Method for producing frozen kidney cells and frozen kidney cells

Abstract

To provide: frozen renal cells which have high cell viability after thawing and in which physiological functions of the kidney are maintained; and a renal cell culture in which the physiological functions of the kidney are maintained.SOLUTION: A method for producing renal cells in a frozen state, comprises: a recovery step of recovering cultured renal cells, and a freezing step of freezing the renal cells recovered in the recovery step, in which the renal cells are recovered as an aggregate in the recovery step, and the aggregate is frozen while an aggregated state of the aggregate is substantially maintained in the freezing step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing frozen kidney cells, and to frozen kidney cells. The present invention further relates to a method for producing kidney cell cultures obtained from frozen kidney cells, and to kidney cell cultures. [Background technology]

[0002] Drugs administered to the body are absorbed into the body and then excreted from the blood into the urine via the proximal tubules of the kidney. Therefore, nephrotoxicity of drugs often leads to kidney damage. In drug discovery research, it is very important to investigate pharmacokinetics in the kidney to clarify the effects of drugs.

[0003] Therefore, there is a need for an assay system that can evaluate pharmacokinetics and toxicity using kidney cells with normal physiological functions as a drug discovery support device. In recent years, many researchers have been developing kidney cells derived from human iPS cells and perfusion culture systems. Because proximal tubule epithelial cells are most susceptible to the effects of drugs, there is a particular demand for proximal tubule epithelial cells that can be used for research.

[0004] It is known that cultured kidney cells lose many of the intrinsic kidney functions when cultured in flat-bottom culture plates, which differ from the in vivo environment. Traditionally, cells collected from human kidneys have typically been dispersed by enzymatic treatment and then cultured two-dimensionally in flat-bottom culture plates. Cultured proximal tubule epithelial cells have not been used in drug discovery research because they no longer exhibit normal kidney pharmacokinetic and toxic responses due to dedifferentiation.

[0005] It has been found that forming aggregates from proximal tubule epithelial cells that have lost their original physiological renal functions and culturing them for a certain period of time can significantly improve renal function (Patent Document 1). In order to use these aggregates of proximal tubule epithelial cells with improved renal function in drug discovery research, it is necessary to produce them in large quantities and store a certain amount.

[0006] Cryopreservation is the best way to preserve cells in a stable state. In a conventional method for freezing cells, intercellular adhesion is first loosened with a digestive enzyme (such as trypsin) to disperse the cells individually. Next, the suspension containing the dispersed cells is centrifuged, and the collected cells are frozen by adding a liquid containing a cryoprotectant (Patent Document 2).

[0007] However, when conventional freezing methods are used to freeze aggregates of proximal tubule epithelial cells, attempts to form aggregates using the same method after thawing have proven difficult, as the cells remain dispersed. Furthermore, cells frozen using this method have reduced expression of genes that control kidney function after thawing, making it impossible to maintain kidney function, and also resulting in low cell viability after culture. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2018 / 186185 [Patent Document 2] Japanese Patent Application Publication No. 6-46840 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide frozen kidney cells that have a high cell survival rate after thawing and that maintain the physiological functions of the kidney, and a kidney cell culture that maintains the physiological functions of the kidney. [Means for solving the problem]

[0010] One embodiment of the present invention is a method for producing frozen kidney cells, comprising a recovery step of recovering cultured kidney cells and a freezing step of freezing the kidney cells recovered in the recovery step, wherein the recovery step recovers the kidney cells as aggregates, and the freezing step freezes the aggregates while substantially maintaining the aggregated state of the aggregates.

[0011] One embodiment of the present invention is a frozen kidney cell produced by a method comprising a recovery step of recovering cultured kidney cells and a freezing step of freezing the kidney cells recovered in the recovery step, wherein the recovery step recovers the kidney cells as aggregates, and the freezing step freezes the aggregates while substantially maintaining the aggregated state of the aggregates. [Effects of the Invention]

[0012] According to the present invention, frozen kidney cells having a high cell survival rate after thawing and maintaining the physiological function of the kidney, and a kidney cell culture in which the physiological function of the kidney is maintained are provided. [Brief explanation of the drawings]

[0013] [Figure 1-1] FIG. 1-1 is a light micrograph showing the morphology of aggregates of proximal tubular epithelial cells frozen in an aggregate state after thawing. [Figure 1-2] FIG. 1-2 shows the ATP content (cell viability) of proximal tubular epithelial cells frozen in the state of aggregates. [Figure 2-1] FIG. 2-1 shows the results of OAT1 gene expression analysis by real-time PCR of proximal tubular epithelial cells frozen in an aggregate state, in comparison with human renal cortex. [Figure 2-2] FIG. 2-2 shows the results of OCT2 gene expression analysis by real-time PCR of proximal tubular epithelial cells frozen in an aggregate state, in comparison with human renal cortex. [Figure 2-3]FIG. 2-3 shows the results of URAT1 gene expression analysis by real-time PCR of proximal tubular epithelial cells frozen in the state of aggregates, in comparison with human renal cortex. [Figure 2-4] Figures 2-4 show the results of OAT1 gene expression analysis by real-time PCR of proximal tubular epithelial cell aggregates after thawing and suspension shaking culture for the periods indicated in the figure, compared with human renal cortex. [Figure 3] FIG. 3 shows the results of OAT1 gene expression analysis by real-time PCR of proximal tubular epithelial cell aggregates frozen at different cell numbers, in comparison with human renal cortex. [Figure 4-1] FIG. 4-1 shows the ATP amount (cell viability) of proximal tubular epithelial cell aggregates when freezing methods were changed. [Figure 4-2] FIG. 4-2 shows the results of OAT1 gene expression analysis by real-time PCR of proximal tubular epithelial cell aggregates obtained by different freezing methods, in comparison with human renal cortex. [Figure 5] FIG. 5 is a graph showing the ATP amount (cell viability) of proximal tubular epithelial cell aggregates after thawing when different cryopreservation solutions were added during freezing. [Figure 6] FIG. 6 shows the results of OAT1 gene expression analysis by real-time PCR of thawed proximal tubular epithelial cell aggregates when the frozen storage period was changed. [Figure 7-1] FIG. 7-1 shows optical micrographs showing the morphology of aggregates of proximal tubular epithelial cells (comparative example) frozen in a dispersed state before freezing and after thawing. [Figure 7-2] FIG. 7-2 is a diagram showing the ATP amount (cell viability) before and after thawing of proximal tubular epithelial cells (comparative example) frozen in a dispersed state. [Figure 8] FIG. 8 shows the results of OAT1 gene expression analysis by real-time PCR of proximal tubular epithelial cells (comparative example) frozen in a dispersed state, in comparison with human renal cortex. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present inventors discovered that by freezing kidney cell aggregates in the aggregate state, they can be stably cryopreserved while maintaining cell viability and morphology, and further discovered that kidney cell cultures that maintain kidney function can be obtained from aggregates that have been cryopreserved in the aggregate state after thawing, thereby completing the present invention.

[0015] A method for producing frozen kidney cells according to one embodiment of the present invention includes a recovery step of recovering cultured kidney cells and a freezing step of freezing the kidney cells recovered in the recovery step. In the recovery step, the kidney cells are recovered as aggregates. In the freezing step, the aggregates are frozen while substantially maintaining their aggregated state.

[0016] The kidney cells used in the present invention may be any cell source as long as they can be cultured. Kidney cells are preferably derived from mammals, and preferably from primates such as humans or monkeys. Depending on the purpose, they may be derived from normal kidneys or diseased kidneys. Examples of kidney cells include cells that constitute the epithelium, cortex, proximal tubules, distal tubules, collecting ducts, and glomeruli, specifically proximal renal tubule epithelial cells (RPTECs) and mesangial cells. Kidney cells may be primary cells or kidney cells derived from stem cells such as iPS cells or ES cells. Kidney cells may also be immortalized kidney cells, established cell lines (e.g., HK-2 cells), cells derived from other animal species (e.g., MDCK cells, LLC-PK1 cells, JTC-12 cells), or cells expressing specific transporter proteins or other proteins through gene transfer. More specifically, examples of kidney cells include human proximal tubule epithelial cells, human distal tubule epithelial cells, and human collecting duct epithelial cells collected and isolated from the kidney, as well as proximal tubule epithelial cells, distal tubule epithelial cells, and collecting duct epithelial cells differentiated from human iPS cells or human ES cells. For use in drug discovery research, proximal tubule epithelial cells, particularly proximal tubule epithelial cells derived from normal human kidneys, are preferred.

[0017] Renal cells can be cultured according to standard methods, for example, at 37°C and 5% CO2, using a medium and culture vessel suitable for the cells to be cultured. Culture may be static, shaking, or agitation culture. While adherent culture may be used, it is preferable to culture the cells in a non-adherent state (e.g., suspension culture) for at least a portion of the period. Renal cells can form aggregates by culturing them in a non-adherent state to the culture vessel. The term "non-adherent state" refers to a state in which all or most of the cells are not attached to the surface of the culture vessel, and includes a state in which all or most of the cells exist away from the surface of the culture vessel, and a state in which, even if the cells are in contact with the surface of the culture vessel, they can easily detach from the surface of the culture vessel due to the coating of the culture vessel or convection of the culture medium without the use of instruments or enzymes.

[0018] For example, in some cases, kidney cell aggregates are formed on the first day (i.e., within 24 hours) of kidney cell culture. By culturing kidney cells in an aggregate state for a certain period, it is possible to restore the physiological function of kidney cells that has been reduced due to dedifferentiation. The period for culturing kidney cells in a non-adherent state to a culture vessel is generally 5 days or longer (i.e., 120 hours or longer). This allows for the production of cultured kidney cells in a state where physiological function is more highly expressed. It is preferable to change the culture medium periodically during the culture period. For example, the medium is changed every two days.

[0019] Any known medium can be used as appropriate. For example, when culturing proximal tubular epithelial cells, commercially available renal tubular cell culture media can be used, and preferred examples of such media include REGM (registered trademark) (LONZA), EpiCM (registered trademark) (ScienCell), and KeratinocyteSFM (registered trademark) (ThermoFisher Scientific).

[0020] 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 culture medium containing collagen I promotes the formation of aggregates. Collagen I is preferably full-length collagen I, but may also be the α1 chain or α2 chain that constitutes collagen I, or collagen peptides obtained by fragmenting each chain. Furthermore, the source of collagen I is not particularly limited, and it may be derived from humans or other animals.

[0021] Any culture vessel can be used, but to promote the formation of cell aggregates, it is preferable that the vessel be treated to be non- (low-) cell-adhesive or be made of a non- (low-) cell-adhesive material. Examples of non- (low-) cell-adhesive treatments include coating the vessel surface with a non-cell-adhesive hydrogel, coating with MPC (2-methacryloyloxyethyl phosphorylcholine), coating with ProteoSave® SS, and mirror polishing. Examples of non- (low-) cell-adhesive materials include glass and polymeric 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.

[0022] When forming a large number of aggregates, a high-density spheroid-forming plate or dish can be used. Furthermore, if necessary, a culture vessel such as a spinner flask may be used. For example, it is preferable to use the ELPLASIA® series of culture vessels (Corning) or the EZSPHERE® series of culture vessels (AGC Technoglass). These culture vessels are available in various types, such as 6-well plates, 24-well plates, 96-well plates, and 384-well plates, as well as dishes of various sizes. The number of spheroids or aggregates that can be produced varies depending on the size of the vessel's bottom area. For example, when using a 96-well plate (V-bottom), 96-well plate (U-bottom), or 384-well plate (U-bottom) that has been subjected to low-adhesion treatment, one cell aggregate is formed per well.

[0023] Aggregates prepared using a high-density spheroid preparation plate or dish can be recovered and cultured in suspension with shaking. When performing suspension with shaking, it is preferable to culture the aggregates in a dish or plate that has been treated to prevent (or reduce) cell adhesion, placed on a shaker. Reciprocating shakers and rotary shakers can be used as the shaker.

[0024] As used herein, the term "aggregate" refers to a mass-like collection of several or more cells. The number of cells constituting the aggregate is, for example, 5 or more, 25 or more, 50 or more, preferably 100 or more, and more preferably 125 or more. The number of cells constituting the aggregate is, for example, 10,000 or less, preferably 5,000 or less, more preferably 2,000 or less, or 1,000 or less. If the number of cells constituting the aggregate is too small, the size of the aggregate may become small and there may be a large variation in the size of the aggregate during production. In addition, there is a tendency for the aggregates to bond together, making it difficult to achieve a uniform size. On the other hand, if the number of cells constituting the aggregate is too large, there is a risk that the expression level of genes characteristic of kidney cells may be low, and it may be difficult to maintain the gene expression level after the aggregate is cryopreserved.

[0025] The size of the aggregates can be controlled by adjusting the number of cells seeded in the culture vessel. For example, when culturing in a multi-well plate, one aggregate is formed per well. Therefore, when the number of kidney cells seeded per well is 500 to 5,000, the number of kidney cells constituting the aggregate is 500 to 5,000. When the number of cells constituting the aggregate is 500 to 5,000, the size of the aggregate is 100 μm to 350 μm.

[0026] When culturing using a culture vessel such as a dish or spinner flask, the diameter of the aggregates or the number of kidney cells constituting one aggregate can be controlled by adjusting the cell density in the vessel. For example, when forming aggregates with a diameter of about 100 μm to about 350 μm or with a constituent cell count of 500 to 5000, the cell density in the vessel is set to 1500 cells / cm. 2 More than 15000 pieces / cm 2 It can be adjusted as follows:

[0027] The size of the aggregate is defined as the maximum width of the aggregate. That is, the size of the aggregate is the length of the longest line connecting two points on the outer edge of the aggregate. Because the aggregate is approximately spherical, the size of the aggregate may hereinafter be referred to as the diameter of the aggregate for convenience.

[0028] The kidney cells cultured in this manner can be recovered in a recovery step. "Recovering as aggregates" means recovering the cells without damaging the morphology of the aggregates, without intentionally disrupting or dispersing the formed aggregates (e.g., trypsin treatment). Therefore, the recovered kidney cells may contain cells in aggregate form, and may also contain dispersed cells, such as cells that did not form aggregates during culture or cells that accidentally detached from aggregates.

[0029] To recover aggregates, it is preferable to use a wide-mouth tip to avoid damaging the aggregates. Wide-mouth tips have a wider tip diameter than standard tips, and by using a tip with an inner diameter larger than the diameter of the aggregates, for example, approximately 1.5 mm (1500 μm), kidney cells can be recovered without damaging the aggregate morphology. An example of the recovery process involves first aspirating the aggregates together with the medium using a wide-mouth tip and transferring them to a centrifuge tube. This centrifuge tube is centrifuged (160 × g, 3 minutes) to collect the aggregates at the bottom. Alternatively, the medium containing the aggregates can be transferred to a 1.5 mL tube or similar, and the aggregates can be collected by natural sedimentation. When freezing the recovered aggregates, it is preferable to aspirate the supernatant and add a cryopreservation medium containing a cryoprotectant, as described below.

[0030] The collected kidney cells can be frozen by cooling them at a low temperature at which the cells can freeze until they are frozen. Kidney cells are preferably frozen by slow freezing. Slow freezing refers to a method of freezing cells by adjusting the temperature drop rate within a predetermined range so that the cells freeze gradually. The temperature drop rate can be adjusted by using commercially available cryopreservation containers, programmable freezers that can set the freezing conditions for cells and tissues, etc. Examples of cryopreservation containers used for slow freezing include BICELL (registered trademark) (Nippon Freezer Co., Ltd.) and CoolCell (registered trademark) (Corning), as well as cryopreservation containers that perform slow freezing using isopropyl alcohol. The temperature drop rate of cells during slow freezing can be, for example, in the range of about 0.2°C to about 3°C ​​per minute, with a rate of about 1°C per minute being preferred.

[0031] Kidney cells can be cooled using a general freezer. For example, when freezing aggregates of proximal tubular epithelial cells, it is preferable to use an ultra-low temperature freezer that can be set to -80°C.

[0032] "Substantially maintaining the aggregated state of the aggregates" in the freezing step means that the morphology of the aggregates is not damaged by not intentionally destroying or dispersing the aggregates contained in the collected kidney cells (e.g., trypsin treatment). Furthermore, "substantially" means that the reduction in the amount of aggregates after freezing is within an insignificant range compared to the amount of aggregates contained in the collected kidney cells before freezing, and does not necessarily mean that the cells that make up the aggregates are not dispersed at all during freezing.

[0033] The method of this embodiment may include any step between the collection step and the freezing step that is beneficial to the cryopreservation of the cells, for example, adding a suitable cryopreservation medium to the collected kidney cells, as long as it does not significantly adversely affect the maintenance of the aggregate morphology.

[0034] Cryopreservation media include cryoprotectants that reduce damage caused by intracellular ice crystals, such as dimethyl sulfoxide (5-15%), mammalian serum, dextran, glycogen, methylcellulose or carboxymethylcellulose, polyethylene glycol, polyvinylpyrrolidone, glucose, and sucrose. The medium can be a liquid, such as a culture medium. A solution containing an appropriate blend of two or more of these cryoprotectants is preferred. Therefore, commercially available cryopreservation solutions, such as CELLBANKER® 1plus (Zenoac Resources), CELLBANKER® 1 (Zenoac Resources), and CELLBANKER® 2 (Zenoac Resources), may also be used.

[0035] Frozen kidney cells can be preserved using a standard freezer. For example, when preserving aggregates of proximal tubular epithelial cells, it is preferable to use an ultra-low temperature freezer that can be set to a temperature of -80°C. Furthermore, when cryopreserving cells for a longer period of time, it is preferable to store them in a lower temperature environment, such as a liquid nitrogen storage vessel (-150°C or lower).

[0036] Frozen kidney cells can be thawed by known methods. For example, a cryovial can be thawed in a 37°C incubator. After thawing, kidney cells can be cultured in the same manner as described above for culturing for aggregate formation. For example, the thawed kidney cells are mixed with medium and centrifuged (160 × g, 3 minutes), and the supernatant is removed. The required amount of medium is added to the collected kidney cells, which are then transferred to a culture vessel such as a culture dish or culture plate and cultured. When culturing a large number of aggregates, the cells are transferred to a low-adhesion dish and cultured in suspension with shaking. The thawed kidney cells are preferably cultured in a non-adherent state to the culture vessel for at least 5 days after thawing, more preferably for at least 7 days. During the culture period, it is preferable to periodically change the medium. In this way, kidney cell cultures with higher renal physiological function or functionally equivalent to human renal cortex can be produced from cryopreserved kidney cells at a desired time point.

[0037] "Functionally equivalent to human renal cortex" means that the expression level is equivalent to that of human renal cortex, at least with respect to the expression of one or more genes related to the physiological functions of the kidney that are expressed in human renal cortex.

[0038] Genes related to renal physiological functions include AQP1, CD13, SGLT2, Na / K ATPase, URAT1, PEPT1, MDR1, OAT1, OCT2, OCTN2, E-cadherin, and ZO-1. AQP1 (aquaporin 1) is a gene encoding a protein involved in water transport. CD13 (alanyl aminopeptidase) is a gene encoding a protein involved in protein peptidation. SGLT2 (sodium glucose cotransporter 2) is a gene encoding a protein involved in sodium and glucose transport. Na / K ATPase is a gene encoding a protein involved in ion transport. URAT1 (urate transporter 1) is a gene encoding a protein involved in uric acid reabsorption. PEPT1 (peptide transporter 1) is a gene encoding a protein involved in peptide transport. MDR1 (multiple drug resistance 1), OAT1 (organic anion transporter 1), OCT2 (organic cation transporter 2), and OCTN2 (organic cation transporter novel 1) are genes encoding proteins involved in drug transport. E-cadherin and ZO-1 (zonula occludens-1) are genes encoding proteins involved in cell-cell junctions.

[0039] The expression levels of one or more of these genes can be measured in human renal cortex and kidney cell cultures using standard real-time PCR (qPCR) techniques, and the equivalent expression levels can be determined by comparing the two. For this purpose, the average values ​​from two or more experiments are used.

[0040] For example, a kidney cell culture is determined to be functionally equivalent to human renal cortex if, after thawing frozen kidney cells and culturing for 7 days, the expression level of any of the above genes is 10% or more of the expression level of that gene in human renal cortex. In a kidney cell culture functionally equivalent to human renal cortex, the expression level of one of the above genes is preferably 10% or more, more preferably 25% or more, of the expression level in human renal cortex. Alternatively, in a kidney cell culture functionally equivalent to human renal cortex, the expression levels of two or more of the above genes are preferably both 10% or more, more preferably 25% or more, of the expression level in human renal cortex. Such expression levels in the kidney cell culture of this embodiment are significantly higher than those in conventional two-dimensionally cultured kidney cell cultures, and in particular, OAT1 and OCT2 can have expression levels comparable to those in human renal cortex. Therefore, in a kidney cell culture functionally equivalent to human renal cortex, the expression levels of one or both of OAT1 and OCT2 are preferably 40% or more, more preferably 55% or more, of the expression level in human renal cortex.

[0041] Similarly, by comparing the expression levels of one or more of these genes in kidney cells before freezing with those in kidney cell cultures after thawing, it can be determined whether the two are functionally equivalent to kidneys.

[0042] For example, a kidney cell culture is determined to be functionally equivalent to a kidney if, after thawing frozen kidney cells and culturing them for 7 days, the expression level of any of the above genes is 40% or more of the expression level of that gene in kidney cells before freezing. A kidney cell culture functionally equivalent to a kidney has the expression level of one of the above genes preferably 40% or more, more preferably 55% or more, of the expression level in kidney cells before freezing. Alternatively, a kidney cell culture functionally equivalent to a kidney has the expression levels of two or more of the above genes preferably both 40% or more, more preferably 55% or more, of the expression level in kidney cells before freezing.

[0043] The production method of this embodiment freezes kidney cells, such as proximal tubule epithelial cells, in an aggregate state, thereby achieving the effect of maintaining cell viability and kidney physiological function after thawing better than the conventional method of freezing cells as a cell suspension. According to this embodiment, kidney cell aggregates in which renal function lost in two-dimensional culture has been restored by three-dimensional culture can be cryopreserved while maintaining the renal function and cell viability, thereby providing kidney cell products and kidney cell cultures that can be used in drug discovery research.

[0044] The present invention is not limited to the above-described embodiments, and various modifications such as design changes may be made based on the knowledge of those skilled in the art, and such modified embodiments are also included in the scope of the present invention. [Example]

[0045] 1. Kidney Cell Culturing, Aggregate Formation, and Freezing Human proximal tubule epithelial cells (Clonetics®, Catalog No. CC-2553, RPTEC-renal proximal tubule epithelial cells) obtained from LONZA were used as kidney cells. The cells were thawed according to the manufacturer's instructions and cultured in the recommended medium (REGM®, LONZA) at 37°C and 5% CO2, with medium changes every two days. The cells were harvested before reaching confluence and cultured in a 96-well V-bottom plate (PrimeSurface® Plate 96V, Sumitomo Bakelite Co., Ltd.) treated with a low cell attachment treatment to form aggregates. The aggregates were cultured with medium changes every two days. The term "confluent" refers to a state in which the cells occupy approximately 100% of the entire culture surface of the culture vessel, i.e., the cells have proliferated over the entire culture surface without any gaps.

[0046] After culturing for 10 days or more, the culture medium containing the aggregates was collected and dispensed into cryovials so that each vial contained approximately 1,000 aggregates. 500 μL / vial of cryopreservation solution (CELLBANKER® 1plus, Zenoac Resources) was added to the aggregates from which the medium had been removed and mixed thoroughly. The aggregates were then placed in a cryopreservation container (BICELL®, Nippon Freezer Co., Ltd.) and slowly frozen at −80°C in an ultra-low temperature freezer.

[0047] The produced frozen cells were cryopreserved for at least one week, then thawed and cultured in suspension with shaking for 7 days, with the medium replaced every two days.

[0048] The cell viability of the cultured cells was measured using the CellTiter-Glo® 3D Cell Viability Assay (Promega), which measures ATP levels by luminescence. Specifically, the aggregates were collected together with the medium, and an amount of CellTiter-Glo 3D Reagent equal to the volume of the medium was added. This mixture was incubated at room temperature for 30 minutes. After thorough mixing, luminescence was measured using a microplate reader (Perkin Elmer).

[0049] The morphology of the thawed aggregates observed under an optical microscope is shown in Figure 1-1. "Thawing Day 0" represents the day of thawing, "Thawing Day 1" represents the day after thawing, and "Thawing Day 5" represents the fifth day after thawing (the number of days since thawing will be expressed in the same way in the following experiments). The results of ATP measurement are shown in Figure 1-2. "Before freezing" represents the time point before freezing after culturing for more than 10 days since aggregate formation, and "after thawing" represents the time point after thawing and shaking culture of the aggregates in suspension for 7 days after frozen storage for more than one week.

[0050] It was confirmed that proximal tubule epithelial cells frozen in aggregate form maintained their aggregate morphology even after thawing. ATP measurement revealed that the majority of cells survived after thawing, confirming that good cell viability was maintained.

[0051] 2. Analysis of gene expression levels Gene expression in proximal tubular epithelial cell aggregates before and after freezing was examined and compared with gene expression in human renal cortex. As in 1. above, kidney cell aggregates were formed and frozen in the aggregate state. The cells were then thawed and cultured in suspension with shaking, with the medium replaced every two days.

[0052] mRNA was extracted and purified from the aggregates before freezing and after thawing and culturing for a predetermined period 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, real-time PCR was performed using the Thermal Cycler Dice® Real Time System 1 (Takara Bio Inc.) to measure the gene expression levels of OAT1 (organic anion transporter 1), OCT2 (organic cation transporter 2), and URAT1 (uric acid transporter 1), which are highly expressed in the proximal tubule. Furthermore, the gene expression level of OAT1 was measured for aggregates thawed and cultured in suspension with shaking for 2, 4, or 7 days. Note that for all experiments, each sample was measured in triplicate.

[0053] For comparison, human renal cortex collected from a human patient donor was used to extract RNA in the same manner as above, and the gene expression levels of OAT1, OCT2, or URAT1 were measured.

[0054] The results are shown in Figures 2-1, 2-2, 2-3, and 2-4. The results for gene expression levels of OAT1 are shown in Figure 2-1, OCT2 in Figure 2-2, and URAT1 in Figure 2-3. In Figures 2-1, 2-2, and 2-3, "before freezing" represents the time point before freezing after culturing for 10 days or more since aggregate formation, and "after thawing" represents the time point after thawing the aggregates after frozen storage for 1 week or more and then culturing them in suspension with shaking for 7 days. The results for gene expression levels of aggregates thawed and cultured in suspension with shaking for 2, 4, or 7 days are shown in Figure 2-4.

[0055] In addition, a comparison of expression between before freezing and human renal cortex (Table 1-1), a comparison of expression between after thawing and human renal cortex (Table 1-2), and a comparison of expression before freezing and after thawing (Table 2) are shown (measured values ​​and their averages from two experiments). In Tables 1-1, 1-2, and 2, "before freezing" refers to the point before freezing after culturing for 10 days or more from the formation of aggregates, and "after thawing" refers to the point after thawing and culturing in suspension with shaking for 7 days after the aggregates have been frozen and stored for 1 week or more.

[0056] [Table 1-1]

[0057] [Table 1-2]

[0058] [Table 2]

[0059] When kidney cells were frozen in aggregate form, the freezing and thawing process did not affect the gene expression levels of OAT1, OCT2, and URAT1 in the kidney cells. After 7 days of thawed suspension culture, the kidney cells showed gene expression levels comparable to those of human renal cortex. Therefore, it was confirmed that the physiological function of proximal tubule epithelial cells frozen in aggregate form was well maintained, and that cell cultures equivalent to those of human renal cortex could be obtained by thawing and culturing the cells.

[0060] 3. Effect of Cell Number on Aggregates Kidney cell aggregates were formed and frozen in the same manner as in 1. However, the number of cells per aggregate was adjusted to 125, 250, 500, or 1000.

[0061] From the aggregates cultured in suspension with shaking for 2, 5, or 7 days before freezing and after thawing, mRNA was extracted and purified, cDNA was synthesized, and the expression level of the OAT1 gene was measured in the same manner as described above in 2.

[0062] The results of gene expression are shown in Figure 3. After 7 days of culture following thawing, aggregates containing 125 to 1,000 cells showed OAT1 expression levels comparable to those before freezing and in human renal cortex, regardless of cell number. On Day 2 after thawing, aggregates with fewer cells tended to maintain OAT1 expression better. However, even aggregates with higher cell numbers were observed to recover expression levels by extending the culture period. Therefore, it was confirmed that good cell cultures could be obtained after thawing for aggregates with a wide range of cell numbers.

[0063] 4. Effect of freezing method Kidney cell aggregates were formed and frozen as aggregates in the same manner as in 1 above. However, instead of slow freezing in a cryopreservation container (BICELL®, Nippon Freezer Co., Ltd.), the media was removed from the aggregates in the 96-well plate, and 20 μL / well of cryopreservation solution (CELLBANKER® 1plus, Zenoac Resources Co., Ltd.) was added to the aggregates, which were then thoroughly mixed and frozen in an ultra-low temperature freezer.

[0064] The cells were thawed and cultured for 7 days with medium changes every two days. The ATP content of the cells was measured and cell viability was determined as described in 1 above. Furthermore, mRNA was extracted and purified from the thawed aggregates, cDNA was synthesized, and the OAT1 gene expression level was measured as described in 2 above.

[0065] The results of ATP measurement are shown in Figure 4-1. The results of gene expression are shown in Figure 4-2. In Figures 4-1 and 4-2, "before freezing" indicates the time point before freezing after culturing for 10 days or more since the formation of aggregates, and "after thawing" indicates the time point after thawing the aggregates after being frozen for 1 week or more and then cultured in suspension with shaking for 7 days.

[0066] Proximal tubule epithelial cell aggregates without slow freezing showed lower cell viability than those with slow freezing. Furthermore, without slow freezing, frozen aggregates showed reduced OAT1 expression compared to those with slow freezing. Therefore, when freezing proximal tubule epithelial cell aggregates in the aggregate state, slow freezing was confirmed to be advantageous in terms of maintaining cell viability and renal function.

[0067] 5. Effects of cryopreservation solution Kidney cell aggregates were formed and frozen as such in the same manner as in 1 above. However, 500 μL / vial of either CELLBANKER® 1plus (Zenoac Resources) or REGM containing 10% dimethyl sulfoxide (Sigma) was added to the aggregates from which the medium had been removed as a cryopreservation medium, mixed thoroughly, and then placed in a cryopreservation container (BICELL®, Nippon Freezer Co., Ltd.) and slowly frozen in an ultra-low temperature freezer at −80°C.

[0068] Each cell type was thawed and cultured in suspension with shaking, with the medium replaced every two days. Seven days after thawing, the ATP content of the cells in the aggregates was measured, and the cell viability was determined in the same manner as in 1. above.

[0069] The results of ATP measurement are shown in FIG.

[0070] Compared with aggregates frozen in REGM containing 10% dimethyl sulfoxide, aggregates frozen in CELLBANKER® 1plus showed higher cell viability after thawing, confirming the advantage of adding a cryopreservation medium containing two or more cryoprotectant components.

[0071] 6. Effects of the frozen period As in 1. above, kidney cell aggregates were formed and frozen in the aggregate state. The cells were thawed after being frozen in a cryogenic freezer at -80°C for one week and after being frozen for more than five months, and then cultured in suspension with shaking for seven days with medium changes every two days, as in 2. above. From the aggregates seven days after thawing, mRNA was extracted and purified, cDNA was synthesized, and the expression level of the OAT1 gene was measured, as in 2. above.

[0072] The results of gene expression levels are shown in FIG.

[0073] Similar levels of OAT1 expression were observed after both short-term (1 week) and long-term (over 5 months) freezing. Therefore, kidney cells can be stably cryopreserved at -80°C for at least several months while maintaining kidney physiological function.

[0074] 7. Cell viability of kidney cells frozen in a dispersed state (Comparative example) The same kidney cells as in 1. above were dispersed with a digestive enzyme, and the cell suspension was frozen (a conventional method for freezing cells), and the cell morphology and viability were examined. Proximal tubule epithelial cells were thawed according to the manufacturer's instructions and cultured in REGM at 37°C and 5% CO2, with medium changes every two days. Before the cells reached confluence, they were dispersed and collected using Accutase® (Innovative Cell Technologies). The cell number was measured, and the required amount was centrifuged to remove the medium. When the cell concentration was 5 × 10 5CELLBANKER (registered trademark) 1plus (Zenoac Resources) was added to the cells to give a cell suspension of ≥ 100 cells / mL. This cell suspension was dispensed into cryovials, placed in a cryopreservation container (BICELL (registered trademark), Nippon Freezer Co., Ltd.), and slowly frozen in an ultra-low temperature freezer at -80°C. After cryopreservation for a certain period, the cells were thawed, diluted with REGM to give aggregates of 1,000 cells, and seeded onto a low-adhesion 96-well V-bottom plate (PrimeSurface (registered trademark) plate 96V, Sumitomo Bakelite Co., Ltd.). After seeding, the cells were cultured with medium changes every two days.

[0075] The cells before freezing and after thawing were seeded at the same cell number and cultured. After seeding, the medium was changed every two days, and ATP content (cell viability) was measured from the aggregates 7 days after thawing using the CellTiter-Glo® 3D Cell Viability Assay (Promega).

[0076] The morphology of the thawed cells observed under an optical microscope is shown in Figure 7-1. The results of ATP measurement are shown in Figure 7-2.

[0077] As shown in Figure 7-1, kidney cells formed aggregates of 1,000 cells before freezing. When these cells were dispersed and frozen as a conventional cell suspension, they did not form aggregates even after thawing and seeding on low-adhesion plates. Furthermore, when seeded with the same number of cells, cells frozen in cell suspension showed lower cell viability than before freezing. Therefore, it was confirmed that conventional cell freezing methods reduced the viability of aggregates and impaired functions such as cell-to-cell adhesion, making it impossible to form aggregates.

[0078] 8. Gene Expression Analysis of Kidney Cells Frozen in a Dispersed State (Comparative Example) The gene expression levels were examined when the same kidney cells as in 1 above were dispersed in a digestive enzyme and frozen (a conventional method for freezing cells). As in 7. above, a cell suspension of proximal tubular epithelial cells was frozen and thawed, and the cells were seeded to form aggregates of 1,000 cells. After seeding, the cells were cultured with medium changes every two days. mRNA was extracted and purified over time from the cells before freezing and after thawing using an RNeasy® Mini Kit (QIAGEN), and cDNA was synthesized using a QuantiTect® Whole Transcriptome Kit (QIAGEN). Using these cDNAs as templates, the expression level of the OAT1 gene was measured by real-time PCR using a Thermal Cycler Dice® Real Time System 1 (Takara Bio).

[0079] The results of gene expression levels are shown in FIG.

[0080] Although kidney cells showed good OAT1 expression before freezing, when frozen as a conventional cell suspension, OAT1 expression was lower than that before freezing and after 7 days of culture after thawing compared with that of human renal cortex. Therefore, freezing aggregates of proximal tubular epithelial cells in the aggregate state was confirmed to be more advantageous in terms of maintaining renal physiological function than conventional cell freezing methods.

[0081] (Embodiments of the invention) A first embodiment of the present invention is a method for producing frozen kidney cells, comprising a recovery step of recovering cultured kidney cells and a freezing step of freezing the kidney cells recovered in the recovery step, wherein the kidney cells are recovered as aggregates in the recovery step, and the aggregated state of the aggregates is substantially maintained during freezing in the freezing step. This has the effect of producing frozen kidney cells that exhibit good aggregate morphology and cell viability after thawing.

[0082] A second embodiment of the present invention is the first embodiment, further comprising a step of adding a cryopreservation medium to the collected kidney cells between the collection step and the freezing step, thereby enhancing the effect of the frozen kidney cells exhibiting good cell viability after thawing.

[0083] A third embodiment of the present invention is the first or second embodiment, further characterized in that the number of kidney cells constituting the aggregate is not less than 100 and not more than 5000. This has the effect of favorably maintaining the physiological functions of the kidney in a kidney cell culture obtained from frozen kidney cells.

[0084] A fourth embodiment of the present invention is any one of the first to third embodiments, further comprising freezing the kidney cells by slow freezing in the freezing step, thereby achieving the effect of allowing the frozen kidney cells to exhibit a good cell viability after thawing and maintaining good physiological functions of the kidney.

[0085] A fifth embodiment of the present invention is any one of the first to fourth embodiments, further comprising the cultured kidney cells comprising aggregates obtained by culturing kidney cells in a non-adherent state in a culture vessel, thereby achieving the effect of the frozen kidney cells exhibiting a good cell survival rate after thawing and maintaining good physiological functions of the kidney.

[0086] A sixth embodiment of the present invention is any one of the first to fifth embodiments, wherein the kidney cells are human primary cells, which has the effect of making it easier to reflect clinical phenomena in humans and making the cells suitable for use in drug discovery research to develop drugs for humans.

[0087] A seventh embodiment of the present invention is any one of the first to sixth embodiments, wherein the kidney cells are derived from proximal tubules, thereby providing the effect that kidney cells and kidney cell cultures are more likely to reflect the effects of drugs on the kidney, making them suitable for use in evaluating pharmacokinetics and the nephrotoxicity of drugs.

[0088] An eighth embodiment of the present invention is kidney cells in a frozen state, produced by the method according to any one of the first to seventh embodiments, which has the effect of easily obtaining a cell culture that maintains kidney physiological functions well by culturing the cells after thawing.

[0089] A ninth embodiment of the present invention is a method for producing a kidney cell culture, comprising a culturing step of thawing frozen kidney cells produced by the method according to any one of the first to seventh embodiments and culturing them in a non-adherent state in a culture vessel. This has the effect of easily producing, whenever necessary, a kidney cell culture that can be used in drug discovery research and that maintains kidney physiological functions well.

[0090] A tenth embodiment of the present invention is the method of the ninth embodiment, further comprising culturing the kidney cells for 5 days or more in the culturing step, which has the effect of favorably maintaining the physiological functions of the kidney in the kidney cell culture and increasing the utility value of the kidney cell culture in drug discovery research.

[0091] An eleventh embodiment of the present invention is a kidney cell culture produced by the method according to the ninth or tenth embodiment. This provides a drug discovery support device that maintains kidney physiological functions well, and has the effect of enabling easy evaluation of pharmacokinetics and nephrotoxicity. [Industrial Applicability]

[0092] The present invention can be used for producing and preserving kidney cells that can be used in drug discovery research and the like.

Claims

1. A method for producing frozen kidney cells, comprising: a recovery step of recovering cultured kidney cells, which are proximal tubule epithelial cells derived from proximal tubules, excluding cells derived from cancer tissue or cancer cells; and a freezing step of freezing the kidney cells recovered in the recovery step, wherein the recovery step does not involve any operation to destroy or disperse the kidney cell aggregates, and the kidney cells are recovered as aggregates having a number of 125 to 5,000 cells, and the freezing step involves freezing the aggregates while maintaining their aggregated state.

2. 10. The method of claim 1, further comprising the step of adding a cryopreservation medium to the collected kidney cells between the collecting and freezing steps.

3. 3. The method according to claim 1, wherein the size of the kidney cells constituting the aggregate is 100 μm or more and 350 μm or less.

4. The method according to any one of claims 1 to 3, wherein the freezing step is carried out by slow freezing.

5. The method according to any one of claims 1 to 4, wherein the cultured kidney cells comprise aggregates obtained by culturing kidney cells in a non-adherent state in a culture vessel.

6. The method of any one of claims 1 to 5, wherein the kidney cells are human primary cells.

7. Kidney cells in a frozen state that are composed of cultured kidney cells that are proximal tubular epithelial cells, excluding cells derived from cancer tissue or cancer cells, and in which the aggregation state of aggregates is maintained, with the number of kidney cells being 125 to 5,000.

8. A method for producing a kidney cell culture, comprising a culturing step of thawing frozen kidney cells produced by the method according to any one of claims 1 to 6 and culturing them in a non-adherent state in a culture vessel.

9. 9. The method of claim 8, wherein the kidney cells are cultured for 5 days or more in the culturing step.

10. The kidney cell according to claim 7, which is a proximal tubule epithelial cell, in which the gene expression level of one or both of OAT1 and OCT2 is 40% or more of the gene expression level in human renal cortex.

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