Glomerulus-on-chip and use thereof

Abstract

The present specification provides a glomerulus-on-a-chip, a method for producing same, and a method for screening for a therapeutic agent for renal diseases, using same. In one aspect, a three-dimensional microfluidic glomerulus-on-a-chip of the present invention can effectively reproduce a major microenvironment in vivo, can be used to develop a kidney disease model, and can rapidly and accurately predict an evaluation of the efficacy and toxicity of therapeutic agents for renal diseases.

Description

Glomerular-mimetic organ-on-a-chip and its uses

[0001] [Cross-reference to related applications]

[0002] This application claims the benefit and priority of Korean Patent Application No. 10-2023-0120726, filed September 11, 2023, entitled “Glomerular mimetic organ chip and use thereof,” the entire contents of which are incorporated herein by reference.

[0003]

[0004] The present specification provides a glomerular mimicking organ chip, a method for manufacturing the same, and a method for screening a treatment for kidney disease using the same.

[0005] Meanwhile, this application was supported by the following national development project.

[0006]

[0007] [National Research and Development Project Supporting This Invention]

[0008] [Project ID] 1415173251

[0009] [Assignment Number] 20008413

[0010] Ministry of Trade, Industry and Energy

[0011] [Name of Project Management (Specialist) Agency] Korea Industrial Technology Evaluation and Planning Institute

[0012] [Research Project Name] 3D Bio-Tissue Chip-Based New Drug Development Platform Construction Project (R&D)

[0013] [Research Project Name] Development of Disease Models and Drug Efficacy Evaluation Technologies Based on a 3D Multi-Organ Microenvironment Integrated Platform

[0014] [Contribution rate] 1 / 2

[0015] [Name of Project Performing Organization] Bundang Seoul National University Hospital

[0016] Research Period: April 1, 2020 - December 31, 2023

[0017]

[0018] [National Research and Development Project Supporting This Invention]

[0019] [Project ID] 1711195966

[0020] [Assignment Number] 00222910

[0021] [Ministry Name] Ministry of Science and ICT

[0022] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0023] [Research Project Name] Bio-Medical Technology Development (R&D)

[0024] [Research Project Name] Development of Medical Field Application Technologies for Five Major Diseases and Training of Physician Scientists through a Customized Future Medical Research Center in the Era of 6P Medicine

[0025] [Contribution rate] 1 / 2

[0026] [Name of Project Performing Organization] Bundang Seoul National University Hospital

[0027] Research Period: April 1, 2023 - December 31, 2026

[0028]

[0029] Chronic renal failure (CKD) is a disease characterized by kidney damage and decreased kidney function. It is rapidly increasing due to an aging society and the increase in the number of people with underlying conditions such as hypertension and diabetes. If left untreated in the early stages, chronic kidney disease can progress to end-stage renal failure requiring dialysis or kidney transplantation. Preserving kidney function during the chronic kidney disease stage is crucial for preventing end-stage renal failure. According to the National Health Insurance Service, the number of new cases of end-stage renal failure reached 11,480 in 2021, a 120.3% increase compared to 2012. The total health insurance cost for end-stage renal failure in 2021 was close to 2 trillion won, an 80.1% increase compared to 2012. Glomerulonephritis (GN), a leading cause of chronic renal failure, accounts for 10% of end-stage renal disease cases in Korea. Compared to other diseases, GN has a younger age at dialysis initiation and a higher long-term survival rate, resulting in a significant socioeconomic burden both personally and nationally. Furthermore, despite the growing demand for treatment for glomerulonephritis, clear treatment guidelines remain inadequate.

[0030] Meanwhile, animal experiments are widely used to model glomerular disease, to study human renal biology, to identify preclinical drug candidates related to glomerular toxicity, and to assess their efficacy and safety. However, animal experiments are expensive, time-consuming, have low reproducibility, and are subject to ethical limitations. Therefore, alternatives to animal models that closely replicate human physiology are urgently needed to predict the efficacy, safety, bioavailability, and toxicity of candidate treatments. Among the various methods proposed as alternatives to animal testing, organ-on-a-chip technology is emerging as a simple yet predictive new approach to clinical research. Conventional two-dimensional cell experiments often fail to accurately reflect the in vivo microenvironment, and recently emerging organoid technology also faces limitations, such as difficulty in replicating urine filtration in vitro.

[0031] Accordingly, to overcome the above problems, the present inventors fabricated a 3D glomerulus-mimicking organ-on-a-chip using microfluidic technology. The 3D microfluidic glomerulus-mimicking organ-on-a-chip effectively implemented a 3D glomerular filtration barrier composed of glomerular podocytes, basement membranes, and endothelial cells by culturing constituent cells within the chip by reproducing the in vivo microenvironment. Therefore, the glomerulus-mimicking organ-on-a-chip can be used to evaluate renal glomerular damage and proteinuria, evaluate the efficacy of kidney disease treatments, and utilize it as a new platform for predicting clinical trial results more quickly and accurately.

[0032]

[0033] One object of the present invention is to provide a glomerular mimicking organ chip.

[0034] Another object of the present invention is to provide a method for manufacturing the glomerular mimicking organ chip.

[0035] Another object of the present invention is to provide a method for screening a treatment agent for kidney disease using the glomerular mimicking organ chip.

[0036]

[0037] To achieve the above object, the present invention provides an insert module including, in one aspect, a podocyte culture unit in which glomerular podocytes are cultured; and an endothelial cell culture unit in which glomerular endothelial cells are cultured.

[0038] In another aspect, the present invention provides a glomerular mimicking organ chip comprising one or more of the above insert modules.

[0039] In another aspect of the present invention, a method for manufacturing a glomerular mimic organ chip is provided, comprising: a step of culturing glomerular podocytes in a podocyte culture unit; and a step of culturing glomerular endothelial cells in an endothelial cell culture unit.

[0040] In another aspect of the present invention, a method for screening a kidney disease treatment agent is provided, comprising: (a) treating a kidney disease-inducing substance to a glomerular mimic organ chip of claim 4; (b) treating a test substance to the glomerular mimic organ chip; and (c) measuring a kidney disease evaluation index in the kidney mimic organ chip treated with the test substance.

[0041]

[0042] In one aspect, the three-dimensional microfluidic glomerular mimic organ chip of the present invention is simple, highly reproducible, and can effectively reproduce major microenvironments in vivo while reducing user dependency compared to existing glomerular mimic organ chips.

[0043] In another aspect, the immortalized human podocytes used in the three-dimensional microfluidic glomerular mimicking organ chip of the present invention have the advantages of easy cell supply, high-throughput use through subculture, and no ethical issues.

[0044] In another aspect, the three-dimensional microfluidic glomerular mimicking organ chip of the present invention allows for the separation and subsequent individual analysis of the two cells as well as stimulation or treatment of one specific cell, since podocytes and endothelial cells are cultured separately.

[0045] In another aspect, the three-dimensional microfluidic glomerular mimicking organ chip of the present invention can be used to develop a kidney disease model, and the efficacy and toxicity evaluation of kidney disease treatment agents can be rapidly and accurately predicted.

[0046] In another aspect, the 3D microfluidic glomerular-mimetic organ-chip of the present invention can easily expand the configuration of the desired organ through multi-cell culture using multiple insert modules at once. For example, the organ-chip of the present invention has a connection structure that facilitates simultaneous culture, enabling the effective simultaneous reproduction of glomeruli and renal tubules.

[0047]

[0048] Figures 1A to 1D are schematic diagrams of a three-dimensional microfluidic glomerular mimicking organ chip structure and cell culture in an insert module.

[0049] Figure 2 is a schematic diagram of bidirectional perfusion within a 3D microfluidic glomerular-mimetic organ chip.

[0050] Figures 3A and 3B show the morphology of undifferentiated (A) and differentiated (B) podocytes in two dimensions (2D).

[0051] Figure 4 shows the expression and morphology of the mature podocyte-specific marker Podocin in two-dimensional (2D) and three-dimensional (3D) organ chips.

[0052] Figure 5 shows the expression and morphology of the podocyte-specific marker WT1 in two-dimensional (2D) and three-dimensional (3D) organ chips.

[0053] Figures 6a and 6b are about a chemical glomerulonephritis (GN) model induced by puromycin aminonucleoside (6-dimethylaminopurine, 3-amino-d-ribose, PAN) in two dimensions (2D). In Figure 6a, the P values ​​are as follows: CON vs PAN 1: P= 0.004, CON vs PAN 5: P< 0.005, CON vs PAN 10: P< 0.005, PAN 1 vs PAN 5: P< 0.005, PAN 1 vs PAN 10: P< 0.005, PAN 5 vs PAN 10: P< 0.005. In Fig. 6b, the P values ​​are as follows: CON vs PAN 1 : P < 0.005, CON vs PAN 2 : P < 0.005, PAN 1 vs PAN 2 : P = 0.038.

[0054] Figure 7 relates to calcineurin inhibition by tacrolimus (TAC) (FK506) and tacrolimus levels over the course of the dosing interval.

[0055] Figures 8A, 8B, 10A to 10C show the recovery of cell viability by tacrolimus and culture medium treatment after tacrolimus treatment in a PAN-induced glomerulonephritis model of a three-dimensional (3D) organ chip.

[0056] Figure 9 is about Calcein-AM cell viability analysis.

[0057] Figure 11 shows the recovery of glomerular filtration rate (GFR) by tacrolimus in a PAN-induced glomerulonephritis model on a three-dimensional (3D) organ chip.

[0058] Figures 12A to 12C show the increase in WT1 expression by tacrolimus in a PAN-induced glomerulonephritis model of a three-dimensional (3D) organ chip. In Figure 12B, the P values ​​are as follows: NS vs PAN : P < 0.005, NS vs PAN+TAC : Statistical analysis was not possible due to difficulty in calculating the average value with only one PAN+TAC confocal IF image result, NS vs PAN+TAC+Media : P < 0.005, PAN vs PAN+TAC : Statistical analysis was not possible, PAN vs PAN+TAC+Media : P < 0.005, PAN+TAC vs PAN+TAC+Media : Statistical analysis was not possible. In Figure 12C, the P values ​​are as follows: NS vs PAN: P< 0.005, NS vs PAN+TAC: Statistical analysis was not possible due to difficulty in calculating the average value with only one PAN+TAC confocal IF image result, NS vs PAN+TAC+Media: P= 0.239, PAN vs PAN+TAC: Statistical analysis was not possible, PAN vs PAN+TAC+Media: P= 0.030, PAN+TAC vs PAN+TAC+Media: Statistical analysis was not possible.

[0059] Figure 13 shows a normal glomerulus and a glomerulus with membranous nephropathy (MN).

[0060] Figures 14A and 14B show the recovery of cell viability and glomerular filtration rate by tacrolimus in a PAN-induced membranous nephropathy model on a three-dimensional (3D) organ chip.

[0061] Figures 15A to 15C show the increase in WT1 expression by tacrolimus in a PAN-induced membranous nephropathy model of a three-dimensional (3D) organ chip. In Figure 15B, the P values ​​are as follows: NS vs MN1 : P= 0.003, NS vs MN1+TAC : P= 0.007, NS vs MN1+TAC+Media : P= 0.032, MN1 vs MN1+TAC : P= 0.019, MN1 vs MN1+TAC+Media : P= 0.009, MN1+TAC vs MN1+TAC=Media : P= 0.050. In Figure 15C, the P values ​​are as follows: NS vs MN1 : P= 0.928, NS vs MN1+TAC : P= 0.006, NS vs MN1+TAC+Media : P= 0.066, MN1 vs MN1+TAC : P= 0.011, MN1 vs MN1+TAC+Media : P= 0.082, MN1+TAC vs MN1+TAC=Media : P= 0.089.

[0062] Figures 16A and 16B show the restoration of cell viability by tacrolimus in a PAN-induced membranous nephropathy model on a three-dimensional (3D) organ chip. In Figure 16B, the P values ​​are as follows: NS vs MN1: P = 0.006, NS vs MN1+TAC: P = 0.040, MN1 vs MN1+TAC: P = 0.006, MN1 vs MN1+TAC+Media: P = 0.019.

[0063]

[0064] Hereinafter, the present invention will be described in detail.

[0065]

[0066] The glomerular basement membrane (GBM) is formed by the fusion of two distinct basement membranes: one synthesized by glomerular podocytes and the other by glomerular endothelial cells (Musah S et al., Nat Biomed Eng. 2017;1:0069). The biological function of podocytes is to prevent albumin and other important proteins from being filtered out of the glomerulus. Additionally, they maintain the shape of glomerular capillaries, express extracellular matrix proteins to maintain the glomerular basement membrane, and selectively produce vascular endothelial growth factor (VEGF) and angiotensin for endothelial cells.

[0067] Meanwhile, the glomerular perfusion rate was measured using a rocker (Organo Flow L rocker, Mimetas, #MI-OFPR-L) at a gradient angle of 7° for 8 minutes, similar to the human body, to be 0.13 dyne / cm. 2 can be mimicked by perfusing (Vormann MK et al., AAPS J. 2018;20(5):90).

[0068] Accordingly, the present inventors fabricated a three-dimensional microfluidic glomerular organ-mimetic chip that can effectively reflect the environmental, functional, and interrelationship characteristics of human organs and tissues by mimicking the glomerular structure and blood flow. Specifically, the morphological characteristics and specific expression markers of podocytes were verified in the glomerular organ-mimetic chip of the present invention, and a puromycin-induced glomerulonephritis model and a tacrolimus treatment efficacy evaluation system for the model were constructed in the glomerular organ-mimetic chip, and a membranous nephropathy glomerular model using the serum of a membranous nephropathy patient and a tacrolimus treatment efficacy evaluation system for the model were constructed, thereby completing the present invention.

[0069]

[0070] Accordingly, the present invention relates to an insert module in which a first cell culture section in which first cells are cultured and a second cell culture section in which second cells are cultured are arranged on different sides of the insert module.

[0071] From another perspective, the present invention relates to an insert module comprising a podocyte culture unit in which glomerular podocytes are cultured; and an endothelial cell culture unit in which glomerular endothelial cells are cultured.

[0072] In an exemplary embodiment, the podocyte culture unit and the endothelial cell culture unit may be arranged on different sides of the insert module, for example, as shown in FIGS. 1A to 1C, the podocyte culture unit and the endothelial cell culture unit may be arranged horizontally on opposite sides of the membrane of the insert module.

[0073] In an exemplary embodiment, the podocyte culture unit and / or the endothelial cell culture unit of the insert module may include an extracellular matrix.

[0074] In an exemplary embodiment, the podocytes and / or endothelial cells may be, but are not limited to, human-derived cells.

[0075] In an exemplary embodiment, the podocytes are immortalized podocytes, so cell supply may be easy.

[0076] In an exemplary embodiment, the podocyte culture unit comprises 1 * 10 glomerular podocytes. 1 ~ 1 * 10 10 cells / mL, or 1 * 10 2 ~ 1 * 10 8 cells / mL, or 1 * 10 4 ~ 1 * 10 6 It may be included at a concentration of cells / mL, but is not limited thereto.

[0077] In an exemplary embodiment, the podocyte culture unit can culture glomerular podocytes at 37°C, 5% CO2 for 2 hours, but is not limited thereto.

[0078] In an exemplary embodiment, the endothelial cell culture unit comprises 2 * 10 glomerular endothelial cells. 1 ~ 2 * 10 10 cells / mL, or 2 * 10 2 ~ 2 * 10 8 cells / mL, or 2 * 10 4 ~ 2 * 10 6 It may be included at a concentration of cells / mL, but is not limited thereto.

[0079] In an exemplary embodiment, the endothelial cell culture unit can culture glomerular endothelial cells at 37°C, 5% CO2 for 2 hours, but is not limited thereto.

[0080] In another aspect, the present invention relates to a glomerular mimicking organ chip comprising one or more of the above insert modules.

[0081] In an exemplary embodiment, the glomerular mimicking organ chip may include a microfluidic plate.

[0082] In an exemplary embodiment, the glomerular mimicking organ chip may be characterized by, but is not limited to, maintaining bidirectional flow at the human blood flow rate. For example, the glomerular mimicking organ chip may be tilted at a 7° angle for 8 minutes every 8 days to maintain a flow rate of 0.13 dyne / cm. 2 It can permeate at high speed.

[0083] From another aspect, the present invention relates to a method for manufacturing a glomerular mimic organ chip, comprising the steps of: culturing glomerular podocytes in a podocyte culture unit; and culturing glomerular endothelial cells in an endothelial cell culture unit.

[0084] In an exemplary embodiment, the method may further include a pretreatment step of coating the insert module membrane with collagen. This pretreatment step enables effective attachment of cells to the insert module membrane.

[0085] In an exemplary embodiment, the podocyte culture unit and the endothelial cell culture unit may be characterized in that the podocytes and endothelial cells are cultured on different sides of the insert module. The different sides of the insert module may be facing sides of the insert module membrane, as shown in FIGS. 1A to 1C.

[0086] In an exemplary embodiment, glomerular podocytes may be cultured on one side of the insert module, and then the insert module may be rotated to culture glomerular endothelial cells on the other side of the insert module.

[0087] In an exemplary embodiment, the method may be characterized by isolating and culturing podocytes and endothelial cells.

[0088] In an exemplary embodiment, the step of culturing the glomerular podocytes and the step of culturing the glomerular endothelial cells can be performed simultaneously or sequentially.

[0089] In another aspect, the present invention relates to a method for screening a treatment agent for kidney disease, comprising the steps of: (a) treating a kidney disease-inducing substance to the glomerular mimic organ chip; (b) treating a test substance to the glomerular mimic organ chip; and (c) measuring a kidney disease evaluation index in the kidney mimic organ chip treated with the test substance.

[0090] In an exemplary embodiment, the renal disease may be characterized as glomerulonephritis, wherein the glomerulonephritis may include, but is not limited to, one or more selected from the group consisting of membranous nephropathy, renal failure, immunoglobulin A nephropathy (IgAN), and minimal change disease (MCD).

[0091] In an exemplary embodiment, the renal disease-inducing substance may be at least one selected from the group consisting of puromycin aminonucleoside (6-dimethylaminopurine, 3-amino-d-ribose, PAN), serum of a patient with glomerulonephritis, and adriamycin, but is not limited thereto.

[0092] In an exemplary embodiment, step (c) may include comparing kidney disease evaluation indicators before and after treating the kidney-mimetic organ chip with a test substance, or when the kidney-mimetic organ chip is not treated with a test substance and when the kidney-mimetic organ chip is treated with a test substance.

[0093] In an exemplary embodiment, step (c) may include comparing kidney disease evaluation indicators in a kidney mimicking organ chip treated with a test substance and a kidney mimicking organ chip that is not treated with a test substance or before treating the test substance.

[0094] In an exemplary embodiment, the kidney disease evaluation index may be at least one selected from the group consisting of cell viability, albumin permeability, WT1 expression rate, RFP expression rate, and calcein-AM expression rate, but is not limited thereto.

[0095] In an exemplary embodiment, the step (c) may further include a step of determining the test substance as a treatment for kidney disease when at least one selected from the group consisting of cell viability, albumin permeability, WT1 expression rate, RFP expression rate, and calcein-AM expression rate increases after treatment with the test substance compared to before treatment or without treatment with the test substance.

[0096]

[0097] Hereinafter, the composition and effects of the present invention will be described in more detail through examples and experimental studies. However, the examples and experimental studies below are provided solely for illustrative purposes to aid understanding of the present invention and are not intended to limit the scope and scope of the present invention.

[0098] [Example]

[0099] [Example 1]

[0100] Fabrication of 3D microfluidic glomerular-mimetic organ-on-a-chip

[0101] The microfluidic plate and insert module (Greiner BIO-ONE, #662641) used are as shown in Fig. 1A. The membrane material of the insert module is polyethylene terephthalate (PET), and the pore size is 0.4 μm. Human glomerular podocytes (Human Podocyte Cell Line, CIHP-1, Ximbio, #CVCL W186) and human glomerular microvascular endothelial cells (RFP Expressing Human Glomerular Microvascular Endothelial Cells, ANGIO-PROTEOMIE, #cAP-0004RFP) were used to fabricate the glomerular mimicking organ chip. RPMI 1640 medium (Gibco, #11875093) supplemented with 10% fetal bovine serum (Fetal Bovine Serum, Gibco, #16000-044), 1% penicillin-streptomycin (Penicillin-Streptomycin, Gibco, #15140-122), and 1% insulin-transferrin-selenium (Insulin-Transferrin-Selenium, Gibco, #41400-045) was used as the human glomerular podocyte culture medium, and Endo-Basal medium (ANGIO-PROTEOMIE, #cAP-03) supplemented with 10% Endo-Growth Supplements (ANGIO-PROTEOMIE, #cAP-04) and 1% Penicillin-Streptomycin (Gibco, #15140-122) was added and used.

[0102] First, to ensure good attachment of cells to the insert module membrane, both sides of the membrane were coated with 100 μg / mL collagen I (Corning, #354265) at 37°C for 1 hour. After removing collagen I, the insert module was turned over and 1 x 10 human glomerular podocytes were seeded on the membrane. 5After seeding at a density of cells / mL, the insert module was cultured at 37°C for 2 hours. After 2 hours, the culture medium was removed, the insert module was inverted, mounted on a 24-well plate (SPL, #30024), and cultured at 37°C for 1 day. Before seeding human glomerular microvascular endothelial cells, 100 μL of coating solution (ANGIO-PROTEOMIE, #cAP-01) was dispensed into the inner part of the insert module to ensure good attachment of the endothelial cells to the membrane. The coating solution was removed, and 2 x 10 human glomerular microvascular endothelial cells were cultured. 5 After seeding cells / mL and culturing at 37°C for 2 hours, the inner medium of the insert module was replaced with a 1:1 mixture of glomerular podocyte medium and endothelial cell medium (Fig. 1B). 200 μL of glomerular podocyte culture medium was added to each of the five chambers of the microfluidic plate (Fig. 1C), and after pipetting to remove air bubbles in all connected channels of the chambers and fill them with the culture medium, the insert modules were sequentially and slowly mounted (Fig. 1D). The fabricated glomerular-mimetic organ chip was placed horizontally on a rocker (Organo Flow L rocker, Mimetas, #MI-OFPR-L) and co-cultured for 8 days. The rocker was tilted at a 7° tilt angle and 8-min interval to enable bidirectional flow (Fig. 2). The medium was replaced every 2-3 days to ensure that each cell type was reached.

[0103]

[0104] [Example 2]

[0105] Identification of morphological characteristics and specific expression markers of podocytes using a 3D microfluidic glomerular-mimetic organ-on-a-chip.

[0106] The morphology of human glomerular podocytes (CIHP-1) used in Example 1 was confirmed. Undifferentiated podocytes at 33°C had a colony shape, whereas differentiated podocytes at 37°C were transformed into round cells with foot processes (black arrows), filopodia-shaped protrusions (white arrows), and enlarged cells with interdigitation between cells (Fig. 3). These morphological changes in cells are consistent with the podocyte differentiation morphology reported in previous studies (Figs. 6-9, Shankland SJ et al, Kidney Int. 2007;72(1):26-36, B nyai E et al., Oxid Med Cell Longev. 2014; 2014:978394, Song B et al., PLos One. 20l2;7(9):e46453, Rauch C et al., PLos One. 2018;13(9):e020869).

[0107] Meanwhile, glomerular capillary endothelial cells, glomerular basement membrane, and filtration slits function as a filtration barrier of the renal glomerulus. Podocin is located on the membrane of the foot-like long processes of podocytes and is a lipoprotein of the podocyte filtration slit. It is known as a podocyte-specific marker that can confirm the degree of podocyte maturation because it is expressed only in differentiated podocytes and not in undifferentiated podocytes (Yin L. et al., Front Cell Dev Biol. 2021;9:771931, Saleem MA et al., J AM Soc Nephrol. 2002;13(3):630-638). The expression of podocin was confirmed in undifferentiated and differentiated podocytes in a two-dimensional culture environment and in a three-dimensional microfluidic organ chip. Consistent with the results of previous studies, it was confirmed that podocin is a mature podocyte-specific marker that is expressed only in differentiated podocytes and not in undifferentiated podocytes (Fig. 4).

[0108] WT1 (Wilms' tumor-1), which is present in the nucleus of podocytes at all stages, is a transcriptional factor of nephrin, an essential regulatory factor in kidney development, and a marker involved in podocyte neogenesis and differentiation (Saleem MA et al., J AM Soc Nephrol. 2002;13(3):630-638, Dong L et al., Kidney Int. 2015;88(4): 684-690, Martinez-Arroyo O et al., Am J Physiol Renal Physiol. 2020;319(2):F178-F191). The expression of WT1 was confirmed in undifferentiated podocytes, differentiated podocytes in a 2D culture environment, and in a 3D microfluidic glomerulus-mimetic organ chip. The highest expression was found in podocytes in the 3D microfluidic glomerulus-mimetic organ chip, and the expression rate increased in the order of undifferentiated podocytes in the 2D culture environment, differentiated podocytes in the 2D culture environment, and differentiated podocytes in the 3D microfluidic glomerulus-mimetic organ chip (Fig. 5). These results are in line with the research results reported by Martinez-Arroyo O et al., which showed that the expression rate of WT1 was higher in differentiated podocytes than in undifferentiated podocytes, and that the amount of WT1 protein expression increased as podocyte differentiation progressed, thus verifying that it is a marker that can confirm the maturity of podocytes.

[0109]

[0110] [Example 3]

[0111] Puromycin drug concentration for inducing glomerulonephritis

[0112] Puromycin aminonucleoside (6-dimethylaminopurine, 3-amino-d-ribose, PAN) is a podocytotoxin that causes glomerular podocyte damage and has been used in studies of human glomerular diseases, glomerular function, and morphology (Luimula P et al., 2002;82(6):713-8, Ha TS et al., Exp Cell Res. 2016;340(1):139-49, Petrosyan A et al., Nat Commun. 2019;10(1):3656, Fukuda H et al., Cell Tissue Res. 2015;360(2):391-400).

[0113] In order to establish a drug treatment evaluation system for glomerulonephritis (GN), we attempted to induce a glomerulonephritis model according to renal function using PAN. In a two-dimensional culture environment, podocytes (1 x 10 5 Cell viability was analyzed after 24 h of treatment with different PAN concentrations (1 × 10 cells / mL), and the viability was statistically significantly reduced in a PAN concentration-dependent manner (Fig. 6a). In a 3D culture environment, podocytes (1 × 10 5 cells / mL) and endothelial cells (2 x 10 5 Cell viability was analyzed after treatment with PAN concentrations for 24 hours at 100 cells / mL, and the viability was statistically significantly reduced in a PAN concentration-dependent manner (Fig. 6b). In a two-dimensional cell culture environment, cell viability was reduced by approximately 60% with 1 mg / mL of PAN treatment, and a previous study reported a PAN injury model for 5 days with 10 mg / mL of PAN. Therefore, the PAN concentration for inducing a glomerulonephritis model by PAN was ultimately determined to be 1 mg / mL (Petrosyan A et al., Nat Commun. 2019;10(1):3656).

[0114]

[0115] [Example 4]

[0116] Establishment of a puromycin-induced glomerulonephritis model and a tacrolimus therapeutic efficacy evaluation system in a 3D microfluidic glomerular-simulating organ-on-a-chip

[0117] Glomerulonephritis is a disease in which symptoms and signs occur when an inflammatory response occurs in the glomeruli due to immune dysfunction. The basic method of treating glomerulonephritis is the administration of immunosuppressants. Tacrolimus (FK506) is a calcineurin inhibitor that selectively suppresses the cell proliferation and activation of T lymphocytes that bind to antigen-presenting cells. It is widely used in combination with mycophenolate mofetil (MMF) and steroids for maintenance immunosuppression to prevent rejection after kidney transplantation (Figure 7). Tacrolimus is rapidly absorbed, reaching peak levels within 3 hours after administration, and has a known half-life of 12–18 hours (Schiff J et al., Clin J Am Soc Nephrol, 2007;2(2):374-84).

[0118] We aimed to evaluate the pharmacological efficacy of tacrolimus in a model of PAN-induced glomerulonephritis. In a glomerular-mimetic organ-chip, 10 μg / mL tacrolimus was administered hourly to PAN-damaged podocytes. The survival rate of podocytes decreased after 6 hours of treatment, which was statistically significantly increased (Figs. 8A and 8B, P<0.05).

[0119] In living cells, nonfluorescent calcein-AM is hydrolyzed by intracellular esterases to acetoxymethyl ester, which is then converted to green fluorescent calcein, a cell-permeable dye used to measure cell viability (Fig. 9). To confirm podocyte damage and resilience induced by PAN and tacrolimus drug treatment in a glomerular mimic organ chip, images of cells stained with green fluorescence after calcein-AM treatment were analyzed. After 24 hours of PAN treatment, cell viability decreased to 70.4%, and when media was treated for 24 hours after 6 hours of tacrolimus treatment, cell viability increased to 90.3% (Figs. 10A to 10C).

[0120] Glomerular filtration rate (GFR) is the most important value for evaluating kidney function. In patients with glomerulonephritis, the glomerular filtration rate decreases due to glomerular damage, causing a large amount of albumin in the blood to be excreted in the urine. This lowers the concentration of albumin in the blood, causing water and electrolytes in the blood to leak out of the blood vessels, resulting in edema, the most common symptom of glomerulonephritis. To confirm the glomerular filtration function of the glomerular-mimetic organ-chip, albumin permeability was analyzed after drug treatment with PAN and tacrolimus. As a result, the albumin permeability was statistically significantly increased to 142.5% after PAN treatment, and decreased to 107.7% and 102.0% after tacrolimus treatment and tacrolimus plus culture medium treatment, respectively (Fig. 11).

[0121] In addition, the expression rate of WT, a podocyte-specific marker, was confirmed, and the expression rate of WT1 was statistically significantly reduced to 66.0% after PAN treatment (P<0.005), and the expression rate increased to 91.2% after tacrolimus treatment. This is thought to be due to the increase in WT1 expression rate due to the recovery of cell viability in cells damaged by PAN by tacrolimus treatment (Figures 12A and 12B). In addition, the expression rate of RFP was also reduced to 19.0%, but increased to 36.1% by tacrolimus, and when the culture medium was treated after tacrolimus treatment, the expression rate of RFP in the glomerular mimic organ chip was significantly increased to 81.2% (Figures 12A and 12C). Taken together, these results suggest that PAN is a podocyte toxicant in a 3D microfluidic glomerular-simulated organ-on-a-chip, and that tacrolimus has therapeutic efficacy in restoring podocytes damaged by PAN treatment.

[0122]

[0123] [Example 5]

[0124] Establishment of a glomerulonephritis model using the serum of patients with membranous nephropathy and establishment of a system for evaluating the efficacy of tacrolimus treatment in a membranous nephropathy glomerular model.

[0125] Membranous nephropathy (MN), also known as membranous glomerulopathy, is an autoimmune disease that is one of many glomerular diseases that can cause nephrotic syndrome. When the immune system attacks the glomeruli in MN, the glomerular filtration function is altered, causing large amounts of protein to be excreted in the urine. If this continues at high levels, it can eventually lead to renal failure. Renal filtration occurs through the glomerular endothelium (yellow), the basement membrane (gray), and podocytes (green) (Figure 13, cross-section of a glomerulus). In a normal person, proteins are blocked by the three layers of the glomerulus (glomerular endothelium, basement membrane, and podocytes) and cannot pass into the urine. However, in the case of primary membranous nephropathy (PMN), which is idiopathic and accounts for 70-80% of the incidence of membranous nephropathy, immune complex deposits (black) accumulate in the basement membrane, thickening the basement membrane and damaging podocytes, which prevents normal filtering of proteins, especially albumin, and causes the disease (Figure 13).

[0126] We aimed to develop a glomerular nephritis organ-on-a-chip model using the serum of patients with membranous nephropathy and to suggest a platform for predicting clinical trials through drug treatment evaluation using the constructed glomerular nephritis organ-on-a-chip. Five patients with severe membranous nephropathy, whose histopathological examination revealed glomerular peripheral IgG deposits of ≥2+ and UPCR test results of ≥2,000 mg / g, were treated with 0.5% of their blood serum collected at the time of diagnosis for 24 hours to induce a membranous nephropathy model (Table 1, Petrosyan A et al., Nat Commun. 2019;10(1):3656).

[0127] [Table 1] General characteristics and outcomes of membranous nephropathy

[0128]

[0129] When we examined the viability of podocytes in response to serum treatment, cell viability was reduced by serum treatment from all patients with membranous nephropathy (Fig. 14A). In particular, cell viability was significantly reduced in patient groups MN1, MN2, and MN3, in which MN1 and MN2 were confirmed to have deposits of highly electron-dense materials on the epithelial side of the basement membrane as determined by pathological subepithelial electron-dense deposits (Table 1). Furthermore, when podocytes damaged by the serum of patients with membranous nephropathy were treated with tacrolimus (10 μg / mL) for 6 hours, cell viability was restored in patient groups MN1, MN2, and MN3 (Fig. 30A). Among the five patients, MN1, MN2, MN3, and MN5 were the patients who showed a drug response when administered tacrolimus. Additionally, examination of the albumin permeability in the glomerular model of membranous nephropathy revealed that the albumin level was significantly increased due to podocyte damage in the glomerular mimic chips treated with serum from patients MN1 and MN2, whereas the albumin level was decreased in the damaged glomerular mimic chips treated with tacrolimus (Fig. 14B).

[0130] The expression rate of WT1 in the glomerular mimicking organ chip treated with MN1 patient serum was confirmed to be statistically significantly reduced to 71.4% after PAN treatment (P<0.005) in podocytes, and the expression rate was significantly increased to 85.5% and 92.9% after tacrolimus treatment and when culture medium was treated after tacrolimus treatment, respectively (P<0.05, Figs. 15A and 15B). This is thought to be due to the increase in WT1 expression rate due to recovery of cell viability in podocytes damaged by MN1 patient serum by tacrolimus treatment.

[0131] In addition, the images of cells stained with green fluorescence after calcein-AM treatment were analyzed, and the cell viability was significantly reduced to 68.3% after 24 hours of treatment with MN1 patient serum, and significantly increased to 122.8% and 136.2% when treated with tacrolimus for 6 hours and the culture medium after tacrolimus treatment for 24 hours, respectively (Figures 16A and 16B). Taken together, these results suggest that the serum of membranous nephropathy patients damages podocytes in the 3D microfluidic glomerular-mimetic organ-chip, and tacrolimus is believed to have therapeutic drug efficacy in restoring podocytes damaged by membranous nephropathy serum.

[0132] Therefore, the present invention has produced results consistent with some clinical findings, and the development of a new type of preclinical nephrotoxicity model can evaluate candidate drugs for the treatment of kidney diseases and elucidate their main mechanisms of action.

Claims

1. An insert module, wherein a first cell culture section in which first cells are cultured and a second cell culture section in which second cells are cultured are arranged on different sides of the insert module.

2. An insert module comprising a podocyte culture section in which glomerular podocytes are cultured and an endothelial cell culture section in which glomerular endothelial cells are cultured.

3. In the second paragraph, the insert module, wherein the podocyte culture unit and the endothelial cell culture unit are arranged on different sides of the insert module.

4. In paragraph 2, An insert module in which the above-mentioned podocyte culture unit and / or endothelial cell culture unit includes an extracellular matrix.

5. In paragraph 2, The above-mentioned podocytes and / or endothelial cells are human-derived cells, insert module.

6. A glomerular mimicking organ chip comprising at least one insert module of any one of claims 2 to 5.

7. In paragraph 6, The above glomerular mimicking organ chip is a glomerular mimicking organ chip including a microfluidic plate.

8. In paragraph 6, The above glomerular mimic organ chip is a glomerular mimic organ chip that maintains bidirectional flow at the blood flow rate of the human body.

9. A method for manufacturing a glomerular mimic organ chip according to Article 6, comprising: a step of culturing glomerular podocytes in a podocyte culture unit; and a step of culturing glomerular endothelial cells in an endothelial cell culture unit.

10. In paragraph 9, A method for manufacturing a glomerular mimicking organ chip, wherein the method further comprises a pretreatment step of coating the insert module membrane with collagen.

11. In paragraph 9, A method for manufacturing a glomerular mimic organ chip, characterized in that the podocyte culture unit and the endothelial cell culture unit culture podocytes and endothelial cells on different sides of the insert module.

12. In paragraph 9, The method is a method for manufacturing a glomerular mimic organ chip, characterized in that the method comprises isolating and culturing the glomerular podocytes and glomerular endothelial cells. 13.(a) A step of treating a kidney disease-inducing substance on the glomerular mimic organ chip of Article 6; (b) a step of treating a test substance on the glomerular mimic organ chip; and (c) a step of measuring a kidney disease evaluation index in the kidney mimic organ chip treated with the test substance. A method for screening a kidney disease treatment agent, comprising:

14. In paragraph 13, A method for screening for a treatment agent for kidney disease, wherein the kidney disease is glomerulonephritis.

15. In paragraph 14, A method for screening a therapeutic agent for a kidney disease, wherein the glomerulonephritis comprises at least one selected from the group consisting of membranous nephropathy, renal failure, immunoglobulin A nephropathy (IgAN), and minimal change disease (MCD).

16. In paragraph 13, A method for screening a renal disease treatment agent, wherein the renal disease-inducing substance is at least one selected from the group consisting of puromycin aminonucleoside (6-dimethylaminopurine, 3-amino-d-ribose, PAN), serum of a patient with glomerulonephritis, and adriamycin.

17. In paragraph 13, A method for screening a treatment agent for kidney disease, wherein the step (c) comprises a step of comparing kidney disease evaluation indicators before and after treating a test substance on a kidney-mimetic organ chip or when the test substance is not treated and when the test substance is treated.

18. In paragraph 13, A method for screening a kidney disease treatment agent, wherein the step (c) comprises a step of comparing kidney disease evaluation indicators in a kidney mimic organ chip treated with a test substance and a kidney mimic organ chip not treated with a test substance or before treating the test substance.

19. In paragraph 13, A method for screening a treatment agent for kidney disease, wherein the above kidney disease evaluation index is at least one selected from the group consisting of cell viability, albumin permeability, WT1 expression rate, RFP expression rate, and calcein-AM expression rate.

20. In paragraph 13, A method for screening a treatment for kidney disease, further comprising a step of determining the test substance as a treatment for kidney disease, if the measurement result of the step (c) above shows that at least one selected from the group consisting of cell viability, albumin permeability, WT1 expression rate, RFP expression rate, and calcein-AM expression rate after treatment with the test substance has increased compared to before treatment or not treating the test substance.

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