Method for storing and transporting kidney cells

Abstract

To provide a method for storing or transporting renal cells, which are usable as a drug evaluation system, have high cell viability and preserve renal physiological function, at a temperature lower than the culture temperature.SOLUTION: One aspect of the present invention is a method for storing or transporting renal cells, the method including a maintenance step for maintaining a liquid containing aggregates of the renal cells at a maintenance temperature which is lower than the culture temperature of the renal cells and at which the liquid does not freeze.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for storing renal cells and a method for transporting renal cells. The present invention further relates to a cell product containing renal cells.

Background Art

[0002] A drug administered to a living body is absorbed in the living body and then excreted from the blood into urine in the proximal renal tubule in the kidney. Therefore, kidney damage is often caused by the nephrotoxicity of drugs. In drug discovery research, it is very important to examine the pharmacokinetics of drugs in the kidney in order to clarify the action of drugs. From this, the development of a drug discovery support device capable of evaluating pharmacokinetics and toxicity using renal cells is desired. In addition, such a drug discovery support device is also useful for the development of therapeutic drugs for kidney-related diseases (for example, kidney cancer, hyperuricemia, etc.).

[0003] When supplying or using a drug discovery support device using such cells, a method for stably storing or transporting the cells is required. Generally, cells are two-dimensionally cultured on a flat culture plate. Conventionally, in order to maintain a normal cell state, a temperature control device for maintaining the temperature of the container containing the cells at the culture temperature is required, and there is a problem that the cost of storing or transporting the cells becomes high.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors have found that when planar cultured kidney cells are maintained at a temperature lower than the culture temperature for a certain period of time, the cell morphology changes and the cell viability decreases. Therefore, based on this finding, the present invention aims to provide a method for storing or transporting kidney cells at a temperature lower than the culture temperature, while maintaining high cell viability and preserving the physiological function of the cells. [Means for solving the problem]

[0006] As a result of various studies conducted to solve the above problems, we found that in kidney cells, it is possible to maintain a temperature lower than the culture temperature for a certain period of time by inducing the formation of aggregates.

[0007] One embodiment of the present invention is a method for storing or transporting kidney cells, comprising a maintenance step of controlling a liquid containing aggregates of kidney cells to a temperature below the culture temperature of the cells and in which the liquid does not freeze.

[0008] One embodiment of the present invention is a method in which a liquid containing aggregates of kidney cells is stored or transported by a method that includes a maintenance step of controlling the temperature of the liquid to be below the culture temperature of the cells and not freezing. [Effects of the Invention]

[0009] The present invention provides a method for storing or transporting kidney cells, which have a high cell viability and maintain the physiological function of the kidney, at a temperature lower than the culture temperature. [Brief explanation of the drawing]

[0010] [Figure 1] This is a block diagram illustrating the schematic configuration of a renal cell storage and transport system. [Figure 2] This is a flowchart illustrating an example of the operation of a renal cell storage and transport system. [Figure 3-1] Figures 3-1(a) to (c) are optical microscope images of proximal tubular epithelial cell aggregates under conditions of 37°C, room temperature, and refrigeration, respectively. [Figure 3-2]Figure 3-2 shows the amount of ATP (cell viability) in proximal tubular epithelial cell aggregates after 24 hours of standing. [Figure 3-3] Figures 3-3(a) to (d) show the amount of ATP (cell viability) of proximal tubular epithelial cell aggregates at 48, 72, 144, and 240 hours after standing, respectively. [Figure 4-1] Figure 4-1 shows the results of real-time PCR analysis of OAT1 gene expression in proximal tubular epithelial cell aggregates after 72 hours of standing at room temperature, compared to human renal cortex. [Figure 4-2] Figure 4-2 shows the results of real-time PCR analysis of OCT2 gene expression in proximal tubular epithelial cell aggregates after 72 hours of standing at room temperature, compared to human renal cortex. [Figure 4-3] Figure 4-3 shows the results of real-time PCR analysis of URAT1 gene expression in proximal tubular epithelial cell aggregates after 72 hours of standing at room temperature, compared with human renal cortex. [Figure 4-4] Figure 4-4 shows the results of real-time PCR analysis of OAT1 gene expression in proximal tubular epithelial cell aggregates after 24 hours of refrigeration, compared to human renal cortex. [Figure 4-5] Figure 4-5 shows the results of real-time PCR analysis of OCT2 gene expression in proximal tubular epithelial cell aggregates after 24 hours of refrigeration, compared to human renal cortex. [Figure 4-6] Figure 4-6 shows the results of real-time PCR analysis of URAT1 gene expression in proximal tubular epithelial cell aggregates after 24 hours of refrigeration, compared to human renal cortex. [Figure 5-1] Figures 5-1(a) to (e) are light microscope images of proximal tubular epithelial cell aggregates with cell counts of 125, 500, 2000, 10000, and 40000, respectively. [Figure 5-2] Figures 5-2(a) to (e) show the amount of ATP (cell viability) in proximal tubular epithelial cell aggregates for cell counts of 125, 500, 2000, 10000, and 40000, respectively. [Figure 6-1]Figs. 6-1(a) to (c) are respectively optical microscope images of proximaltubular epithelial cells cultured in a planar manner under conditions of 37°C, normal temperature, and refrigeration. [Figure 6-2] Figs. 6-2(a) to (c) are respectively the amounts of ATP (cell viability) of proximaltubular epithelial cells cultured in a planar manner at elapsed times of 24 hours, 4 hours, and 72 hours. [Figure 7-1] Figs. 7-1(a) to (c) are respectively optical microscope images of human iPS cell aggregates under conditions of 37°C, normal temperature, and refrigeration. [Figure 7-2] Figs. 7-2(a) to (c) are respectively the amounts of ATP (cell viability) of human iPS cell aggregates at elapsed times of 24 hours, 48 hours, and 72 hours.

Mode for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in detail. A method for storing or transporting renal cells according to an embodiment of the present invention includes a maintenance step of controlling a liquid containing aggregates of the renal cells to a temperature that is lower than the culture temperature of the cells and at which the liquid does not freeze.

[0012] The kidney cells used in this invention can be cultured, and their source is not restricted. The kidney 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 kidney cells include cells that constitute the epithelium, cortex, proximal tubule, distal tubule, collecting duct, glomerulus, etc. Specifically, proximal tubular epithelial cells (RPTECs) and mesangial cells. The kidney cells may be primary cells or kidney cells derived from stem cells such as iPS cells or ES cells. Furthermore, the kidney cells may be immortalized kidney cells, established cell lines (such as HK-2 cells), cells derived from other animal species (such as MDCK cells, LLC-PK1 cells, and JTC-12 cells), or forced-expression cells into which genes have been introduced to express specific transporters or other 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.

[0013] Kidney cells can be cultured 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. Culture may be static culture, shaking culture, or agitated culture. Culture may be adherent culture, but it is preferable to culture in a non-adherent state for at least a portion of the period (e.g., suspension culture). Kidney cells can form aggregates by being cultured in a non-adherent state in the culture vessel. "Non-adherent 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 even if they are in contact with the surface of the culture vessel, a state in which they can be easily separated from the surface of the culture vessel by coating of the culture vessel or convection of the culture medium without the use of instruments or enzymes.

[0014] 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 non-adherent 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 function. During the culture period, it is preferable to change the culture medium regularly. For example, the medium is changed every two days.

[0015] 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).

[0016] 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.

[0017] Any culture vessel can be used, but to promote aggregate formation, it is preferable that the vessel is treated to be non-(or low-)adherent or composed of a non-(or low-)adherent material. Examples of non-(or low-)adherent treatments include non-(or low-)adherent hydrogel coating on the vessel surface, MPC (2-methacryloyloxyethyl phosphorylcholine) coating, Proteosave® SS coating, and mirror polishing. Examples of non-(or 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.

[0018] 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.

[0019] 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.

[0020] In this specification, the term "aggregate" of cells refers to a clump-like collection of several or more cells. It is also called an aggregate or spheroid. The number of cells constituting the aggregate is, for example, 5 or more, 25 or more, 50 or more, preferably 100 or more, more preferably 125 or more, and even more preferably 500 or more. The number of cells constituting the aggregate is, for example, 4000 or less, preferably 10000 or less, more preferably 2000 or less, and 1000 or less. When the number of cells constituting the aggregate is within this range, there is less variation during aggregate formation, and the aggregates are less likely to join with each other, so the aggregate size tends to be uniform. Furthermore, the cells tend to maintain a high viability rate.

[0021] 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 in each well. Therefore, when the number of kidney cells seeded per well is between 125 and 10,000, the number of kidney cells constituting the aggregate will be between 125 and 10,000.

[0022] The diameter of the aggregate is preferably, for example, 100 μm to 800 μm. The volume of the aggregate is, for example, 0.001 mm³. 3 Above, 0.300mm 3 The following are preferred.

[0023] The diameter of an aggregate is defined as its maximum width. That is, the diameter of the aggregate is the length of the longest straight line connecting two points on the outer edge of the aggregate. Furthermore, since aggregates are approximately spherical, their volume can be calculated from the measured diameter. The diameter of an aggregate can be measured, for example, using photographs taken with a phase-contrast microscope. A BZ-X710 (Keyence) can be used as the phase-contrast microscope, and analysis software can be used to measure the diameter.

[0024] The liquid used for storing or transporting kidney cells is not particularly limited as long as it does not adversely affect the cells, and may be the culture medium used to form aggregates, fresh culture medium, or a liquid other than culture medium (e.g., physiological saline or buffer solution). This liquid may contain salts, buffers, serum, vitamins, amino acids, glucose (sugar), electrolytes, antibiotics, and growth factors (compounds, proteins). If storage or transport lasts for more than 48 hours, it is preferable to change the liquid periodically. For example, the liquid may be changed every two days.

[0025] Any container can be used for storing and transporting liquids containing aggregates of kidney cells, similar to the culture vessels described above, but it is preferable that the container is treated to be non-(or low-)adherent or composed of a non-(or low-)adherent material.

[0026] A method for storing or transporting kidney cells according to one embodiment of the present invention includes a maintenance step in which, when storing or transporting kidney cells, the liquid containing aggregates of kidney cells is controlled to a temperature below the culture temperature of the kidney cells and at which the liquid does not freeze (hereinafter, for convenience, may be referred to as the "maintenance temperature").

[0027] The temperature below the culture temperature of the kidney cells and at which the liquid does not freeze can be appropriately selected as the optimal temperature depending on the type of cell, its intended use, and / or the period for which storage or transport is required. The culture temperature is the temperature suitable for cell survival, and the cells may or may not proliferate. The culture temperature of cells is typically 37°C, so it is desirable that the maintenance temperature be lower than this. On the other hand, if the maintenance temperature is too low, partial freezing may occur within the cells, which may reduce the viability of the cells in the aggregate. Therefore, it is desirable that the maintenance temperature be 0°C or higher. For example, the maintenance temperature can be 0°C or higher and less than 37°C. If maintenance at a relatively high temperature is desirable, it can preferably be 10°C or higher and less than 37°C, and more preferably 20°C or higher and less than 28°C. If maintenance at a relatively low temperature is desirable, it can preferably be 0°C or higher and less than 10°C, and more preferably 3°C or higher and less than 8°C. In this specification, for convenience, the temperature range of 10°C or higher and less than 37°C may be referred to as "room temperature," and the temperature range of 0°C or higher and less than 10°C may be referred to as "refrigerated."

[0028] For storage, incubators, constant temperature rooms, refrigerators, etc., that can be adjusted to the desired temperature can be used during the maintenance process.

[0029] For transportation, it is preferable to use an insulated container such as polystyrene foam for the maintenance process. The size of the insulated container is not particularly limited as long as it can accommodate the storage and transport containers. If there is a gap between the insulated container and the storage and transport containers, it is preferable to include cushioning material to prevent the storage and transport containers from moving. Paper containers or corrugated cardboard containers can also be used as insulated containers.

[0030] Furthermore, the maintenance process does not necessarily have to be carried out by leaving the material still. Therefore, it can be performed under conditions where there is vibration that does not cause the aggregates to break down, such as on a shaker or during transport.

[0031] When storing or transporting kidney cells, the number of aggregates in the liquid can be limited to one to prevent the aggregates from joining together and becoming uneven in size. In this case, it is preferable that the lower limit of the volume ratio of aggregates to liquid be 0.001% or more, 0.002% or more, or 0.003% or more. Furthermore, it is preferable that the upper limit of the volume ratio of aggregates to liquid be 0.800% or less, 0.500% or less, or 0.200% or less. By keeping the volume ratio of aggregates to liquid within these ranges, it is possible to prevent the liquid from drying out and exposing the aggregates to the air during storage or transport, and to ensure sufficient oxygen supply to the aggregates.

[0032] When storing or transporting kidney cells, the lower limit of the specific gravity of the aggregates relative to the liquid is preferably 1.00 or higher. This specific gravity allows the aggregates to remain in the liquid without floating on the surface, thus making them less susceptible to changes in the extracellular environment. Therefore, the upper limit of the specific gravity of the aggregates is preferably 1.20 or lower, 1.15 or lower, or 1.10 or lower.

[0033] The maintenance period in the maintenance process is the period during which the cell viability rate in the kidney cell aggregates exceeds a predetermined value. Cell viability is defined as the ratio of the number of viable cells at the maintenance temperature to the number of viable cells at the culture temperature. The cell viability rate is preferably 80% or higher, and more preferably 90% or higher. Kidney cells with such a cell viability rate can be used for drug evaluation.

[0034] The inventors have found that there is the following relationship between the maintenance period and the maintenance temperature in the maintenance process. Specifically, the maintenance period during which the cell viability can be maintained above a predetermined value can be calculated using the following formula. (Sustainable period (days))=A / {(Culture temperature (℃))-(Maintenance temperature (℃))} A: A coefficient determined according to the predetermined value and the number of cells in the aggregate (days / °C). From the above formula, it is possible to calculate the upper limit of the maintenance period according to the desired maintenance temperature and cell viability.

[0035] Similarly, the maintainable temperature at which cell viability can be maintained above a predetermined value can be calculated using the following formula. (Maintenance period (days)) = A / {(Culture temperature (℃)) - (Sustainable temperature (℃))} A: A coefficient determined according to the predetermined value and the number of cells in the aggregate (days / °C). From the above formula, it is possible to calculate the lower limit of the maintenance temperature according to the desired maintenance period and cell viability.

[0036] Here, for example, when the predetermined value is 90% and the number of cells in the aggregate is 1000, the value of A can be set to 34.5, and when the predetermined value is 80% and the number of cells in the aggregate is 1000, the value of A can be set to 69.

[0037] From the above, for example, when the number of cells in the aggregate is 1000, and the culture temperature is 37°C and the maintenance temperature is 25°C, the maintenance period is 5.75 days when the predetermined value is 80% and 2.88 days when the predetermined value is 90%, respectively. That is, when the culture temperature is 37°C and the maintenance temperature is 25°C, the maintenance period is preferably within 5.75 days, and more preferably within 2.88 days.

[0038] One embodiment of the present invention involves renal cells that have higher physiological function than the kidney, or are functionally equivalent to or comparable to human renal cortex.

[0039] "Functionally equivalent to the human renal cortex" means that, with respect to the expression of at least one gene related to the physiological function of the kidney that is expressed in the human renal cortex, the expression level is equivalent to that of the human renal cortex.

[0040] Genes related to the physiological function of the kidney include AQP1, CD13, SGLT2, Na / K ATPase, URAT1, PEPT1, MDR1, OAT1, OCT2, OCTN2, E-cadherin, and ZO-1. AQP1 (aquaporin 1) is a gene that codes for a protein involved in water transport. CD13 (alanyl aminopeptidase) is a gene that codes for a protein involved in the peptide formation of proteins. SGLT2 (sodium glucose cotransporter 2) is a gene that codes for a protein involved in the transport of sodium and glucose. Na / K ATPase is a gene that codes for a protein involved in ion transport. URAT1 (urate transporter 1) is a gene that codes for a protein involved in uric acid reabsorption. PEPT1 (peptide transporter 1) is a gene that codes for 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 that encode proteins involved in drug transport. E-cadherin and ZO-1 (zonula occludens-1) are genes that encode proteins involved in intercellular junctions.

[0041] The expression levels of one or more of these genes can be measured in human renal cortex and kidney cell cultures using a common real-time PCR (qPCR) method, and whether the two are equivalent can be determined by comparing them. When using this method for determination, the average of measurements from two or more experiments should be used.

[0042] For example, if the expression level of any of the above genes in kidney cells stored or transported for a predetermined period is 10% or more of the expression level of that gene in the human renal cortex, then the cells are determined to be functionally equivalent to the human renal cortex. A kidney cell culture functionally equivalent to the human renal cortex has an expression level of one of the above genes that is preferably 10% or more of the expression level in the human renal cortex, and more preferably 25% or more. Alternatively, kidney cells functionally equivalent to the human renal cortex have an expression level of two or more of the above genes that are preferably all 10% or more of the expression level in the human renal cortex, and more preferably 25% or more.

[0043] One embodiment of the present invention, renal cells, can be provided as a drug evaluation system or a cell product. Examples of drug evaluation systems include those for evaluating pharmacokinetics and nephrotoxicity in renal cells. Furthermore, such renal cells can be used for analyzing the mechanisms of kidney-related diseases such as renal cancer and hyperuricemia, as well as as a tool for drug discovery.

[0044] (Kidney cell storage and transport system) Figure 1 is a block diagram showing a schematic configuration of the renal cell storage system 10 according to an embodiment. As shown in Figure 1, the renal cell storage system 10 includes a renal cell storage device 20 and a temperature control device 30.

[0045] The renal cell storage device 20 is a storage device capable of maintaining the temperature inside at a predetermined temperature, such as an incubator, a constant temperature room, or a refrigerator. Culture vessels 22 are stored inside the renal cell storage device 20. The culture vessels 22 contain liquid 24 and aggregates 26 of renal cells. When the temperature inside the renal cell storage device 20 is constant at the predetermined maintenance temperature, the temperature of the liquid 24 inside the culture vessels 22 can be considered to be at the predetermined maintenance temperature.

[0046] The temperature control device 30 comprises a storage information input unit 40, a temperature control unit 50, a maintenance period calculation unit 60, and a display unit 70. The temperature control device 30 may be built into the kidney cell storage device 20.

[0047] The storage information input unit 40 can be used by the user to input information such as information about the liquid 24 in the culture vessel 22, information about the culture temperature of the kidney cells, information about the maintenance temperature of the liquid 24, information about the storage period (maintenance period) of the kidney cells, information about the cell viability, and information about the number of cells in the aggregates. Furthermore, the storage information input unit 40 may also receive information regarding the components and concentrations (or content) of the liquid 24 in which the aggregates 26 are stored. For example, if the liquid is physiological saline, "sodium chloride" is entered as a component, and the concentration of "sodium chloride" is also entered. Additionally, the storage information input unit 40 may receive information regarding the freezing temperature of the liquid 24.

[0048] The temperature control unit 50 includes a maintenance temperature setting unit 52 and a temperature control unit 54. The maintenance temperature setting unit 52 sets the maintenance temperature inside the kidney cell storage device 20, that is, the maintenance temperature of the liquid 24 in the culture vessel 22, to be lower than the culture temperature of the kidney cells entered into the storage information input unit 40, and to a temperature at which the liquid 24 does not freeze. The temperature at which the liquid 24 does not freeze may be higher than the temperature at which the liquid 24 freezes, as entered into the storage information input unit 40, or it may be calculated considering the freezing point depression from the components and concentration of the liquid 24. Specifically, the maintenance temperature may be set by the user via the storage information input unit 40, or it may be determined according to the desired cell viability, the number of cells in the aggregate, the maintenance period, etc. When the maintenance temperature is set based on factors such as cell viability and the number of cells in aggregates, the maintenance temperature is set to be equal to or higher than the maintainable temperature calculated by the following formula. In other words, the lower limit of the maintenance temperature is determined according to the maintenance period, which is the period for which the kidney cells are stored. (Maintenance period (days)) = A / {(Culture temperature (℃)) - (Sustainable temperature (℃))} A: A coefficient determined according to the predetermined value and the number of cells in the aggregate (days / °C). Here, the maintainable temperature is the temperature at which the cell viability rate, which is the ratio of the number of viable cells at the maintenance temperature to the number of viable cells at the culture temperature of kidney cells, can be maintained at or above a predetermined value.

[0049] The temperature control unit 54 controls the temperature inside the kidney cell storage device 20 so that it reaches the maintenance temperature set by the maintenance temperature setting unit 52. Specifically, the temperature control unit 54 and the temperature control device 30 are connected wirelessly or via wired communication, and the temperature control unit 54 transmits information regarding the maintenance temperature to the temperature control device 30. Upon receiving the information regarding the maintenance temperature, the kidney cell storage device 20 manages the temperature so that the temperature inside the storage device reaches the specified maintenance temperature. Note that temperature control within the renal cell storage device 20 may be started after the culture vessels 22 are placed in the renal cell storage device 20, or it may be started before the culture vessels 22 are placed in the renal cell storage device 20. By performing temperature control within the renal cell storage device 20 in advance and then placing the culture vessels 22 in the renal cell storage device 20, the temperature of the liquid 24 in the culture vessels 22 can be quickly brought to the desired maintenance temperature.

[0050] The maintenance period calculation unit 60 sets the maintenance period for which the renal cell storage device 20 maintains the liquid temperature at the maintenance temperature to a minimum or less than the maintenance period (days) calculated by the following formula, as needed. In other words, the upper limit of the maintenance period is determined according to the maintenance temperature. (Sustainable period (days))=A / {(Culture temperature (℃))-(Maintenance temperature (℃))} A: A coefficient determined according to the above predetermined value and the number of cells in the aggregate (days / °C). Here, the maintenance period (days) is the period during which the cell viability rate, which is the ratio of the number of viable cells at the maintenance temperature to the number of viable cells at the culture temperature of kidney cells, can be maintained at or above a predetermined value.

[0051] The display unit 70 displays information necessary for a display (not shown). Information displayed by the display unit 70 includes the maintenance period calculated by the maintenance period calculation unit 60 (maintenance (storage) start date and time and maintenance (storage) end date and time), and the maintenance temperature set by the maintenance temperature setting unit 52.

[0052] The renal cell storage system 10 can be installed on means of transport such as trucks, freight cars, and ships. In this case, the renal cell storage system 10 can be read as the renal cell transport system 10.

[0053] Figure 2 is a flowchart illustrating an example of the operation of the renal cell storage system 10. The following example shows a case where the storage temperature is set according to the storage period of the renal cells. First, the user inputs information regarding the storage of kidney cells into the storage information input unit 40. Specifically, the maintenance period for the kidney cells, the culture temperature of the kidney cells, and the freezing temperature of the liquid are entered (S10). Next, the maintenance temperature setting unit 52 sets the maintenance temperature of the liquid 24 according to the setting method described above (S20). The maintenance temperature is required to be below the culture temperature of kidney cells and below the freezing temperature of the liquid 24. Next, with the culture vessel 22 stored in the kidney cell storage device 20, the temperature control unit 54 controls the temperature inside the kidney cell storage device 20, i.e., the temperature of the liquid 24, so that it reaches the maintenance temperature (S30). Next, it is determined whether the time elapsed since the temperature inside the kidney cell storage device 20 was set to the maintenance temperature has reached the maintenance period (S40). If the elapsed time has reached the maintenance period (yes in S40), the storage or temperature control of the kidney cells is terminated. If the elapsed time has not reached the maintenance period (no in S40), the process returns to the determination in S40.

[0054] One aspect of the present invention is a renal cell storage or transport system. The renal cell storage or transport system includes a cell storage device that houses culture vessels containing liquid and aggregates of renal cells, and is capable of maintaining the temperature of the liquid at a predetermined temperature. A temperature control device maintains the temperature of the liquid maintained by the cell storage device at a temperature below the culture temperature of kidney cells and at a maintenance temperature that prevents the liquid from freezing. The above embodiment includes the following: In the above embodiment, the lower limit of the maintenance temperature may be determined according to the maintenance period, which is the period for which the kidney cells are stored. Furthermore, another aspect of the present invention, a renal cell storage or transport system, includes a cell storage device capable of housing a culture vessel containing a liquid and aggregates of renal cells, and maintaining the temperature of the liquid at a predetermined temperature. A temperature control device maintains the temperature of the liquid maintained by the cell storage device so that it is below the culture temperature of kidney cells and at a maintenance temperature that prevents the liquid from freezing. The system may be equipped with the above-mentioned maintenance temperature and an upper limit on the maintenance period may be determined accordingly. According to one embodiment of the present invention, it is possible to calculate an upper limit for the maintenance period according to the maintenance temperature of kidney cells, or to calculate a lower limit for the maintenance temperature according to the maintenance period. In other words, one embodiment of the present invention can be used as a storage and transport system for kidney cells to manage the maintenance temperature and maintenance period.

[0055] The present invention is not limited to the embodiments described above, and various modifications such as design changes can be made based on the knowledge of those skilled in the art, and such modified embodiments are also included within the scope of the present invention. [Examples]

[0056] 1. The effect of temperature on proximal tubular epithelial cell aggregates <1-1. Morphology and cell viability of aggregates> Human proximal tubular epithelial cells (RPTEC-kidney proximal tubular epithelial cells) obtained from LONZA were used as kidney cells. Frozen vials stored in a liquid nitrogen storage container 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 cultured in a culture dish. The cells were cultured at 37°C and 5% CO2, with the culture medium changed every two days. The cells were harvested before confluence and seeded into low-adhesion 96-well V-bottom plates (PrimeSurface® plate 96V, Sumitomo Bakelite) at a rate of 1000 cells per well to form aggregates. The aggregates were cultured with the culture medium changed every two days. "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 leaving any gaps on the culture surface.

[0057] After culturing for more than 240 hours, plates containing the culture medium with aggregates were left standing at room temperature or refrigerated conditions, or placed in a 37°C incubator under 5% CO2 conditions. Specifically, room temperature conditions involved placing the plates in a polystyrene container and leaving them standing in a room (air conditioning set to 25°C). Specifically, refrigerated conditions involved placing the plates in a refrigerator (set to 4°C).

[0058] The aggregates were observed using an optical microscope 24 hours after the start of standing at various temperature conditions. The number of viable cells in the aggregates was also measured using the CellTiter-Glo® 3D Cell Viability Assay (Promega), which measures ATP levels by luminescence. Specifically, the aggregates were collected along with the culture medium, and an equal volume of CellTiter-Glo 3D Reagent was added. This mixture was incubated at room temperature for 30 minutes. After thorough mixing, the luminescence value was measured using a microplate reader (Perkin Elmer).

[0059] Figure 3-1 shows the morphology of aggregates under each temperature condition {(a) 37°C; (b) room temperature; (c) refrigerated conditions}. Figure 3-2 shows the results of ATP content measurement after 24 hours of standing.

[0060] Aggregates of proximal tubular epithelial cells were found to maintain their morphology even after being maintained at temperatures lower than the culture temperature (37°C) (room temperature or refrigeration) for 24 hours. ATP levels were measured, and the number of surviving cells at low temperatures was comparable to that at culture temperature. Since the number of surviving cells at low temperatures fell within the range of in-house data for surviving cells at culture temperature, survival was confirmed.

[0061] <1-2. Changes in cell viability over time> The aggregates were cultured in the same manner as described in 1-1 above, and the plates containing the culture medium with the aggregates were left standing at room temperature or refrigerated conditions, or placed in a 37°C incubator under 5% CO2 conditions. After standing, the culture medium was changed every two days.

[0062] After a predetermined time had elapsed since the start of standing, the amount of ATP in the aggregates under each temperature condition was measured in the same manner as described in 1-1 above.

[0063] The results of the number of surviving cells after 48 hours, 72 hours, 144 hours, and 240 hours of standing are shown in Figure 3-3 {(a) 48 hours; (b) 72 hours; (c) 144 hours (6 days); (d) 240 hours (10 days)}.

[0064] Regarding the number of viable cells in aggregates of proximal tubular epithelial cells, it was confirmed that there was almost no difference between the number of viable cells under culture temperature conditions at 48 and 72 hours after standing at room temperature. On the other hand, a decrease in the number of viable cells was observed after 144 hours of standing at room temperature conditions and after 48 hours of standing under refrigerated conditions. The cell viability (ratio of the number of viable cells under each temperature condition to the number of viable cells under culture temperature) at this time is shown in Table 1. In Table 1, cell viability is expressed as follows: "A" = 90% or more, "B" = 80% or more and less than 90%, "C" = 50% or more and less than 80%, and "D" = less than 50%. The numbers in parentheses represent the difference from 100% for each cell viability.

[0065] [Table 1]

[0066] From these results, it was confirmed that kidney cell aggregates can be maintained for more than 24 hours at temperatures lower than the culture temperature. Furthermore, it was confirmed that at temperatures with a small difference from the culture temperature (room temperature), aggregates can be stably maintained for a longer period compared to lower temperatures (refrigeration).

[0067] 2. Analysis of gene expression levels in renal cells <2-1. Gene expression levels during storage at room temperature> Gene expression in proximal tubular epithelial cell aggregates was examined after standing at room temperature and at culture temperature, and compared with gene expression in the human renal cortex. The aggregates were cultured in the same manner as described in 1-1 above, and the plates containing the culture medium with the aggregates were left standing at room temperature or in an incubator at 37°C under 5% CO2 conditions. After standing, the culture medium was changed every two days.

[0068] mRNA was extracted and purified from each aggregate 72 hours after the start of standing 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 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 tubules, 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 each experiment.

[0069] For comparison, RNA was extracted from human renal cortex collected from human patient donors in the same manner as described above, and the gene expression levels of OAT1, OCT2, or URAT1 were measured.

[0070] The gene expression levels for OAT1 are shown in Figure 4-1, for OCT2 in Figure 4-2, and for URAT1 in Figure 4-3. Table 2-1 shows the culture temperature conditions under normal temperature conditions and a comparison of expression levels with human renal cortex.

[0071] [Table 2-1]

[0072] At culture temperature, aggregates of proximal tubular epithelial cells showed comparable gene expression levels for OAT1, OCT2, and URAT1 compared to human renal cortex. Furthermore, under room temperature conditions, there was no change in gene expression levels compared to culture temperature conditions or human renal cortex, confirming that renal physiological function was maintained even after 72 hours of standing at room temperature.

[0073] <2-2. Gene expression levels during refrigerated storage> Gene expression in proximal tubular epithelial cell aggregates was examined after refrigeration and at culture temperature, and compared with gene expression in the human renal cortex. The aggregates were cultured in the same manner as described in 1-1 above, and the plates containing the culture medium with the aggregates were left standing under refrigerated conditions, or placed in an incubator at 37°C under 5% CO2 conditions. After standing, the culture medium was changed every two days.

[0074] The gene expression levels of OAT1, OCT2, or URAT1 were measured in each aggregate 24 hours after the start of standing, and in human renal cortex collected from human patient donors, in the same manner as described in 2-1. above.

[0075] The gene expression levels for OAT1 are shown in Figure 4-4, OCT2 in Figure 4-5, and URAT1 in Figure 4-6. Table 2-2 shows the culture temperature conditions under refrigerated conditions and a comparison of expression levels with human renal cortex.

[0076] [Table 2-2]

[0077] These results confirm that kidney function is maintained even when the samples are left undisturbed in the refrigerator for 24 hours.

[0078] 3. Effect of aggregate cell number <3-1. Number of aggregate cells> After culturing and harvesting kidney cells in the same manner as described in 1-1 above, the cells were seeded into 96-well V-bottom plates treated for low cell adhesion, with cell counts of 125, 500, 2000, 10000, or 40000 per well, respectively, and cultured to form aggregates. The aggregates were cultured with the culture medium changed every two days.

[0079] After incubation for more than 240 hours, plates containing the culture medium with aggregates were left standing at room temperature or refrigerated conditions, or placed in a 37°C incubator under 5% CO2 conditions. The appearance of the aggregates after incubation under each temperature condition was also observed using an optical microscope.

[0080] The amount of ATP in the aggregates was measured at each temperature condition after a predetermined time had elapsed since the start of standing.

[0081] Figure 5-1 shows the morphology of aggregates at various cell counts {(a) 125 cells; (b) 500 cells; (c) 2000 cells; (d) 10000 cells; (e) 40000 cells}. Figure 5-2 shows the results of measuring ATP levels over time in aggregates at various cell counts {(a) 125 cells; (b) 500 cells; (c) 2000 cells; (d) 10000 cells; (e) 40000 cells}.

[0082] The number of surviving cells in aggregates of proximal tubular epithelial cells did not differ significantly between room temperature / refrigerated conditions and culture temperature conditions, regardless of the maintenance period, when the cell count ranged from 125 to 10,000 cells. This confirmed that cells in aggregates with a cell count of 125 to 10,000 cells could survive even after 72 hours of standing. On the other hand, when the cell count was 40,000 cells, a decrease in the number of surviving cells was observed under refrigerated conditions for 48 hours after standing.

[0083] Table 3-1 shows the cell viability in aggregates of each cell number at this time. Cell viability is expressed as follows: "A" = 90% or more, "B" = 80% or more and less than 90%, "C" = 50% or more and less than 80%, and "D" = less than 50%. The numbers in parentheses represent the difference between each cell viability and 100%.

[0084] [Table 3-1]

[0085] <3-2. Method for calculating the maintenance period and maintenance temperature> From the results in sections 1-2 and 3-1 above, we found a tendency for the period until cell viability decreases to be longer as the difference between the culture temperature and the maintenance temperature decreases. In other words, we found the following relationship between the maintenance period and the maintenance temperature in the maintenance process. Specifically, the maintenance period during which cell viability can be maintained above a predetermined value can be calculated using the following formula. (Sustainable period (days))=A / {(Culture temperature (℃))-(Maintenance temperature (℃))}

[0086] Here, the value of A is determined according to the desired cell viability and the number of cells in the aggregate. For example, from Table 1, when the number of cells in the aggregate is 1000 and the culture temperature is 37°C, the period during which a cell viability of 90% or more can be maintained at a maintenance temperature of 25°C is 72 hours (i.e., 3 days), so A' = 36. The period during which a cell viability of 90% or more can be maintained at a maintenance temperature of 4°C is 24 hours (i.e., 1 day), so A'' = 33. The average of these values, 34.5, is set as A.

[0087] Furthermore, for example, according to Table 1, when the aggregate has 1000 cells and the culture temperature is 37°C, the period during which a cell viability of 80% or more can be maintained at a maintenance temperature of 25°C is 144 hours (i.e., 6 days), so A' = 72. The period during which a cell viability of 80% or more can be maintained at a maintenance temperature of 4°C is 48 hours (i.e., 2 days), so A'' = 66. The average of these values, 69, is defined as A.

[0088] Similarly, the maintainable temperature at which cell viability can be maintained above a predetermined value can be calculated using the following formula. (Maintenance period (days)) = A / {(Culture temperature (℃)) - (Sustainable temperature (℃))} A: A coefficient determined according to the predetermined value and the number of cells in the aggregate (days / °C). From the above formula, it is possible to calculate the lower limit of the maintenance temperature according to the desired maintenance period and cell viability.

[0089] <3-3. Measurement of the diameter and volume of aggregates> Similar to 3-1 above, kidney cells were seeded and cultured in such a way that the number of cells per well reached a predetermined number, thereby forming aggregates, which were then cultured.

[0090] After culturing for more than 10 days, the appearance of each aggregate was observed and imaged using a phase-contrast microscope BZ-X710 (Keyence). At this time, it was confirmed that all aggregates were submerged in the culture medium; that is, the specific gravity of each aggregate relative to the culture medium was greater than 1. Using analysis software (Keyence), the diameter was measured from the morphological images. Furthermore, the volume was calculated from the obtained diameter, and the volume ratio of the aggregate to the culture medium was calculated. In addition, for aggregates with 10,000 and 40,000 cells, 48 ​​of each aggregate were collected, their weight was measured, and the specific gravity relative to the culture medium was calculated.

[0091] Table 3-2 shows the diameter and volume of proximal tubular epithelial cell aggregates at each cell count, as well as their volume ratio to the culture medium and specific gravity.

[0092] [Table 3-2]

[0093] 4. Effects of temperature on planar cultured proximal tubular epithelial cells <4-1. Morphology of planar cultured cells> After culturing and harvesting kidney cells in the same manner as described in 1-1 above, the number of cells per well is 2 × 10⁶. 4 The cells were seeded individually into 96-well flat-bottom cell culture plates. After seeding, they were cultured until confluence.

[0094] After culturing, plates containing the culture medium with planar cultured cells were left standing at room temperature or refrigerated conditions, or placed in a 37°C incubator under 5% CO2 conditions. After 24 hours of standing, the appearance of the planar cultured cells under each temperature condition was observed using a light microscope.

[0095] Figure 6-1 shows the morphology of planar cultured cells under various temperature conditions {(a) 37°C; (b) room temperature; (c) refrigerated conditions}. In planar cultured proximal tubular epithelial cells, it was confirmed that the intercellular spaces increased and the cell morphology changed under both room temperature and refrigerated conditions. In planar culture of proximal tubular epithelial cells, it was confirmed that the cell morphology changed with decreasing culture temperature.

[0096] <4-2. Changes in cell viability over time> The cells were cultured until confluence, as described in 4-1 above. After culturing, the plates containing the culture medium with the planar cultured cells were left standing at room temperature or refrigerated, or placed in a 37°C incubator under 5% CO2 conditions. After standing, the culture medium was changed every two days.

[0097] The amount of ATP in the aggregates was measured at each temperature condition after a predetermined time had elapsed since the start of standing. Figure 6-2 shows the results of the time-dependent measurement of the amount of ATP in the aggregates at each temperature condition at each elapsed time {(a) 24 hours; (b) 48 hours; (c) 72 hours}.

[0098] The number of viable proximal tubular epithelial cells cultured in a planar manner was found to be significantly lower under both ambient temperature and refrigerated conditions after 24 hours of standing compared to the number of viable cells under culture temperature conditions. The cell viability (ratio of the number of viable cells under each condition to the number of viable cells under culture temperature) is shown in Table 4. Cell viability is expressed as follows: "A" = 90% or more, "B" = 80% or more and less than 90%, "C" = 50% or more and less than 80%, and "D" = less than 50%. The numbers in parentheses represent the difference from 100% for each cell viability.

[0099] [Table 4]

[0100] 5. Effects of temperature on human iPS cell aggregates Human iPS cells (201B7 strain) were used. The human iPS cells were removed from frozen vials stored in a liquid nitrogen storage container and 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 {StemFit®, Ajinomoto Co., Inc.} and cultured in a culture dish. The cells were cultured at 37°C and 5% CO2, with the culture medium changed once a day. The cells were harvested before confluence and seeded into low-adhesion 96-well V-bottom plates {PrimeSurface® Plate 96V, Sumitomo Bakelite Co., Ltd.} with 1000 cells per well, and cultured to form aggregates. The aggregates were cultured with the culture medium changed every two days.

[0101] After culturing for more than 240 hours, plates containing the culture medium with aggregates were left standing at room temperature or under refrigerated conditions, or placed in a 37°C incubator under 5% CO2 conditions. After 24 hours of standing, the appearance of the cells under each temperature condition was observed using a light microscope.

[0102] The amount of ATP in the aggregates was measured at each temperature condition after a predetermined time had elapsed since the start of standing.

[0103] Figure 7-1 shows the morphology of human iPS cell aggregates under various temperature conditions {(a) 37°C; (b) room temperature; (c) refrigerated conditions}. Figure 7-2 shows the results of measuring ATP levels over time in human iPS cell aggregates under each temperature condition at various elapsed times {(a) 24 hours; (b) 48 hours; (c) 72 hours}.

[0104] It was confirmed that the number of viable cells in human iPS cell aggregates decreased significantly after 24 hours of standing under both room temperature and refrigerated conditions compared to the number of viable cells under culture temperature conditions. The cell viability (ratio of the number of viable cells under each condition to the number of viable cells under culture temperature) is shown in Table 5. Cell viability is expressed as follows: "A" = 90% or more, "B" = 80% or more and less than 90%, "C" = 50% or more and less than 80%, and "D" = less than 50%. The numbers in parentheses represent the difference from 100% for each cell viability.

[0105] [Table 5]

[0106] (Embodiments of the Invention) A first embodiment of the present invention is a method for storing or transporting kidney cells, comprising a maintenance step of controlling a liquid containing aggregates of the kidney cells to a maintenance temperature that is below the culture temperature of the kidney cells and does not freeze. This provides the effect of storing or transporting kidney cells with high cell viability and maintaining the physiological function of the kidney at a temperature lower than the culture temperature.

[0107] A second embodiment of the present invention is the same as the first embodiment, but further wherein the maintenance temperature is 0°C or higher and less than 37°C. This enhances the effect of cells exhibiting a high cell viability rate.

[0108] A third embodiment of the present invention is a method in which, in the first or second embodiment, the number of cells in a single aggregate is between 125 and 10,000. This results in uniform aggregate size and further enhances the effect of cells exhibiting a high cell viability rate.

[0109] A fourth embodiment of the present invention is a method in which, in the first to third embodiments, the diameter of the aggregates is 100 μm or more and 800 μm or less. This results in uniform aggregate size and further enhances the effect of cells exhibiting a high cell viability rate.

[0110] A fifth embodiment of the present invention is the first to fourth embodiments, further comprising the following: 3 Above 0.300mm 3 The method is as follows. This results in uniform aggregate size and further enhances the effect of cells exhibiting a high cell viability.

[0111] A sixth embodiment of the present invention is a method in which, in the first to fifth embodiments, the number of aggregates in the liquid is further one. This has the effect of reducing the risk of the aggregates joining together and resulting in non-uniform size.

[0112] A seventh embodiment of the present invention is a method in which, in the first to sixth embodiments, the volume ratio of the aggregate to the liquid is 0.001% or more and 0.200% or less. This has the effect of preventing the liquid from drying out and exposing the aggregate to the air during storage or transportation, and ensuring sufficient oxygen supply to the aggregate.

[0113] An eighth embodiment of the present invention is a method in which, in the first to seventh embodiments, the specific gravity of the aggregate with respect to the liquid is 1.00 or more and 1.20 or less. This has the effect of making the aggregate less susceptible to changes in the extracellular environment.

[0114] A ninth embodiment of the present invention is a method in which, in the first to eighth embodiments, the renal cells are proximal tubular epithelial cells. This has the effect of producing aggregates that can be used for pharmacokinetic studies in renal cells and for drug discovery research related to renal diseases.

[0115] A tenth embodiment of the present invention is a method in which, in the first to ninth embodiments, the maintenance period of the maintenance step is less than or equal to the maintenance period in which the cell viability rate, which is the ratio of the number of viable cells at the maintenance temperature to the number of viable cells at the culture temperature of the kidney cells, can be maintained at or above a predetermined value, and the maintenance period is calculated from the following formula. (Sustainable period (days))=A / {(Culture temperature (℃))-(Maintenance temperature (℃))} A: A coefficient determined according to the predetermined value and the number of cells in the aggregate (days / °C). This has the effect of allowing us to calculate the upper limit of the maintenance period according to the desired maintenance temperature and cell viability.

[0116] An eleventh embodiment of the present invention is a method in which, in the first to ninth embodiments, the maintenance temperature is greater than or equal to a maintenance temperature that can maintain the cell viability rate, which is the ratio of the number of viable cells at the maintenance temperature to the number of viable cells at the culture temperature of the kidney cells, at or above a predetermined value, and the maintenance temperature is calculated from the following formula. (Maintenance period (days)) = A / {(Culture temperature (℃)) - (Sustainable temperature (℃))} A: A coefficient determined according to the predetermined value and the number of cells in the aggregate (days / °C). This has the effect of allowing us to calculate the lower limit of the maintenance temperature according to the desired maintenance period and cell viability.

[0117] A twelfth embodiment of the present invention is a method in which, in the tenth or eleventh embodiment, the value of A is 34.5 when the predetermined value is 90% and the number of cells in the aggregate is 1000. This has the effect of being able to calculate the maintenance period at a desired maintenance temperature and the maintenance temperature at a desired maintenance period, such that the cell viability rate is 90% or more when the number of cells in the aggregate is 1000.

[0118] A thirteenth embodiment of the present invention is a method in which, in the tenth or eleventh embodiment, the value of A is 69 when the predetermined value is 80% and the number of cells in the aggregate is 1000. This has the effect of being able to calculate the maintenance period at a desired maintenance temperature and the maintenance temperature at a desired maintenance period, such that the cell viability rate is 80% or more when the number of cells in the aggregate is 1000.

[0119] The fourteenth embodiment of the present invention is kidney cells obtained according to the first to thirteenth embodiments. This provides the effect of obtaining kidney cells with a high cell viability and that maintain the physiological function of the kidney.

[0120] A fifteenth embodiment of the present invention is a drug evaluation system including renal cells obtained according to the fourteenth embodiment. This provides the effect of obtaining a drug evaluation system that can be used for drug discovery research on pharmacokinetics in renal cells and for kidney diseases.

[0121] A sixteenth embodiment of the present invention is a cell product containing renal cells obtained according to the fifteenth embodiment. This provides the effect of obtaining a cell product that can be used for pharmacokinetic studies in renal cells and for drug discovery research related to renal diseases. [Industrial applicability]

[0122] This invention can be used for the storage and transport of renal cell aggregates that can be used in drug evaluation systems. [Explanation of Symbols]

[0123] 10 Renal cell storage system, 20 Renal cell storage device, 22 Culture vessel, 24 Liquid, 26 Aggregates, 30 Temperature control device, 40 Storage information input unit, 50 Temperature control unit, 52 Maintenance temperature setting unit, 54 Temperature control unit, 60 Maintenance period calculation unit, 70 Display unit

Claims

1. A method for storing or transporting renal cells while maintaining the expression of OAT1, OCT2, and URAT1, When storing or transporting kidney cells, the method includes a maintenance step in which the culture medium containing aggregates of the kidney cells is maintained at a temperature that prevents the medium from freezing, wherein the maintenance temperature and maintenance period in the maintenance step are either (i) or (ii) below: (i) the maintenance temperature is 10°C or higher and less than 37°C, and the maintenance period is 72 hours or less; (ii) the maintenance temperature is 0°C or higher and less than 10°C, and the maintenance period is 24 hours or less. The number of cells in one aggregate is between 125 and 10,000. A method for storing or transporting renal cells, wherein the aggregate consists of proximal tubular epithelial cells.

2. A method for storing or transporting renal cells while maintaining the expression of OAT1, OCT2, and URAT1, When storing or transporting kidney cells, the method includes a maintenance step in which the culture medium containing aggregates of the kidney cells is maintained at a temperature that prevents the medium from freezing, wherein the maintenance temperature and maintenance period in the maintenance step are either (i) or (ii) below: (i) the maintenance temperature is 10°C or higher and less than 37°C, and the maintenance period is 72 hours or less; (ii) the maintenance temperature is 0°C or higher and less than 10°C, and the maintenance period is 24 hours or less. The diameter of the aggregate is 100 μm or more and 800 μm or less. A method for storing or transporting renal cells, wherein the aggregate consists of proximal tubular epithelial cells.

3. The method according to claim 2, wherein the number of cells in one aggregate is 125 or more and 10,000 or less.

4. The method according to claim 1, wherein the diameter of the aggregate is 100 μm or more and 800 μm or less.

5. The volume of the aggregate is 0.001 mm ３ Above 0.300 mm ３ The method according to any one of claims 1 to 4, as follows:

6. The method according to any one of claims 1 to 5, wherein the number of aggregates in the culture medium is one.

7. The method according to claim 6, wherein the volume ratio of the aggregate to the culture medium is 0.001% or more and 0.200% or less.

8. The method according to any one of claims 1 to 7, wherein the specific gravity of the aggregate with respect to the culture medium is 1.00 or more and 1.20 or less.

9. The method according to claim 1 or 2, wherein the aggregate consists solely of kidney cells.

10. A renal cell storage and transport system for storing or transporting renal cells while maintaining the expression of OAT1, OCT2, and URAT1, comprising a renal cell storage device capable of maintaining a culture medium containing aggregates of the renal cells at a predetermined temperature during storage or transport of the renal cells, The renal cell storage device comprises a temperature control device that maintains the culture medium at a temperature at which the culture medium does not freeze, The temperature control device controls the maintenance temperature and the maintenance period for maintaining the maintenance temperature to be either (i) or (ii) below: (i) the maintenance temperature is 10°C or higher and less than 37°C, and the maintenance period is 72 hours or less; (ii) the maintenance temperature is 0°C or higher and less than 10°C, and the maintenance period is 24 hours or less. The number of cells in one aggregate is between 125 and 10,000. The aggregates consist of proximal tubular epithelial cells, forming a renal cell storage and transport system.

11. A renal cell storage and transport system for storing or transporting renal cells while maintaining the expression of OAT1, OCT2, and URAT1, comprising a renal cell storage device capable of maintaining a culture medium containing aggregates of the renal cells at a predetermined temperature during storage or transport of the renal cells, The renal cell storage device includes a temperature control device that maintains the temperature of the culture medium at a temperature at which the culture medium does not freeze. The temperature control device controls the maintenance temperature and the maintenance period for maintaining the maintenance temperature to be either (i) or (ii) below: (i) the maintenance temperature is 10°C or higher and less than 37°C, and the maintenance period is 72 hours or less; (ii) the maintenance temperature is 0°C or higher and less than 10°C, and the maintenance period is 24 hours or less. The diameter of the aggregate is 100 μm or more and 800 μm or less. The aggregates consist of proximal tubular epithelial cells, forming a renal cell storage and transport system.

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