homeostasis in addition to the functions of proximal tubule epithelial cells.

[0049] The LTL-positive cells obtained as described above may be expanded in a flask, dish, or the like in a medium typically used to culture epithelial cells, such as DMEM / F12 medium containing REGM (Lonza™ Renal Epithelial Growth Medium, CC-3190), RELAR (registered trademark, serum-free medium for human renal proximal tubular epithelial cell culture, Cell Science Institute, Inc., 2112P05), or hTERT Immortalized RPTEC Growth Kit (ATCC™, ACS-4007), for 1 to 20 days, preferably 2 to 16 days, more preferably 5 to 14 days, even more preferably 7 to 12 days, and particularly preferably 8 to 11 days, before being used in step (3) described below.

[0050] [Process (3)] Step (3) is a step of seeding the LTL-positive cells obtained in the above step (2) into the first chamber and culturing them to allow the LTL-positive cells to adhere to the first surface of the porous membrane.

[0051] The total number of LTL-positive cells to be seeded into one chamber is, for example, 1.2 x 10 5 ~6×10 5 / cm 2 and preferably 3.6 × 10 5 / cm 2 The cells can adhere to the porous membrane within about one day of the start of culture, and are then cultured while growing. The medium and culture conditions are as described above in the section <Proximal tubule biomimetic system>. The number of days for culture is not particularly limited as long as it is sufficient for the formation of proximal tubule epithelial-like tissue by LTL-positive cells on the porous membrane, but is, for example, 2 to 20 days, preferably 3 to 14 days, more preferably 4 to 10 days, and even more preferably 5 to 7 days.

[0052] When the proximal tubule epithelial-like tissue formed from LTL-positive cells is observed under a microscope, if the epithelial tissue completely fills the entire channel without any gaps, the biomimetic system can be considered complete. The biomimetic system can be evaluated by measuring the substrate transport capacity, glucose or albumin reabsorption capacity, etc. Specific methods for measuring the substrate transport capacity, etc., include the methods specifically described in the Examples.

[0053] <Proximal tubule biomimetic system construction kit> The present invention also includes a kit for producing the proximal tubule biomimetic system according to the present invention. The proximal tubule biomimetic system production kit of the present invention includes a microfluidic device and LTL-positive cells derived from pluripotent stem cells, and the microfluidic device (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) A porous membrane is located between the first chamber and the second chamber, separating the first chamber from the second chamber, and a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix.

[0054] The pluripotent stem cells are preferably iPS cells or ES cells, more preferably iPS cells. Furthermore, the LTL-positive cells are preferably LTL-positive cells derived from kidney organoids prepared from iPS cells or ES cells. The proximal tubule biomimetic system of the present invention can be prepared by using the proximal tubule biomimetic system preparation kit of the present invention and following the explanation in the above section <Method for producing a proximal tubule biomimetic system>. The explanation in the above section <Proximal tubule biomimetic system> can be applied to the microfluidic device constituting the proximal tubule biomimetic system preparation kit of the present invention and the LTL-positive cells derived from pluripotent stem cells. [Example]

[0055] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0056] 1. Microfluidic Device Fabrication The proximal tubule biomimetic system of the present invention is shown schematically in Figure 1. A microfluidic device, which is one of its components, was fabricated using the following procedure.

[0057] The microfluidic device 1 shown in Figure 2 was fabricated using polydimethylsiloxane (PDMS). Two PDMS layers with identical S-shaped chambers were cast on a silicon wafer with an embossed chamber pattern. The chamber pattern was formed using standard SU-8 photolithography (SU-8 3050 (MicroChem, USA)) and had a height of approximately 100 μm. Each layer was identical except for its thickness. To facilitate microscopic observation, the upper layer (containing the first chamber 2) was 4 mm thick, and the lower layer (containing the second chamber 3) was approximately 2 mm thick. PDMS polymer (PDMS base:curing agent = 10:1 (weight ratio)) (Dow Corning Toray Co., Ltd., Japan) was cured overnight at 65 °C, after which the slab was cut and peeled off from the silicon wafer. The lower layer, chamber side down, was gently placed on a glass slide and first spin-coated with a thin layer of uncured PDMS, which was then immediately removed. This thin PDMS layer served as an adhesive layer after the porous membrane 4 was sandwiched between the layers. A porous membrane (Culture Insert, Falcon) with pores approximately 3 μm in diameter was cut to a 3 mm x 20 mm piece and placed on the bottom layer, covering the entire width and length of the chambers and extending slightly beyond the inflection point to completely isolate the first chamber 2 and the second chamber 3. The thin PDMS layer was allowed to cure overnight to securely bond the top and bottom layers. The resulting microfluidic device 1 had dimensions of 20 mm x 30 mm x 10 mm, with the first chamber 2 and second chamber 3 measuring 1 mm x 15 mm x 0.35 mm.

[0058] Before cell seeding, the first chamber 2 and the second chamber 3 were filled with 70% ethanol for sterilization, dried overnight at 60°C, and then irradiated with UV light for one hour. The first chamber 2 and the second chamber 3 were filled with a coating solution containing an extracellular matrix and incubated for five hours to coat the first surface 41 of the porous membrane 4.

[0059] 2. Generation of iPSC-PToC using organoid-derived LTL-positive cells A test was carried out using a proximal tubule model using LTL-positive cells derived from human iPS cells, according to the scheme shown in Figure 3. The following explains each step in turn.

[0060] 2-1. Generation of LTL-positive cells derived from iPS cells Renal organoids were generated using human iPS cell line CRL1502.3 (female CRL1502.3 fetal fibroblasts derived from ATCC CRL-1502) according to the protocols of Takasato et al. (Nat Protoc, vol. 11, pp. 1681-1692, 2016) (upper scheme in Figure 3) and Tsujimoto et al. (Cell Reports 31, 10476 April 7, 2020) (lower scheme in Figure 3).

[0061] In Takasato et al.'s protocol, human induced pluripotent stem cells (hiPSCs) (female CRL1502.3 fetal fibroblasts derived from ATCC CRL-1502) were differentiated into the posterior primitive streak by activating canonical WNT signaling using the GSK-3 inhibitor CHIR99021 (R&D, catalog no. 4423 / 10) in APEL2 medium (Stem Cell Technologies, catalog no. 5210). To induce intermediate mesoderm, the GSK-3 inhibitor CHIR99021 was switched to FGF9 (R&D, catalog no. 273-F9-025) on day 5. On day 7, 3D self-organization was initiated by culturing aggregates in low-attachment U-bottom 96-well plates (MS-9096M, Sumitomo Bakelite). The cell density was 3.0 × 10 5 Cells / well. Nephrogenesis was promoted by pulsing with 8 μM CHIR99021 for 24 hours. The next day, aggregates were transferred to Transwell cell culture plates (Corning, Cat. No. 3450), and growth factors FGF9 and Heparin were removed on Day 12.

[0062] In the protocol by Tsujimoto et al., human induced pluripotent stem cells (hiPSCs) 4A6 cultured under feeder-independent conditions were differentiated into SIX2+ nephron progenitor cells (NPCs) by Day 11. The induced NPCs were dispersed by incubation in Accumax (Innovative Cell Technologies, Inc.) and plated at 2.0 × 10 cells in a low-attachment M-bottom 96-well plate (MS-9096M, Sumitomo Bakelite). 5 After 24 hours of incubation, on day 13, the aggregates were transferred to a transwell cell culture plate (Corning, Cat. No. 3450) and cultured at the air-liquid interface for 10 days.

[0063] LTL-positive cells were selected from the kidney organoids obtained by the above method using the following method.

[0064] For each experiment, approximately 40 kidney organoids were prepared and dissociated using the enzymes included in the Tumor Dissociation Kit (Miltenyi Biotec, #130-095-929). Dissociation buffer was prepared by dissolving the enzymes in the dissociation buffer. Dissociation buffer was prepared by mixing DPBS (1X) (Gibco®), 2 mM EDTA (Fisher, 15575020), and 0.5% (v / v) FBS. Dissociated cells were kept on ice throughout the entire process before sorting. To remove cell clumps, they were passed through a 10 μm pluriStrainer® (pluriSelect, #43-50010-03). The cells were then centrifuged and mixed with biotinylated Lotus tetragonolobus lectin (LTL), a proximal tubule brush border marker, at a 1:100 ratio. The sample was thoroughly mixed and incubated at 4°C for 30 minutes. After centrifugation and two washes to completely remove unbound antibody, monoclonal antibiotin microbeads (Miltenyi Biotec, #130-105-637) were added to the mixed sample at a 1:5 ratio for indirect magnetic labeling. The suspension was then incubated at 4°C for 30 minutes. The cell suspension was slowly applied to an MS column (Miltenyi Biotec, #130-042-201) connected to one of the eight slots of an OctoMACS Separator (Miltenyi Biotec, #130-042-108). The column was then washed with 500 μL of separation buffer (total volume 1.5 mL) to completely remove the negative fraction (LTL-). The column was then placed in a collection tube, and the positive fraction (LTL+) was immediately collected with 1 mL of separation buffer. LTL+ cells were grown in REGM (renal epithelial growth medium, Lonza, CC-3190) at 1.5 × 10 7 The LTL cells were then resuspended at 15 × 10 cells / ml. The LTL cells were then seeded onto a 6-well plate (3335, Corning) coated with iMatrix-511 (0.5 mg / mL, Nippi, 892011 / 892012). After 4–5 days of culture, when the cells reached approximately 90% confluence, they were trypsinized and resuspended in REGM at 15 × 10 cells / ml. 6The cells were resuspended at a concentration of 1000 cells / mL and used for device seeding.

[0065] Hereinafter, LTL+ cells obtained according to the protocol of Takasato et al. will be referred to as "T.LTL+", and LTL+ cells obtained according to the protocol of Tsujimoto et al. will be referred to as "A.LTL+" to distinguish them.

[0066] 2-2. Cultivation of LTL-positive cells on porous membranes (comparison of porous membrane coating agents) The first surface of the porous membrane of a sterilized microfluidic device was coated with iMatrix-511 for stem cells (0.5 mg / mL, Nippi, 892011 / 892012) or FNC Coating Mix® (AthenaES, 0407H) and incubated for 5 hours. The coating solution was aspirated before seeding with LTL-positive cells. A negative control without coating was also prepared. 200 μL of REGM was added to the lower channel of each device, and 40 μL of the cell suspension was slowly injected into the upper channel. The devices were incubated at 37°C and 5% CO2 for 2 hours until the cells had fully settled and attached to the membrane. Then, 100 μL of REGM was added to the upper channel reservoir. The devices were maintained in an incubator, and the channel medium was replaced with fresh medium daily. After culturing for 5 days under each of the above coating conditions, the cells were observed and photographed under an optical microscope. In addition, the expression of EpCAM, LTL, and LRP2 was confirmed for cells cultured on iMatrix-511-coated porous membranes using the antibodies listed in Table 1 below. Nuclear staining was also performed with DAPI. The results are shown in Figure 4.

[0067] [Table 1]

[0068] As shown in Figure 4, when iMatrix-511 was used as a coating agent, uniform epithelial tissue was formed on the porous membrane. However, when FNC was used, the seeded LTL+ cells formed cell aggregates, failing to form an epithelial barrier, as in the case without coating. Furthermore, when iMatrix-511 was used as a coating agent, expression of the adhesion molecule EpCAM and LRP2, which is involved in endocytosis, was observed, and it was confirmed that LTL expression was also maintained. In subsequent tests, iMatrix-511 was used as an extracellular matrix coating agent.

[0069] 2-3. Evaluation of iPSC-PToC (1) Confirmation of epithelial tissue barrier function using fluorescent inulin In a microfluidic device, 3.5 × 10 LTL+ cells (T.LTL+ or A.LTL+) or RPTEC (proximal tubular epithelial cells) were plated on a porous membrane coated with iMatrix-511 for stem cells (Nippi, 892011 / 892012) using a method similar to that described in 2-2 above. 5 / cm 2 The cells were seeded and cultured in REGM at 37°C in a 5% CO2 atmosphere to form epithelial tissue. Inulin was introduced into the apical side, and leakage to the basal side was measured over time. Specifically, FITC-labeled inulin was dissolved in preheated REGM medium at a concentration of 100 μg / mL and then applied to the upper channel under all conditions. The device was maintained at 37°C, and 100 μL samples were taken from the lower channel at intervals of 1, 2, and 3 hours. These samples were transferred to individual wells of a black-bottom 96-well plate (Corning, CLS3925). Each well was pre-filled with REGM to a volume of 200 μL according to the microplate reader manufacturer's protocol. Immediately after sampling, the lower channel was refilled with 100 μL of REGM. Before sampling, a row of the 96-well plate was filled with a known concentration of FITC-labeled inulin solution to obtain calibration data. The fluorescence emission of the samples was measured by a microplate reader (Molecular Devices, SpectraMax® iD5).

[0070] The insulin concentration over time, reflecting the amount eliminated, was calculated from the concentrations of samples taken at each interval using the following formulas (1) and (2):

[0071]

number

[0072] where M n and M n-1 and represent the amount of insulin transported in the channel in μg at the current and previous sampling times, respectively. C n and C n-1 is the corresponding sample concentration, all in μg / mL. 200 μL and 100 μL refer to the total channel and sampling volume, respectively. Pd is the diffusional transmittance ratio; if the membrane were completely permeable, half of the added insulin would diffuse into the lower channel when diffusion was complete.

[0073] The results are shown in Figure 5 (change in inulin leakage over time) and Figure 6 (inulin leakage after 3 hours). As shown in Figures 5 and 6, it was confirmed that leak-free epithelial tissue could be produced in the microfluidic device.

[0074] (2) Evaluation of OAT1 and OAT3 by immunohistochemistry In a microfluidic device, 3.5 × 10 LTL+ cells (T.LTL+ or A.LTL+) were plated on a porous membrane coated with iMatrix-511 (Nippi, 892011 / 892012) for stem cells, using a method similar to that described in 2-2 above. 5 / cm 2 The RPTECs were seeded onto a porous membrane coated with FNC Coating Mix (registered trademark) (Athena 0407) at 3.5 × 10 5 / cm 2The cells were seeded and cultured in REGM at 37°C in an environment of 5% CO2 to form epithelial tissue.

[0075] The RPTECs were prepared as follows: 10 cryopreserved RPTEC / TERT1 cells (ATCC® CRL-4031®). 6 Cells / vial were cultured in DMEM / F12 (Gibco 11320033) supplemented with hTERT-immortalized RPTEC proliferation kit (ATCC® ACS4007™) in a T25 cell culture flask until approximately 90% confluent. After trypsinization, cells were cultured in REGM medium (Lonza CC-3190) at 15 × 10 6 The cells were resuspended at a concentration of 1000 cells / mL and seeded into a microfluidic device.

[0076] The formed epithelial tissue was examined for the expression of LTL, OAT1, OAT3, and ZO-1 by immunostaining. Specifically, the cells were fixed in 4% paraformaldehyde in DPBS for 15 minutes. The channels were then washed three times with DPBS to remove residual fixative and permeabilized with 0.1% Triton X-100 for 10 minutes. Both channels were then incubated in 10% donkey serum-containing blocking buffer for 1 hour at room temperature with shaking. Freshly prepared primary antibodies in blocking buffer were applied to the upper microchannel and incubated overnight at 4°C. The next day, the channels were washed five times with DPBS for 5 minutes each, followed by the addition of secondary antibodies and incubation for 1 hour at room temperature on a shaker. Finally, the porous membrane on the upper channel was treated with DAPI for 10 minutes, treated with Antifade mountant (Fisher, S36937), and mounted on a microscope slide. Confocal fluorescence microscopy was performed using an Olympus FV3000 microscope. Image analysis was performed using FV31S-SW (v2.5.1.228, Olympus) viewer software and Image J (v1.53f51, NIH, USA). The concentrations of the antibodies used are shown in Table 1. The results are shown in Figures 7 to 9.

[0077] As shown in Figure 7, the epithelial tissue formed was positive for LTL, the transporters OAT1 and OAT3, and ZO-1, which indicates tight junction formation. Conventional methods using RPTEC (proximal tubule epithelial cells) failed to produce epithelial tissue positive for OAT1 and OAT3. However, the method of the present invention, which uses human iPS cell-derived LTL cells and a porous membrane coated with iMatrix-511 (Nippi, 892011 / 892012) for stem cells, was able to form epithelial tissue positive for both OAT1 and OAT3.

[0078] Figure 8 shows cross-sectional fluorescence images of epithelial tissue formed on a porous membrane. Figure 9 quantifies the results shown in Figure 8, showing that a smaller peak difference (Delta_D) indicates greater basal expression of the target protein, OAT1. For OAT1, a clear peak was observed closer to the basal membrane in T.LTL+ cells, while in A.LTL+ cells, OAT1 expression was predominantly near the basal membrane but also in the cytoplasm. In contrast, in RPTEC cells, a peak was observed near the apical membrane, and although OAT1 expression was present, functional localization was not observed. This protein expression location is thought to support the higher polarized transport of T.LTL+ cells. Furthermore, as shown in Figure 10, OAT1 was clearly basolaterally localized in T.LTL+ PToC cells (Δd < 0). In contrast, OAT1 was distributed both basally and apically in A.LTL+ PToC cells, whereas in RPTEC PToC cells, OAT1 expression was mainly apically localized. A similar trend was observed for OAT3, with a basolateral orientation in T.LTL+ PToC. A.OAT3 was expressed on both the apical and basolateral sides in LTL+ PToC, whereas it was only weakly expressed on the apical side in RPTEC PToC. This diverse expression pattern may explain the lack of functional OAT uptake and transport in RPTEC PToC.

[0079] (3) Evaluation of OAT1 / 3-mediated fluorescein uptake To evaluate the transport ability of OAT1 / OAT3 expressed in epithelial tissue formed in a microfluidic device, 2 μM fluorescein was used as the substrate, and substrate uptake was evaluated for the cell type and inhibitor combinations shown in Table 1 below. 100 μM probenecid was used as a competitive inhibitor. MK571 was also used as an inhibitor of MRP4, an excretory transporter. Figure 11 shows a schematic diagram of the experimental method. Figure 12 shows a schematic diagram of transport from the vascular side (B: basal) to the primary urine side (A: apical).

[0080] [Table 2]

[0081] Specifically, PToCs were incubated with 2 μM fluorescein in the presence or absence of specific drug transporter inhibitors under a confocal fluorescence microscope (Olympus FV3000) at 37°C and 5% CO2 in a microscope incubator (TOKAI HIT, STXG-IX3WX-SET). Background-corrected data were normalized to fluorescein uptake at 0 min. Cell location was confirmed using Hoechst staining, and images were taken every 2 min for 30 min, starting from the apical side of the cell layer. The field of view was divided into six equally sized and positioned segments for all devices, and each segment was analyzed as an individual region of interest. Measurement data were collected using at least N = 3 independent devices (chips) per condition.

[0082] To measure the amount of fluorescein transported after 30 minutes, a 100 μL sample was taken from the top channel. The fluorescence concentration was then measured using a microplate reader, similar to the diffuse permeability measurement. The results of Test Examples 1-2 and 4-7 are shown in Figures 13-16. The results of Test Example 3 are shown in Figures 18 and 19.

[0083] As shown in Figures 13 to 16, fluorescein uptake was observed in Test Examples 1 and 4, while a decrease in uptake due to the inhibitor was observed in Test Examples 2 and 5. On the other hand, in Test Examples 6 and 7, fluorescein uptake was almost nonexistent, and no effect of the inhibitor was observed. Furthermore, differences were observed between the two organoid-derived cells in transport to the primary urine side (A: apical).

[0084] To investigate the cause of the difference in fluorescein transport to the apical side (A), we compared gene expression of the excretory transporter MRP4 in the two organoid-derived cells. The results are shown in Figure 17.

[0085] As shown in Figure 17, there was a difference in the expression level of MRP4 between the two organoid-derived cells, and it was revealed that in A.LTL+, where fluorescein transport to the apical side (A) was almost nonexistent, MRP4 expression was significantly lower than in T.LTL+.

[0086] Furthermore, as shown in Figure 18, the uptake of fluorescein into epithelial tissue was inhibited by probenecid. Furthermore, the addition of MK571 increased the amount of fluorescein remaining in the epithelial tissue. As shown in Figure 19, when examining the overall transport of fluorescein from the vascular side (B: basal) to the urinary side (A: apical), it was inhibited by probenecid and MK571. These results suggest that MRP4 is deeply involved in the transport of fluorescein to the urinary side (A: apical).

[0087] (4) OAT1 / 3-mediated substrate transport The extracellular transport activity of transporters was evaluated by measuring the transport rate of 10 μM specific drug transporter substrates in the presence or absence of specific drug transporter inhibitors. After removing and washing the medium, the upper and lower channels were filled with 200 μL of HBSS pH 6 and HBSS pH 7.4, respectively, and the device was preincubated at 37°C for 30 minutes. Sampling was performed similarly to the procedure for diffusional permeability measurements. However, because we were interested in the efflux rate (basolateral to apical), the substrate was added primarily to the bottom channel and measured in the upper channel. 100 μL samples were taken from the upper channel at regular intervals and transferred to individual 1.5 ml Eppendorf tubes. Immediately after sampling, the upper channel was replenished with 100 μL of HBSS pH 6. Prior to sampling, a substrate solution with a known concentration was prepared, and a calibration curve was constructed. To quantify the reverse transport rate, the substrate was dissolved in HBSS pH 6 and administered through the lower channel. All samples were analyzed using an LCMS system (LCMS-8060NX Shimadzu).

[0088] The concentration of the substrate over time was calculated from the concentration of the collected samples using the following formula.

[0089]

number

[0090] M n and M n-1 and represent the amount of transported substrate in the channel at the current sampling time point and the previous sampling time point, respectively, in μg. C n and C n-1 indicates the corresponding sample concentration, all units are μg / ml. The volumes 200 μL and 100 μL indicate the total volume of the channel and the sampling, respectively. ΔQ / Δt is the filtration rate (nmol / min) and is the slope of the regression line of the transported substrate (nmol) versus time (min), C0 is the initial concentration in the donor channel (μM), and A is the surface area of ​​the channel (cm 2 )

[0091] The sample was collected from the supernatant and immediately mixed with an equal volume of acetonitrile (Fujifilm Wako). This mixture was centrifuged at 15,000 g for 5 minutes and then filtered through a CosmoNice® filter (aqueous, 0.45 μm, 4 mm, Nacalai Tesque). The concentrations of adefovir, para-aminohippuric acid (PAH), rosuvastatin Ca, quinidine, and metformin were measured using a standard curve. LC / MS analysis was performed using an LCMS-8060NX (Shimadzu Corporation). The dwell time for each MRM transition was 100 msec. Table 3 below summarizes the test details and results. Figures 20–23 show the test results.

[0092] [Table 3]

[0093] As shown in Figure 20 (Adefovir) and Figure 21 (PAH), OAT1-mediated transport was measured in both organoid-derived cells according to cell polarity, and the inhibitor probenecid inhibited substrate transport in a concentration-dependent manner. However, the order of substrate transport was higher in RPTEC than in TERT.

[0094] As shown in Figure 22 (Rosuvastatin), transport from the basal (B) to the apical (A) side via OAT3 was stronger than transport from the basal (A) side to the basal (B) side. In both organoid-derived cells, probenecid inhibited substrate transport in a concentration-dependent manner. Neither polarized transport nor the inhibitor effect was observed in the T.LTL+>A.LTL+>>RPTEC / TERT system.

[0095] As shown in Figure 23, inhibition of MRP4 by MK571 inhibited the transport of OAT1 substrates but not the transport of OAT3 substrates, demonstrating that MRP4 selectively excretes OAT1 substrates but not OAT3 substrates.

[0096] (5) Polar transport of the OCT2 substrate metformin OCT2 activity in iPSC-PToCs (hiPSC-derived kidney organoid-based) and RPTEC PToCs (immortalized RPTEC / TERT1-based PToCs) was examined by quantifying metformin (OCT2 substrate, Tocris Bioscience, 2864 / 100). The iPSC-PToCs or RPTEC PToCs were washed with HBSS. The upper and lower channels of the iPSC-PToCs or RPTEC PToCs were filled with 200 μL of HBSS (pH 6) and HBSS (pH 7.4), respectively, and preincubated at 37°C for 30 min. After preincubation, 10 μM drug transporter substrate was added to the upper or lower channel. 100 μL samples were taken from the upper or lower channel at regular intervals and transferred to individual 1.5 ml Eppendorf tubes. Immediately after sampling, the samples were refilled with 100 μL of HBSS (pH 6) or HBSS (pH 7.4). All samples were analyzed using an LCMS system (LCMS-8060NX Shimadzu). The results are shown in Figures 24 and 25.

[0097] (6) Polar transport of the P-gp substrate quinidine P-gp activity in iPSC-PToCs (hiPSC-derived kidney organoid-based) and RPTEC PToCs (immortalized RPTEC / TERT1-based PToCs) was examined by quantifying quinidine (ABCB1 substrate, Tokyo Chemical Industry Co., Ltd., Q0006). The iPSC-PToCs or RPTEC PToCs were washed with HBSS. The upper and lower channels of the iPSC-PToCs or RPTEC PToCs were filled with 200 μL of HBSS (pH 6) and HBSS (pH 7.4), respectively, and preincubated at 37°C for 30 min. After preincubation, 10 μM drug transporter substrate was added to the upper or lower channel. 100 μL samples were taken from the upper or lower channel at regular intervals and transferred to individual 1.5 ml Eppendorf tubes. Immediately after sampling, the samples were refilled with 100 μL of HBSS (pH 6) or HBSS (pH 7.4). All samples were analyzed using an LCMS system (LCMS-8060NX Shimadzu). The results are shown in Figures 26 and 27.

[0098] All reservoirs and channels were supplied with fresh medium before the start of the toxicity test. Cisplatin (100 μM) or aristolochic acid (50 μM) was then manually introduced into the lower channel and incubated at 37°C. After 24 or 48 h, the medium from both the upper and lower channels was collected in 1.5 mL Eppendorf tubes. The collected medium samples were centrifuged at 5000 rcf for 5 min to remove cellular debris. Each supernatant was transferred to a new tube and stored at -20°C for LDH assay analysis. For inhibitor-pretreated samples, cimetidine (10 mM) was first added to both the upper and lower channels, followed by incubation at 37°C for 30 min. Cisplatin was then introduced into both channels in the presence of cimetidine, and sampling was performed using the same procedure as for the cisplatin group. A control group, referred to as the untreated group, was not exposed to any drugs.

[0099] The LDH assay was performed according to the manufacturer's data sheet (Dojindo Laboratories, Cytotoxicity LDH Assay Kit-WST, 343-91753). Thawed media samples were prepared at 4°C and freeze-thawed once. 50 μL of media from the upper and lower channels were combined to a total volume of 100 μL and transferred to a clear, flat-bottom 96-well plate. 100 μL of working solution was then added to each well and incubated at room temperature for 30 minutes, protected from light. 50 μL of stop solution was added, air bubbles were removed with a syringe, and absorbance at 490 nm was measured using a plate reader. Normalized LDH release was adjusted based on the absorbance measured after 24 hours from untreated A.LTL+ cells.

[0100] Aristolochic acid is a potent nephrotoxicant that is primarily transported to the basolateral side by OAT1 and OAT3. To evaluate its effects on A.LTL+ and T.LTL+ PToCs, aristolochic acid (AA) was introduced into the basolateral reservoir and allowed to diffuse across the membrane for 24 hours. As shown in Figure 28, upon introduction of 50 μM AA, both A.LTL+ and T.LTL+ PToCs exhibited significantly higher normalized lactate dehydrogenase (LDH) release compared to the control group without aristolochic acid. Interestingly, T.LTL+ exhibited significantly higher LDH release compared to A.LTL+. However, the untreated T.LTL+ group exhibited higher baseline normalized LDH release compared to the A.LTL+ group, indicating an inherently higher rate of cell death in the T.LTL+ model, consistent with our observations of limited viability in long-term culture. These findings highlight the high physiological relevance of iPSC-PToC for drug screening and toxicity testing applications.

[0101] Cisplatin, known for its accumulation in the proximal tubule and consequent nephrotoxicity, is primarily taken up by the basolateral OCT2 transporter, causing tubular injury through the generation of reactive oxygen species. Cisplatin, introduced through the basolateral channel, readily diffused and was taken up by proximal tubule cells. Administration of 100 μM cisplatin resulted in higher LDH production in both A.LTL+ and T.LTL+ groups compared with their respective untreated counterparts. Treatment with cimetidine (10 mM), an OCT2 inhibitor, for 24 hours significantly reduced LDH production in T.LTL+, whereas the effect was less pronounced in A.LTL+ under similar conditions. However, when the medium was refreshed and exposure to cimetidine was extended to 48 hours, a significant decrease in LDH production was observed in A.LTL+ PToCs. In contrast, in T.LTL+ PToC, LDH production suddenly decreased when the medium was refreshed and exposure to cimetidine was extended to 48 hours, with no significant difference observed across conditions (Figure 29). The high sensitivity of T.LTL+ PToC to cisplatin-induced nephrotoxicity is evident from the fact that most cells were already dead after 24 hours. The LIVE / DEAD cell viability assay showed a similar trend to the LDH assay, with significantly more dead cells in the cisplatin-treated group than in the untreated group, and cell death was reduced by the introduction of the inhibitor, cimetidine (Figure 30).

[0102] As shown in the above test results, we successfully generated highly functional proximal tubule MPS (iPSC-PToC) using human iPS cells. In the highly functional proximal tubule MPS (iPSC-PToC) using human iPS cells of the present invention, uniform epithelial tissue was formed by culturing organoid-derived LTL+ cells on a porous membrane coated with extracellular matrix. The epithelial tissue formed by this method expressed the transporters SLC22A6 (OAT1) and SLC22A8 (OAT3), which are not expressed when using conventional RPTECs. We then confirmed the intracellular uptake of fluorescein by the expressed OAT1 / 3, B-to-A transport, and probenecid-mediated uptake inhibition. We also confirmed the polar transport of adeforvir and PAH by OAT1, and the polar transport of rosuvastatin (B-to-A transport, A-to-B transport) by OAT3, and the probenecid-mediated uptake inhibition. Furthermore, we confirmed the inhibition of MRP4 efflux transporter by MK571. We also confirmed the polar transport of metformin, a substrate of SLC22A2 (OCT2), and quinidine, a substrate of MDR1 (P-gp).Furthermore, we confirmed the inhibition of aristoloxic acid by OAT1 / 3 and the inhibition of cisplatin-induced nephrotoxicity by OCT2. [Industrial Applicability]

[0103] The biomimetic system of the present invention enables in vitro evaluation of human proximal tubules that is closer to that of human proximal tubules than conventional biomimetic systems. This not only makes it possible to easily evaluate the efficacy, nephrotoxicity, and pharmacokinetics of candidate drugs in preclinical trials in drug discovery, but also serves as an alternative to animal experiments and cultured cell experiments, thereby saving resources such as experimental animals and leading to a reduction in the dropout of candidate drugs in clinical trials and a reduction in drug discovery costs. [Explanation of symbols]

[0104] 1...device body, 1a...first substrate, 1b...second substrate, 2...first chamber, 3...second chamber, 4...porous membrane, 5...first supply section, 6...second supply section, 6a...through hole, 6b...recess, 7...cell layer, 41...first surface of porous membrane, 42...second surface of porous membrane.

Claims

1. 1. A proximal tubule biomimetic system comprising: A method for producing a pluripotent stem cell-derived LTL-positive cell culture medium comprising: a microfluidic device; and LTL-positive cells derived from pluripotent stem cells contained in the microfluidic device. The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber, separating the first chamber from the second chamber; and the LTL-positive cells are contained in the first chamber and adhere to a first surface of the porous membrane facing the first chamber; The first surface of the porous membrane is coated with an extracellular matrix. A proximal tubule biomimetic system characterized by:

2. The proximal tubule biomimetic system of claim 1 , wherein the pluripotent stem cells are iPS cells or ES cells.

3. The proximal tubule biomimetic system of claim 2, wherein the LTL-positive cells are LTL-positive cells derived from kidney organoids produced from iPS cells or ES cells.

4. The proximal tubule biomimetic system of claim 3, wherein the LTL-positive cells adhering to the first surface of the porous membrane facing the first chamber express OAT1 and OAT3.

5. A kit for producing a proximal tubule biomimetic system, comprising: A microfluidic device and LTL-positive cells derived from pluripotent stem cells, The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber, separating the first chamber from the second chamber; and A first surface of the porous membrane facing the first chamber is coated with an extracellular matrix. A kit for producing a proximal tubule biomimetic system, comprising:

6. The kit for producing a proximal tubule biomimetic system according to claim 5 , wherein the pluripotent stem cells are iPS cells or ES cells.

7. The proximal tubule biomimetic system production kit according to claim 6, wherein the LTL-positive cells are LTL-positive cells derived from kidney organoids produced from iPS cells or ES cells.

8. 1. A microfluidic device for a proximal tubule biomimetic system, comprising: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber, separating the first chamber from the second chamber; and A microfluidic device for a proximal tubule biomimetic system, wherein a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix.

9. 1. A method for manufacturing a proximal tubule biomimetic system, comprising: (1) providing a microfluidic device; (2) preparing LTL-positive cells derived from pluripotent stem cells; and (3) Culturing LTL-positive cells derived from pluripotent stem cells in a microfluidic device Including, The microfluidic device comprises: (A) the device itself; (B) a first chamber provided in the device body; (C) a second chamber provided in the device body; and (D) a porous membrane located between the first chamber and the second chamber, separating the first chamber from the second chamber; and a first surface of the porous membrane facing the first chamber is coated with an extracellular matrix; In the step (3), LTL-positive cells derived from pluripotent stem cells are seeded in the first chamber and cultured, and the LTL-positive cells are allowed to adhere to the first surface of the porous membrane. A method for producing a proximal tubule biomimetic system, comprising:

10. The method for producing a proximal tubule biomimetic system according to claim 9, wherein the pluripotent stem cells are iPS cells or ES cells.

11. The method for producing a proximal tubule biomimetic system according to claim 10, wherein the LTL-positive cells are LTL-positive cells derived from kidney organoids produced from iPS cells or ES cells.

Images