Aggregates of kidney cells and methods for producing aggregates of kidney cells

Abstract

This invention provides aggregates of kidney cells in which the expression level of transporter genes is enhanced. [Solution] One aspect of the present invention is an aggregate of kidney cells. The aggregate has a ratio R (gene expression level of OAT1 / gene expression level of MDR1) of 0.01 or higher between the gene expression levels of OAT1 and MDR1.

Description

[Technical Field]

[0001] This invention relates to aggregates of kidney cells and a method for producing aggregates of kidney cells. [Background technology]

[0002] Drugs administered to a living organism are absorbed into the body and then excreted from the blood into the urine via the proximal tubules in the kidneys. Therefore, nephrotoxicity of drugs often causes kidney damage. In drug discovery research, investigating pharmacokinetics in the kidneys is crucial for elucidating the effects of drugs. For this reason, the development of drug discovery support devices capable of evaluating pharmacokinetics and toxicity using renal cells is highly desirable. Furthermore, such drug discovery support devices would also be useful in developing treatments for kidney-related diseases (e.g., diabetes, renal cancer, hyperuricemia, etc.).

[0003] Renal cell aggregates have been proposed as a useful tool for confirming pharmacokinetics and for drug discovery (see Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2021-191305 [Overview of the project] [Problems that the invention aims to solve]

[0005] Conventional renal cell aggregates have insufficient expression levels of transporter genes necessary for evaluating pharmacokinetics and toxicity, leaving room for the development of novel renal cell aggregates.

[0006] In view of the above-mentioned problems, the present invention aims to provide kidney cell aggregates with improved transporter gene expression levels and a method for producing kidney cell aggregates. [Means for solving the problem]

[0007] One aspect of the present invention is an aggregate of renal cells. The aggregate of renal cells satisfies at least one or more of the following conditions. (a) The expression level of the OAT1 gene is 1×10 -3 or more (relative to GAPDH). (b) The expression level of the OAT3 gene is 1×10 -4 or more (relative to GAPDH). (c) The expression level of the OCT2 gene is 1×10 -3 or more (relative to GAPDH). (d) The expression level of the MATE1 gene is 1×10 -4 or more (relative to GAPDH). (e) The expression level of the MATE2 gene is 1×10 -5 or more (relative to GAPDH). (f) The expression level of the MDR1 gene is 1×10 -3 or more (relative to GAPDH). (g) The expression level of the URAT1 gene is 1×10 -6 or more (relative to GAPDH).

[0008] The diameter of the renal cell aggregate according to the above aspect may be 150 µm or more and 350 µm or less. The number of renal cells constituting the aggregate may be 400 or more and 2100 or less. Further, the number of cell passages of said renal cells may be 3 or 4. The doubling time of said renal cells may be 20 to 36 hours.

[0009] Another aspect of the present invention is a method for producing a renal cell aggregate. The method for producing a renal cell aggregate comprises: a pre-culture step of culturing renal cells under conditions such that the doubling time of the cultured renal cells is 20 to 36 hours; an aggregate forming step of forming an aggregate of said renal cells that have undergone said pre-culture step; and a standing step of allowing the formed aggregate to stand still for 24 hours or more.

[0010] In said pre-culture step in the method for producing a renal cell aggregate according to the above aspect, renal cells are seeded at a seeding density of 2.1×10 3 to 4.4×10 3 cells / cm 2The culture may be carried out for 48 to 120 hours.

Effects of the Invention

[0011] According to the present invention, a technique relating to aggregates of renal cells with increased expression level of a transporter gene can be provided.

Brief Description of Drawings

[0012] [Figure 1] This is a graph showing the expression level (ratio to GAPDH) of each OAT1 gene in human renal cortex in Examples 1 to 4.

Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described in detail. In the present specification, the notation "a to b" in the description of a numerical range means that it is not less than a and not more than b, unless otherwise specified.

[0014] (Aggregates of renal cells) The renal cells used in this disclosure are not limited to those that are cultureable and are of any source. The renal cells are preferably of mammalian origin, and preferably of primate origin such as humans or monkeys. Depending on the purpose, they may also be derived from normal kidneys or from kidneys with disease. Examples of renal cells include cells that constitute the epithelium, cortex, proximal tubule, distal tubule, collecting duct, glomerulus, etc., specifically proximal tubular epithelial cells (RPTECs), mesangial cells, etc. The renal cells may also be stem cell-derived renal cells such as iPS cells or ES cells. Furthermore, the renal cells may be immortalized renal cells, established cell lines (such as HK-2 cells), cells derived from other animal species (such as MDCK cells, LLC-PK1 cells, JTC-12 cells), or forced-expression cells into which genes have been introduced to express specific transporter proteins. More specifically, examples of kidney cells include, for example, human proximal tubular epithelial cells, human distal tubular epithelial cells, and human collecting duct epithelial cells collected and isolated from the kidney, as well as proximal tubular epithelial cells, distal tubular epithelial cells, and collecting duct epithelial cells differentiated from human iPS cells or human ES cells. For use in drug discovery research, proximal tubular epithelial cells, in particular, proximal tubular epithelial cells derived from normal human kidneys are preferred.

[0015] Here, a "spheroid" of kidney cells refers to a clump-like collection of several or more cells. It is also called a spheroid.

[0016] The kidney cell aggregates according to this embodiment (hereinafter, kidney cell aggregates may be simply referred to as "aggregates") satisfy at least one of the following conditions. (a) Expression level of the OAT1 gene is 1 × 10 -3 The above is the result (GAPDH ratio). (b) Expression level of the OAT3 gene is 1 × 10 -4 The above is the result (GAPDH ratio). (c) Expression level of the OCT2 gene is 1 × 10 -3 The above is the result (GAPDH ratio). (d) Expression level of the MATE1 gene is 1 × 10 -4 The above is the result (GAPDH ratio). (e) Expression level of the MATE2 gene is 1 × 10 -5 The above is the result (GAPDH ratio). (f) Expression level of the MDR1 gene is 1 × 10 -3 The above is the result (GAPDH ratio). (g) Expression level of the URAT1 gene is 1 × 10 -6 The above is the result (GAPDH ratio).

[0017] The expression levels of each transporter gene in kidney cell cultures can be measured using a common real-time PCR (qPCR) method. The "GAPDH ratio" is a value obtained by correcting the expression levels of each transporter gene with the housekeeping gene GAPDH, and is obtained by dividing the expression level of each transporter gene by the expression level of the GAPDH gene. By calculating the "GAPDH ratio," the relative gene expression levels of each transporter can be measured regardless of differences in the number of cells collected. This value corrects for sample errors during sample preparation in the qPCR method.

[0018] OAT1 (Organic Anion Transporter 1), OAT3 (Organic Anion Transporter 3), OCT2 (Organic Cation Transporter 2), MATE1 (Multidrug And Toxin Extrusion 1), MATE2 (Multidrug And Toxin Extrusion 2), and MDR1 (Multiple Drug Resistance 1) are genes that encode proteins involved in drug transport. URAT1 (Urate Transporter 1) is a gene that codes for a protein involved in uric acid reabsorption.

[0019] OAT1 gene expression level 1 × 10 -3 The above (GAPDH ratio) (satisfying condition (a)) makes it easier to induce Tenofovir-induced toxicity, which is OAT1-mediated toxicity, and allows for the acquisition of kidney cell aggregates suitable for evaluating Tenofovir-induced toxicity.

[0020] OAT3 gene expression level 1 × 10 -4 The above (GAPDH ratio) (satisfying condition (b)) makes it easier to induce drug uptake and toxicity via OAT3. Because OAT3 uses compounds with larger molecular weights and higher lipophilicity as substrates than OAT1, it has the effect of enabling uptake of a wider range of drugs.

[0021] OCT2 gene expression level is 1 × 10⁻⁶ -3 The above (GAPDH ratio) (satisfying condition (c)) makes it easier to induce cisplatin-induced toxicity, which is OCT2-mediated toxicity, and allows for obtaining renal cell aggregates suitable for evaluating cisplatin-induced toxicity.

[0022] The expression level of the MATE1 gene is 1 × 10⁻⁶ -4 By meeting the above criteria (GAPDH ratio) (satisfying condition (d)), aggregates of renal cells that have the function of excreting drugs taken into the bloodstream into the urine can be obtained in the kidney.

[0023] The expression level of the MATE2 gene is 1 × 10⁻¹⁶ -5 By meeting the above criteria (GAPDH ratio) (satisfying condition (e)), aggregates of renal cells with the function of excreting drugs taken up by the kidney into the urine can be obtained. By expressing MATE1 simultaneously, aggregates of renal cells with the function of excreting a wide range of drugs into the urine can be obtained.

[0024] MDR1 gene expression level 1 × 10 -3 By meeting the above criteria (GAPDH ratio) (satisfying condition (f)), aggregates of renal cells that have the function of excreting drugs taken up by the kidney into the urine can be obtained. Because it is a transporter with broad substrate recognition, aggregates of renal cells that have the function of excreting various types of drugs can be obtained.

[0025] The expression level of the URAT1 gene is 1 × 10⁻⁶. -6By meeting the above conditions (GAPDH ratio) (satisfying condition (g)), aggregates of renal cells with uric acid reabsorption function can be obtained.

[0026] The kidney cell aggregates according to the embodiment preferably satisfy two, three, four, five, six, or seven (all) of the above-mentioned conditions. If two or more conditions are satisfied, it is more preferable that at least conditions (a) and (f) are satisfied. OAT1 is a transporter responsible for the uptake of drugs from the blood vessels after drug administration. On the other hand, MDR1 is a transporter that excretes the uptaken drug towards the ureter. Therefore, by satisfying conditions (a) and (f), nephrotoxicity or nephrophatic activity in drug discovery can be appropriately evaluated.

[0027] The ratio R (gene expression level of OAT1 / gene expression level of MDR1) of the gene expression level of OAT1 to MDR1 is preferably 0.01 or higher, more preferably 0.1 or higher, and even more preferably 0.5 or higher. The ratio R is greater than or equal to the lower limit mentioned above, and the closer it is to "1", the more normally drug uptake and excretion in the kidneys occur, resulting in human kidney cells that function similarly to those of the kidneys in the human body, and thus cells that can be appropriately evaluated for nephrotoxicity or pharmacokinetics in drug discovery. Furthermore, the more the above ratio R is greater than or equal to the lower limit mentioned above and approaches "1", the more the kidney can perform a function similar to that of a normal kidney, such as excreting drugs absorbed from the blood vessels into the ureter.

[0028] The diameter of the aggregate is preferably 150 μm or more and 350 μm or less, more preferably 200 μm or more and 350 μm or less, and even more preferably 250 μm or more and 350 μm or less.

[0029] The number of kidney cells constituting the aggregate is preferably between 400 and 2100, and more preferably between 900 and 1600. Having the number of kidney cells constituting the aggregate within this range allows for higher homogeneity among multiple aggregates.

[0030] The number of cell passages of kidney cells used in the pre-culture step is preferably 3 or 4. By using 3 or 4 cell passages, it becomes possible to create aggregates of kidney cells with activated metabolic functions, and aggregates that satisfy at least one of the above conditions (a) to (g) can be obtained. Most human somatic cells do not proliferate indefinitely, and when human kidney cells are cultured in vitro, they cease to proliferate after a certain number of passages. Cell chromosomes contain repeating sequences called telomeres, and the shortening of these telomeres due to cell proliferation causes cellular aging. Cellular aging can also be caused by external environmental factors such as oxidative stress, so cellular aging is observed in in vitro culture conditions outside the body. When cells age, DNA damage occurs, making it difficult for them to transition to the proliferation cycle in the cell cycle. Therefore, it is thought that kidney cells age with repeated passages, and their proliferative capacity decreases (doubling time increases). In addition, when cellular aging occurs, a decrease in protein expression and the expression of abnormal proteins due to DNA damage are induced, so it is thought that when aggregates are formed using aged kidney cells, the function of increasing the expression of drug transporters itself is reduced. Based on these findings, it is possible to obtain kidney cell aggregates with high drug transporter expression by forming aggregates using unsenescent kidney cells with three or four passages. Furthermore, since it is difficult to collect the necessary number of cells to form aggregates from kidney cells with reduced proliferative capacity, a sufficient amount of kidney cell aggregates can be produced by using kidney cells in a proliferation cycle with a doubling time of 20 to 36 hours.

[0031] The roundness of the aggregate is preferably 0.3 to 1.0, and more preferably 0.4 to 1.0. The aspect ratio of the aggregate is preferably 1.0 to 2.0, and more preferably 1.0 to 1.5. The roundness and aspect ratio of the aggregate can be calculated by recognizing a single aggregate as an image {slice image (cross-sectional view)} using a CQ1 (confocal image cytometer, manufactured by Yokogawa Electric Corporation) based on a well-known method.

[0032] (Method for producing aggregates of kidney cells) The method for producing kidney cell aggregates according to this embodiment includes a pre-culture step, an aggregate formation step, and a standing step. Details of each step will be described below.

[0033] (Preculture step) In the pre-culture step, kidney cells are cultured in a culture vessel such as a culture dish under conditions that result in a doubling time of 20 to 36 hours, preferably 24 to 32 hours. Specific pre-culture conditions include a kidney cell seeding density of 2.1 × 10⁻⁶. 3 ~4.4×10 3 cells / cm 2 The culture time is typically 48 to 120 hours. At this time, cell confluence is 50-80%. Any known culture medium can be used as appropriate. For example, in the case of culturing proximal tubular epithelial cells, commercially available tubular cell culture media can be used, and examples of preferred media include REGM® (LONZA), EpiCM® (ScienCell), and KeratinocyteSFM® (Thermo Fisher Scientific).

[0034] By performing pre-culturing so that the doubling time of renal cells after culture is 20 to 36 hours, the proliferative capacity of the renal cells is maximized, making it possible to create aggregates of renal cells with activated metabolic function, and aggregates that satisfy at least one of the above conditions (a) to (g) can be obtained.

[0035] (Agglomerate formation process) In the aggregate formation process, aggregates of kidney cells that have undergone the pre-culture process are formed. In the aggregate formation process, the culture of kidney cells can be carried out according to conventional methods, for example, under conditions of 37°C and 5% CO2, using a culture medium and culture vessel suitable for the cells to be cultured. The culture may be static culture, shaking culture, or agitated culture. The culture may be adherent culture, but it is preferable to culture in a low-adhesion state for at least a portion of the period (for example, suspension culture). Kidney cells can form aggregates by being cultured in a low-adhesion state in the culture vessel. "Low-adhesion state" refers to a state in which all or most of the cells are not adhered to the surface of the culture vessel, and includes a state in which all or most of the cells are away from the surface of the culture vessel, and a state in which, even if in contact with the surface of the culture vessel, the cells can be easily separated from the surface of the culture vessel by coating the culture vessel or convection of the culture medium without the use of instruments or enzymes.

[0036] For example, in some cases, aggregates of kidney cells form within 24 hours of starting kidney cell culture. Culturing kidney cells in aggregate form for a period of time can restore the physiological function of dedifferentiated kidney cells. Generally, it is desirable to culture kidney cells in a low-adhesion state in the culture vessel for 120 hours or more. This allows for the acquisition of cultured kidney cells with a higher level of physiological expression. During the culture period, it is preferable to change the culture medium regularly. For example, the medium is changed every two days.

[0037] Any known culture medium can be used as appropriate. For example, in the case of culturing proximal tubular epithelial cells, commercially available tubular cell culture media can be used, and examples of preferred media include REGM® (LONZA), EpiCM® (ScienCell), and KeratinocyteSFM® (Thermo Fisher Scientific).

[0038] Furthermore, conventionally known materials and additives useful for cell culture can be used as appropriate. For example, collagen I (type I collagen) can be added to the culture medium. Collagen I has the effect of adhering kidney cells to each other. Therefore, culturing kidney cells in a medium containing collagen I promotes the formation of aggregates. It is preferable that the collagen I be full-length collagen I, but it may also be the α1 chain or α2 chain that constitutes collagen I, or even collagen peptides obtained by fragmenting each chain. The source of collagen I is not particularly limited and may be derived from humans or other animals.

[0039] Any culture vessel can be used. Here, in order to promote aggregate formation, it is preferable that the vessel is treated to be non-(low)adherent or composed of a non-(low)adherent material. Examples of non-(low)adherent treatments include a non-(low)adherent hydrogel coating treatment on the vessel surface, an MPC (2-methacryloyloxyethyl phosphorylcholine) coating treatment, a Proteosave® SS coating treatment, and a mirror polishing treatment. Examples of non-(low)adherent materials include glass, as well as polymer materials such as low-density polyethylene, medium-density polyethylene, polyvinyl chloride, polyethylene-vinyl acetate copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, poly(ethylene vinyl acetate) copolymer, and mixtures of two or more of these polymers.

[0040] When forming a large number of aggregates, high-density spheroid production plates or dishes can be used. Furthermore, culture vessels such as spinner flasks may be used as needed. For example, it is preferable to use culture vessels from the ELPLASIA® series (Corning Corporation) or the EZSPHERE® series (AGC Technoglass Corporation). These culture vessels come in types such as 6-well plates, 24-well plates, 96-well plates, 384-well plates, and dishes of various sizes, and the number of aggregates that can be produced differs depending on the size of the bottom area of ​​the vessel. For example, when using a 96-well plate (V-bottom), 96-well plate (U-bottom), or 384-well plate (U-bottom) that has been treated to reduce adhesion, one aggregate is formed in each well.

[0041] Aggregates prepared using high-density spheroid production plates or dishes can be collected and cultured by suspension shaking. When culturing by suspension shaking, it is preferable to place dishes or plates that have been treated to prevent (or reduce) cell adhesion on a shaker and culture the aggregates. Reciprocating shakers and swirling shakers can be used.

[0042] (Standing process) In the standing stage, the kidney cell aggregates obtained in the aggregate formation stage are preferably left to stand for 24 hours or more, and more preferably for 48 hours or more, after seeding, under conditions such as 37°C and 5% CO2. The upper limit of the standing time is the number of days for which the effects of standing can be obtained, and it is preferable that there is no shortage of culture medium (nutrients) supplied to the cells. For example, it is preferable that it be within 5 days, and more preferably within 4 days. The culture medium is not changed during the standing stage of the kidney cells. By leaving the obtained aggregates to stand for the above period, the adhesion between cells in the aggregates can be made stronger. In addition, spherical human kidney cell aggregates can be formed during the culture process without applying loads such as shear stress to the kidney cells during aggregate formation. In other words, by going through the standing stage, the roundness and / or aspect ratio of the kidney cell aggregates will be within the above range and approach 1. As a result, it is presumed that the expression levels of each transporter gene described above will increase. The period during which the roundness and aspect ratio approach 1 is called the standing stage. After the standing stage, the culture medium is changed every two days to regularly remove waste products and supply nutrients to the medium.

[0043] (Uses of kidney cell aggregates) The kidney cell aggregates of this embodiment have enhanced expression levels of the transporter genes described above, and can therefore be provided as kidney cell products for drug evaluation systems. Examples of such drug evaluation systems include those for evaluating pharmacokinetics in kidney cells and nephrotoxicity in drug discovery. Furthermore, such kidney cells can be used for analyzing the mechanisms of kidney-related diseases such as diabetes, kidney cancer, and hyperuricemia, as well as as a tool for drug discovery.

[0044] The embodiments of the present invention have been described above, but these are merely examples, and various other configurations can also be adopted. [Examples]

[0045] The present invention will be described below with reference to examples and comparative examples, but the present invention is not limited thereto.

[0046] (Example 1) <Manufacturing of renal cell aggregates> Human proximal tubular epithelial cells (RPTEC-renal proximal tubular epithelial cells) obtained from LONZA were used as renal cells. Frozen vials stored in a liquid nitrogen storage unit were thawed by immersion in a 37°C constant temperature bath. After thawing, the cell suspension in the frozen vial was mixed with the recommended culture medium (REGM®, LONZA), and the pre-culture process was carried out in a culture dish. The pre-culture conditions were a seeding density of 4.2 × 10⁶ cells. 3 cells / cm 2 The cells were cultured for 72 hours and passed through 3 passages. Pre-culture resulted in approximately 70% cell confluence. The kidney cells that had undergone the pre-culture process were harvested, and the doubling time was determined to be 27.5 hours based on the number of cells before seeding and the number of cells at harvesting. Kidney cells that had undergone a pre-culture process were collected, and an aggregate formation process was carried out. Specifically, kidney cells were cultured at 37°C and 5% CO2, with the culture medium being changed every two days. The cells were collected before they became confluent and seeded onto a 96-well V-bottom plate {PrimeSurface® plate 96V, Sumitomo Bakelite Co., Ltd.} treated for low cell adhesion, with 1000 cells per well, and cultured to form aggregates. For two days (48 hours) after aggregate formation, the culture plate was left undisturbed in an incubator without any culture operations such as medium changes. After the undisturbed period, the aggregates were cultured for more than 240 hours, with the culture medium being changed every two days. Note that "confluent" means that the area occupied by cells relative to the entire culture surface of the culture vessel is approximately 100%, that is, the cells have proliferated without any gaps on the culture surface.

[0047] (Example 2) Renal cell aggregates were prepared in the same manner as in Example 1, except that the number of passages was set to 9.

[0048] (Example 3) Renal cell aggregates were prepared in the same manner as in Example 1, except that the number of passages was set to 12.

[0049] (Example 4) Renal cell aggregates were prepared in the same manner as in Example 1, except that the number of passages was set to 17.

[0050] The following evaluations or measurements were performed on each renal cell aggregate in Examples 1 to 4.

[0051] <Measurement of transporter gene expression levels> mRNA was extracted and purified from each aggregate after standing for two days using the RNeasy® Mini Kit (QIAGEN). Furthermore, cDNA was synthesized from this mRNA using the QuantiTect® Whole Transcriptome Kit (QIAGEN). Using these cDNAs as templates, the expression levels of the GAPDH, OAT1, OAT3, OCT2, MATE1, MATE2, MDR1, and URAT1 genes were measured by real-time PCR using the Thermal Cycler Dice® Real Time System 1 (Takara Bio). For all experiments, each sample was measured with n=3 in a single experiment. Table 1 shows the primer names and oligo sequences of each gene used in the real-time PCR method.

[0052] [Table 1]

[0053] The expression levels of each transporter gene were divided by the expression level of the GAPDH gene to obtain the expression levels (GAPDH ratio) of each transporter gene. The results of calculating the expression levels (GAPDH ratio) of each transporter gene are shown in Table 2.

[0054] (Diameter, roundness, and aspect ratio of aggregates) Based on a well-established method, the appearance of the obtained renal cell aggregates was recognized as an image {slice image (cross-sectional view)} of a single aggregate using a CQ1 (confocal image cytometer, Yokogawa Electric Corporation), and the diameter, roundness, and aspect ratio of the aggregate were calculated. The results obtained for the diameter, roundness, and aspect ratio of the renal cell aggregates are shown in the table.

[0055] [Table 2]

[0056] In Table 2 above, items marked with "-" indicate that the action has not been taken. Furthermore, the expression level of the OAT1 gene (GAPDH ratio) was measured in human proximal tubular epithelial cells used to form renal cell aggregates. Figure 1 shows graphs of the OAT1 gene (GAPDH ratio) expression levels for Example 1, Example 3, Example 4, and human renal cortex.

[0057] (Changes in cell morphology and gene expression levels during aggregate formation) Following the method for producing renal cell aggregates described above, the diameter, roundness, aspect ratio, and OAT1 expression level of the aggregates were calculated for 0 days (immediately after seeding), 1 day, 2 days, and 11 days after the start of aggregate formation. It was confirmed that the closer the roundness and aspect ratio were to 1, the higher the expression level of the OAT1 gene.

[0058] [Table 3] [Industrial applicability]

[0059] The kidney cell aggregates of this embodiment have enhanced expression levels of the transporter genes described above, and can therefore be used as kidney cell products for drug evaluation systems. Furthermore, the kidney cells of this embodiment can be used for analyzing the mechanisms of kidney-related diseases such as diabetes, kidney cancer, and hyperuricemia, as well as as a tool for drug discovery.

Claims

1. Agglutinary cell aggregates in which the ratio R (gene expression level of OAT1 / gene expression level of MDR1) of the gene expression level of OAT1 is 0.01 or higher.

2. The kidney cell aggregate according to claim 1, wherein the ratio R is 0.1 or greater.

3. The kidney cell aggregate according to claim 1, wherein the ratio R is 0.5 or more.

4. The expression level of the OAT1 gene is 1 × 10⁻⁶ －３ Furthermore, the expression level of the MDR1 gene is 1 × 10⁻⁶ －３ The above is the aggregate of kidney cells according to any one of claims 1 to 3.

5. The kidney cell aggregate according to any one of claims 1 to 3, wherein the kidney cells are proximal tubular epithelial cells derived from a normal human kidney.

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