Plate comprising kidney cell aggregate, and kit for pharmacokinetic inspection, kit for toxicity inspection, and kit for pharmaceutical development using same

Abstract

And a means for further improving the evaluation precision is provided in the aspect of evaluating pharmacokinetics / toxicity and efficacy by using the cell aggregate of a plurality of samples. A plate having a plurality of holes in which a liquid and an aggregate of kidney cells are accommodated, the openings of the holes being covered with a film, and the ratio of the total volume of the aggregate and the liquid relative to the total volume of the holes being a predetermined ratio.

Description

Technical Field

[0001] The present invention relates to a plate containing renal cell aggregates, a kit for pharmacokinetic examination using the same, a kit for toxicity examination, and a kit for pharmaceutical development. Background Art

[0002] After a medicament administered to an organism is absorbed in the organism, it is excreted from the blood into urine through the proximal convoluted tubule in the kidney. Therefore, renal toxicity of the medicament often causes kidney damage. In pharmaceutical research, it is very important to clarify the pharmacokinetics of the medicament in the kidney for the study of the action of the medicament. Therefore, development of a pharmaceutical support device using renal cells that can evaluate pharmacokinetics and toxicity is desired. In addition, such a pharmaceutical support device is also useful in the development of therapeutic drugs for kidney-related diseases (for example, diabetes, renal cancer, hyperuricemia, etc.).

[0003] Prior Art Documents

[0004] Patent Documents

[0005] Patent Document 1: JP-A-2021-191305 Summary of the Invention

[0006] Problems to be Solved by the Invention

[0007] Here, cell aggregates of renal cells (for example, Patent Document 1) are proposed as those useful for confirmation of pharmacokinetics and in pharmacy. And generally, for a medicament to be tested, cell aggregates of multiple samples (typically, a plate having multiple wells and cell aggregates disposed in each well) are used for confirmation and evaluation of pharmacokinetics / toxicity and pharmacodynamic effects. The problem of the present invention is to provide a means for further improving the accuracy of these evaluations in evaluating pharmacokinetics / toxicity and pharmacodynamic effects using such cell aggregates of multiple samples.

[0008] Means for Solving the Problems

[0009] One aspect of the present invention is a plate having a plurality of wells, characterized in that

[0010] the aforementioned wells accommodate aggregates of renal cells and a liquid,

[0011] an opening portion of the aforementioned wells is covered with a covering member (for example, a membrane, a lid, etc.),

[0012] the ratio of the total volume of the aforementioned aggregates and the aforementioned liquid to the total volume of the aforementioned wells is 35% or more and 75% or less.

[0013] In addition, one aspect of the present invention may be the aforementioned plate, wherein the specific gravity of the aforementioned aggregates with respect to the aforementioned liquid is 1.00 or more and 1.20 or less.

[0014] In addition, one embodiment of the present invention may be the aforementioned plate, wherein the water vapor permeability of the aforementioned covering member is 500 g / m 2 / 24 h or more.

[0015] In addition, one embodiment of the present invention may be a plate for freezing the aforementioned aggregate and the aforementioned liquid.

[0016] In addition, one embodiment of the present invention is a kit for pharmacokinetic examination containing the aforementioned plate.

[0017] In addition, one embodiment of the present invention is a kit for toxicity examination containing the aforementioned plate.

[0018] In addition, one embodiment of the present invention is a kit for pharmaceutical development containing the aforementioned plate.

[0019] Advantages of the Invention

[0020] According to the present invention, there is provided a plate for accommodating aggregates of renal cells in a plurality of wells, which can further improve the accuracy of evaluating pharmacokinetics / toxicity and pharmacodynamic effects by using aggregates of a plurality of samples of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 is a schematic view of the plate according to this embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0022] <<Plate Containing Aggregates of Renal Cells>>

[0023] The plate according to this embodiment has a plurality of wells. And each well contains a liquid and one or more aggregates of renal cells in the liquid. Further, for the plurality of wells, the openings are covered with a covering member. For example, Figure 1 is a culture vessel of a well plate according to an example of this embodiment. As Figure 1 shown on the left (up and down), the culture vessel has a well plate (in the figure, a 96-well, V-bottom is exemplified) whose surface is covered with a covering member (a membrane in the figure), and a well plate lid. In addition, as will be described later, as Figure 1 shown on the right, the culture medium and the aggregates are accommodated in the wells to such an extent that they do not fill the wells, and there is an air layer between the upper surface of the culture medium and the covering member (a membrane in the figure). Each element will be described in detail below.

[0024] <<Aggregates of Renal Cells>>

[0025] ​​The renal cells used in this method can be cultured regardless of their supply source. The renal cells are preferably derived from mammals, preferably primates such as humans and monkeys. In addition, according to the purpose, it can be derived from normal kidneys or kidneys with diseases. As renal cells, for example, cells constituting epithelium, cortex, proximal convoluted tubules, distal convoluted tubules, collecting ducts, glomeruli, etc. are listed, specifically, proximal convoluted tubule epithelial cells (RPTEC), mesangial cells, etc. The renal cells can be primary cells or renal cells derived from cells such as iPS cells or ES cells. In addition, the renal cells can be immortalized renal cells, cell lines (HK-2 cells, etc.), cells derived from other animal species (MDCK cells, LLC-PK1 cells, JTC-12 cells, etc.), and forced expression cells introduced into renal cell genes in order to express proteins such as specific transporters. More specifically, as renal cells, for example, human proximal tubule epithelial cells, human distal tubule epithelial cells and human collecting duct epithelial cells collected and separated from kidney, and proximal tubule epithelial cells, distal tubule epithelial cells and collecting duct epithelial cells differentiated and induced from human iPS cells or human ES cells are exemplified. For use in pharmaceutical research, proximal tubule epithelial cells are preferred, particularly proximal tubule epithelial cells derived from normal human kidney.

[0026] Here, the "aggregate" of cells refers to a mass-like collection of more than one cell. Aggregates are also called spheres. The number of cells constituting the aggregates is, for example, more than 5, more than 25, more than 50, preferably more than 100, more preferably more than 125, further preferably 200, and particularly preferably more than 500. The number of cells constituting the aggregates is, for example, less than 4,000, preferably less than 10,000, more preferably less than 2,000, less than 1,000. When the number of cells constituting the aggregates is within such a range, almost all cells in the culture medium remain viable, and higher homogeneity between multiple aggregates in the plate becomes possible.

[0027] (Morphological parameters)

[0028] Examples of morphological parameters include diameter, volume, cross-sectional area, perimeter, compactness, circularity, and aspect ratio of aggregates. Each parameter is described in detail below.

[0029] The diameter of the aggregate is preferably, for example, 100 μm or more and 800 μm or less. In addition, the diameter of the aggregate is defined as the maximum width of the aggregate. That is, the diameter of the aggregate is the length of the maximum straight line among the straight lines connecting two points on the outer edge of the aggregate. The diameter of the aggregate can be measured, for example, based on a known method, using a photograph taken by a phase contrast microscope. As a phase contrast microscope, BZ-X710 (Keyence Company) can be used, and the diameter can be measured using analysis software.

[0030] The volume of the aggregate is preferably, for example, 0.001 mm 3Above, 0.300 mm 3 Below. Since the aggregate is approximately spherical, the volume of the aggregate can be calculated from the measured diameter.

[0031] The cross-sectional area of the aggregate is preferably 40,000 μm 2 or more and 100,000 μm 2 or less, more preferably 45,000 μm 2 or more and 90,000 μm 2 or less. Here, the cross-sectional area (similarly, the perimeter, compactness, circularity, and aspect ratio described below) is calculated by identifying one aggregate from a CQ1 (confocal image cytometer, manufactured by Yokogawa Electric Corporation) image {section image (cross-sectional view)} based on a known method.

[0032] The perimeter of the aggregate is preferably 600 μm or more and 1800 μm or less, more preferably 800 μm or more and 1600 μm or less.

[0033] In addition, the compactness of the aggregate is preferably 1.0 or more and 3.0 or less, more preferably 1.2 or more and 2.5 or less.

[0034] In addition, the circularity of the aggregate is preferably 0.3 or more and 1.0 or less, more preferably 0.4 or more and 1.0 or less.

[0035] In addition, the aspect ratio of the aggregate is preferably 1.0 or more and 2.0 or less, more preferably 1.0 or more and 1.5 or less.

[0036] (Coefficient of variation of ATP)

[0037] The coefficient of variation of ATP in multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and most preferably 10% or less. The lower limit is 0%.

[0038] (Coefficient of variation of morphological parameters)

[0039] The coefficient of variation of the cross-sectional area in multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and further preferably 10% or less. The coefficient of variation of the perimeter in multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and further preferably 10% or less. The coefficient of variation of the compactness in multiple cell aggregates is preferably 25% or less, more preferably 20% or less, and further preferably 15% or less. The coefficient of variation of the circularity in multiple cell aggregates is preferably 25% or less, more preferably 20% or less, and further preferably 15% or less. The coefficient of variation of the aspect ratio in multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and further preferably 10% or less.

[0040] <Plate>

[0041] The plate containing the aggregate population of renal cells is not particularly limited as long as it has a plurality of wells in which one or more aggregates can be accommodated in one well, and any type can be used. As these culture vessels, for example, there are types such as 6-well plates, 24-well plates, 48-well plates, 96-well plates, 384-well plates, dishes of various sizes, etc. In addition, the shape of the bottom is not particularly limited, and there are types such as flat bottom, V-bottom, U-bottom, etc.

[0042] <Liquid>

[0043] The liquid present in each well is not particularly limited and can be the culture medium when preparing the aggregates, or can be fresh culture medium, or can be a liquid other than the culture medium (such as physiological saline, buffer solution, etc.). This liquid can contain salts, buffers, serum, vitamins, amino acids, glucose (sugar), electrolytes, antibiotics, growth factors (compounds, proteins).

[0044] <Covering member>

[0045] The preferred covering member (for example, a membrane, such as a sheet-like membrane) preferably has a water vapor permeability (measured under the conditions of a temperature of 40 °C and a humidity of 90% RH according to JIS Z0208 method) of 500 g / m 2 / 24 h or more, more preferably 2000 g / m 2 / 24 h or more, most preferably 4000 g / m 2 / 24 h or more. In addition, the upper limit value of the water vapor permeability is, for example, 100000 g / m 2 / 24 h or less, 50000 g / m 2 / 24 h or less, 25000 g / m 2 / 24 h or less, 10000 g / m 2 / 24 h or less. In addition, the preferred covering member has a pore size of 10 μm to 50 μm. In addition, the covering member can cover all the wells in one covering plate, or can cover each well group on the basis of dividing into a plurality of well groups, or can cover each well. In addition, in the case where the covering member is not flat, for example, when it forms an uneven shape based on the plane of the opening part, the "volume of the hole" described in the scope of this specification and claims takes into account the volume of this uneven shape (that is, in the case where the covering member is concave, the "volume that becomes concave" based on the plane of the opening part is subtracted from the "volume of the hole", on the other hand, in the case where the covering member is convex, the "volume that becomes convex" based on the plane of the opening part is added to the "volume of the hole").

[0046] <Volume ratio of aggregate to liquid>

[0047] In the one hole, the volume ratio of the aggregate to the liquid is preferably 0.001% or more, 0.002% or more, or 0.003% or more, and preferably 0.800% or less, 0.500% or less, or 0.200% or less.

[0048] <Volume ratio of the content (liquid + aggregate) to the volume of the hole>

[0049] <Volume ratio of the content (liquid + aggregate) to the volume of the hole> is preferably 35% or more, 37.5% or more, or 40% or more, and preferably 75% or less, 70% or less, or 60% or less. In addition, the volume of the aggregate here is the total volume of the multiple aggregates in the case where there are multiple aggregates in the hole. Here, the reason why it is more preferable when the volume ratio is equal to or higher than the above lower limit value is described. First, the amount of dissolved oxygen per cell increases, and the cells can be maintained more stably. In addition, by reducing the concentration of cell waste, the damaging effect on the cells can be reduced. Further, the risk of attracting aggregates during medium replacement is further reduced, and high uniformity in the culture plate can be maintained. In addition, the situation where cell culture cannot be maintained due to drying of the medium can be further avoided. On the other hand, the reason why it is more preferable when the volume ratio is equal to or lower than the above upper limit value is described. First, since the supply of air (oxygen) to the medium in the hole can be ensured, the amount of dissolved oxygen can be increased, and the reduction of cell function can be prevented. In addition, since an air layer is formed between the aggregate and the liquid medium in the hole, appropriate air (oxygen) can be supplied to the medium via the ventilation sheet. Further, since this air layer is formed, in the case where vibrations such as transportation are assumed, the aggregate does not adhere to the sheet, the loss of the aggregate can be effectively prevented, and a culture plate with more uniform arrangement of the aggregate can be obtained.

[0050] For example, the hole volume of a 96-well plate is generally 280 - 310 mm 3 (For example, PrimeSurface 96V made by Sumitomo Bakelite is about 310 mm 3 , and the EZ BindShut SP 96-well plate made by AGC Techno Glass is about 280 mm 3 ). At this time, the volume of the content (cells and liquid medium) is preferably 120 - 200 mm 3 , more preferably 130 - 160 mm 3 .

[0051] <Specific gravity of the aggregate relative to the liquid>

[0052] In the pores, the lower limit of the specific gravity of the aggregates relative to the liquid is preferably 1.00 or more, preferably 1.20 or less, 1.15 or less, or 1.10 or less. With such a specific gravity, the aggregates do not float on the liquid surface and are not overly pressed against the bottom due to their own weight, so the aggregates are less likely to be affected by changes in the extracellular environment (especially during transportation). As a result, higher homogeneity among multiple aggregates in the plate becomes possible.

[0053] <Number in the pores>

[0054] The number of aggregates in the pores is one or more. Here, in the case of multiple aggregates, it is preferably two or more and 1000 or less. For example, assume a configuration of 500 to 1000 per well in a 6-well plate.

[0055] <<Method for preparing cell aggregates>>

[0056] Cultivation of renal cells can be carried out by applying a culture medium and a culture vessel suitable for the cells to be cultivated, according to a conventional method, for example, at 37 °C, 5% CO 2 conditions. The cultivation can be any one of static cultivation, shaking cultivation, or stirring cultivation, etc. The cultivation can be adherent cultivation, but it is preferably carried out in a non-adherent state for at least a part of the period (for example, suspension cultivation). Renal cells can form aggregates by being cultivated in a non-adherent state in a culture vessel. The "non-adherent state" means a state in which all or most of the cells do not adhere to the surface of the culture vessel, including 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 when in contact with the surface of the culture vessel, they can easily leave the surface of the culture vessel without using tools, enzymes, etc., through coating of the culture vessel, convection of the culture medium, etc.

[0057] For example, in a certain case, aggregates of renal cells are formed within 24 hours after the start of renal cell cultivation. And, by cultivating renal cells in the state of aggregates for a part of the period, the physiological functions of renal cells with reduced dedifferentiation can be restored. The period of cultivating renal cells in a non-adherent state in a culture vessel is generally expected to be 120 hours or more. Thus, cultivated renal cells with a higher expression state of physiological functions can be obtained. During the cultivation period, it is preferable to replace the culture medium regularly. For example, the culture medium is replaced every 2 days.

[0058] Any known one can be appropriately used as the culture medium. For example, in the case of cultivating proximal tubular epithelial cells, commercially available renal tubular cell culture media can be used. As examples of preferred culture media, REGM (registered trademark) (LONZA), EpiCM (registered trademark) (ScienCell), and KeratinocyteSFM (registered trademark) (Thermo Fisher Scientific) are listed.

[0059] In addition, conventionally well-known materials and additives useful in cell culture can be appropriately used. For example, collagen I (type I collagen) can be added to the culture medium. Collagen I has the effect of causing renal cells to adhere to each other. Therefore, by culturing renal cells in a culture medium containing collagen I, the formation of aggregates is promoted. Collagen I is preferably full-length collagen I, but may also be the α1 chain or α2 chain constituting collagen I, or a collagen peptide obtained by further fragmenting each chain. In addition, the source of collagen I is not particularly limited and may be of human origin or of other animal origin.

[0060] Any type of culture vessel can be used. Here, in order to promote the formation of aggregates, it is preferable to perform a cell non-(low)-adhesion treatment or to be made of a cell non-(low)-adhesion material. Examples of the cell non-(low)-adhesion treatment include a cell non-adhesive hydrogel coating treatment on the surface of the vessel, an MPC (2-methacryloyloxyethyl phosphorylcholine) coating treatment, a Proteosave (registered trademark) SS coating treatment, a mirror polishing treatment, etc. Examples of the cell non-(low)-adhesion material include glass and high molecular materials such as low density polyethylene, medium density polyethylene, polyvinyl chloride, ethylene-vinyl acetate copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methyl methacrylate) copolymer, poly(ethylene vinyl acetate) copolymer, and mixtures of two or more of these polymers. The aggregates cultured by the cell non-(low)-adhesion treatment can be transferred to another culture vessel for use. In this case, a culture vessel that has been subjected to a cell non-(low)-adhesion treatment or a cell adhesion treatment can be used. In addition, the shape of the bottom of the well of the transferred culture vessel is not particularly limited, and examples include a flat bottom, a V bottom, and a U bottom.

[0061] In the case of forming a large number of aggregates, a high-density spherical preparation plate or dish can be used. Further, if necessary, a culture vessel such as a rotary flask can also be applied. For example, it is preferable to use culture vessels of the ELPLASIA (registered trademark) series (Corning Inc.), the EZSPHERE (registered trademark) series (AGC Techno Glass Co., Ltd.), etc. These culture vessels are available 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 prepared varies depending on the size of the bottom area of the vessel. For example, in the case of applying a low-adhesion treatment to a 96-well plate (V bottom), a 96-well plate (U bottom), or a 384-well plate (U bottom), one aggregate is formed in one well.

[0062] Aggregates prepared using a plate or dish made of high-density spheres can be recovered and cultured by shaking in suspension. In the case of culturing by shaking in suspension, it is preferable to place a dish or plate that has been subjected to a cell non-(low)-adhesion treatment on an oscillator to culture the aggregates. As the oscillator, a reciprocating oscillator and a rotary oscillator can be used.

[0063] <<Storage / Transportation Method>>

[0064] The storage / transportation method of the plate according to this method includes a maintenance step of managing the temperature of the plate containing aggregates of renal cells at a temperature lower than the culture temperature of the aforementioned renal cells and at which the aforementioned liquid does not freeze (hereinafter, sometimes referred to as the "maintenance temperature" for convenience) during the storage or transportation of renal cells. By adopting this step, higher homogeneity among multiple aggregates in the plate becomes possible.

[0065] The temperature lower than the culture temperature of the aforementioned renal cells and at which the aforementioned liquid does not freeze can be appropriately selected as the optimum temperature according to the type of cells, usage, and / or the period required for storage or transportation, etc. In addition, the culture temperature is a temperature suitable for the survival of cells, and these cells may or may not proliferate. Since the typical culture temperature of cells is 37°C, it is desirable that the maintenance temperature be a lower temperature than this. On the other hand, if the maintenance temperature is too low, there is a concern that the survival rate of cells in the aggregates may decrease due to partial freezing occurring inside the cells, etc. Therefore, it is desirable that the maintenance temperature be 0°C or higher. The maintenance temperature can be, for example, 0°C or higher and lower than 37°C. In the case of maintaining at a relatively high temperature, it is preferably 10°C or higher and lower than 37°C, more preferably 20°C or higher and lower than 28°C. In the case of maintaining at a relatively low temperature, it is preferably 0°C or higher and lower than 10°C, more preferably 3°C or higher and lower than 8°C. In addition, in this specification, for convenience, the temperature range of 10°C or higher and lower than 37°C is sometimes referred to as "room temperature" and the temperature range of 0°C or higher and lower than 10°C is sometimes referred to as "refrigeration" respectively.

[0066] In the case of storage, in the maintenance step, an incubator, a constant temperature room, a refrigerator, etc. that can be adjusted to the desired temperature can be used.

[0067] In the case of transportation, the maintenance step preferably uses a heat-insulating container such as styrofoam. The size of the heat-insulating container is not particularly limited as long as it can package the storage and transportation containers. When there is a gap between the heat-insulating container and the storage and transportation containers, it is preferable to co-package buffer materials in such a way that the storage and transportation containers do not move. In addition, as the heat-insulating container, a paper container or a corrugated cardboard container can also be used.

[0068] In addition, the maintenance step does not necessarily need to be carried out by standing still. Therefore, it can be carried out on an oscillator or during transportation under conditions where vibrations that do not damage the aggregates occur.

[0069] <<Freezing Method>>

[0070] One embodiment of this method can be a plate containing aggregates of renal cells in a frozen state. In this case, the method for preparing the plate in a frozen state includes, for example, the steps of preparing aggregates of renal cells, substantially maintaining the aggregated state of the aggregates and then freezing them, placing the frozen aggregates and a liquid (preferably the cryopreservation medium detailed below) into wells, and freezing the liquid in the wells. Hereinafter, the freezing step of the aggregates will be described in detail.

[0071] The freezing of the aggregates is carried out by cooling at a low temperature at which cells can be frozen until the cells are frozen. Here, the freezing of renal cells is preferably carried out while adjusting the temperature drop rate of the cells to be frozen to a specified range. The temperature drop rate can be adjusted by using commercially available cryopreservation containers, programmable refrigerators capable of setting the freezing conditions of cells and tissues, etc. The temperature drop rate of the cells during freezing can be set to minimize the damage to the cells during freezing. For example, in the case of slow freezing, it can be in the range of about 0.2°C to about 3°C per minute, preferably at a rate of about 1°C per minute.

[0072] As a general freezing method, a refrigerator or liquid nitrogen can be used for the cooling of renal cells. For example, in the case of freezing aggregates of proximal tubular epithelial cells, a ultra-low temperature refrigerator that can be set to a temperature of -80°C is preferably used. In the case of storing in a liquid nitrogen container, it can be stored at a temperature of -196°C in the liquid phase and -150°C to -196°C in the gas layer.

[0073] "Substantially maintaining the aggregated state of the aggregates" in the freezing step means that no operation (such as trypsin treatment) is performed to deliberately disrupt or disperse the aggregates contained in the recovered renal cells, and the morphology of the aggregates is not damaged. In addition, "substantially" means that the reduction in the amount of the aggregates after freezing is not a problem compared to the amount of the aggregates contained in the recovered renal cells before freezing, and it does not necessarily mean that the dispersion of the cells constituting the aggregates does not occur at all during freezing.

[0074] In addition, as long as it does not have a significant adverse effect on the maintenance of the morphology of the aggregates, any steps can be included between the recovery step and the freezing step, for example, other steps beneficial to the cryopreservation of cells. For example, it can include the step of adding a preferred cryopreservation medium to the recovered renal cells.

[0075] As a medium for cryopreservation, cryoprotective agent components that reduce damage caused by intracellular ice crystals are listed. For example, a medium containing any one of dimethyl sulfoxide (5 - 15%), mammalian-derived serum, dextran, glycogen, methylcellulose or carboxymethylcellulose, polyethylene glycol, polyvinylpyrrolidone, glucose, sucrose, etc. The medium can be a liquid such as a culture medium. A solution appropriately formulated with two or more of these cryoprotective agents is preferred. Therefore, as commercially available cryopreservation media, commercially available cryopreservation solutions such as CELLBANKER (registered trademark) 1plus (Xenoac Resource), CELLBANKER (registered trademark) 1 (Xenoac Resource), CELLBANKER (registered trademark) 2 (Xenoac Resource) can be used.

[0076] <<Use>>

[0077] The plate containing cell aggregates according to this method can be provided as a drug evaluation system or a cell product. As a drug evaluation system, for example, a pharmacokinetics and nephrotoxicity evaluation system in renal cells is listed. In addition, such renal cells can also be used as a tool for analyzing the mechanisms of diseases related to the kidney such as diabetes, renal cancer, and hyperuricemia and exploring therapeutic drugs.

[0078] The present invention is not limited to the above-described embodiments, and various design changes and other modifications can be made based on the knowledge of those skilled in the art. Embodiments incorporating such modifications are also included within the scope of the present invention.

[0079] Examples

[0080] <Preparation of cell aggregates>

[0081] As renal cells, human proximal tubular epithelial cells {Clonetics (registered trademark), catalog number CC-2553, RPTEC - renal proximal tubular epithelial cells} obtained from LONZA were used. The cryovial stored in the liquid nitrogen storage was immersed in a 37°C constant temperature bath for thawing. After thawing, the cell suspension in the cryovial was mixed with the recommended culture medium {REGM (registered trademark), LONZA} and cultured in a petri dish. The cells were cultured at 37°C, 5% CO 2Under the condition of, the cells were cultured while changing the culture medium at a frequency of once every two days. The cells were recovered before confluence and cultured by inoculating them in a 96-well V-bottom plate for low cell adhesion treatment {PrimeSurface (registered trademark) plate 96V, Sumitomo Bakelite Co., Ltd.} so that the number of cells per well was 1000 to form aggregates. The aggregates were cultured while changing the culture medium at a frequency of once every two days. In addition, "confluence" means that the proportion of the area occupied by the cells relative to the entire culture surface of the culture vessel is approximately 100%, that is, the state in which the cells proliferate without gaps on the culture surface. After culturing for 240 hours or more, the surface of the plate containing the culture solution with the aggregates was covered with a film {Aera Seal (Excel Scientific Co., Ltd.) / water vapor permeability: 4200 g / m 2 / 24hr; pore size: 10 μm to 50 μm}. At this time, the specific gravity of the cell aggregates relative to the culture medium was 1.00 to 1.07. In addition, the volume ratio of the content (liquid + aggregates) to the volume of the well was 48%. Further, the volume ratio of the aggregates in the well to the liquid was 0.013%. Thereafter, the plate was placed in a styrene foam container and left standing in the room (air conditioner set temperature: 25°C) for 72 hours.

[0082] <ATP amount>

[0083] Regarding the cell aggregate population (96) after standing for 72 hours, CellTiter-Glo (registered trademark) 3D Cell Viability Assay (Promega Corporation) was applied, and the ATP amount was measured by luminescence method. Specifically, the aggregates were recovered from each culture medium, and an equal volume of CellTiter-Glo 3D Reagent as the volume of the culture medium was added. The mixture was incubated at room temperature for 30 minutes. After thorough mixing, the luminescence value was measured using a microplate reader (Perkin Elmer).

[0084] <Measurement of aggregate diameter, volume and surface area>

[0085] Regarding the cell aggregate population (96) after standing for 72 hours, the situation of each aggregate in the well was observed and photographed through a phase-contrast microscope BZ-X710 (Keyence Corporation). Using analysis software (Keyence Corporation), the diameter was measured based on the morphological photographs. Further, the volume was calculated from the obtained diameter, and the volume ratio of the aggregates to the culture medium was calculated. In addition, the cross-sectional area, perimeter, compactness, circularity and aspect ratio were calculated by identifying one aggregate based on a known method through a CQ1 (confocal image cytometer, Yokogawa Electric Corporation) image {section image (cross-sectional view)}.

[0086] The results are shown in the tables. Table 1 shows the measured values and coefficients of variation of various morphological parameters. In addition, Table 2 shows the measured values and coefficients of variation of ATP per aggregate. From these tables, it was found that after a long period, it was also possible to confirm the suppression of the variation deviation of morphological parameters and ATP between multiple aggregates. In addition, regarding a freezing plate containing aggregates obtained by freezing aggregates prepared by the same method as in the examples, the same evaluation was also performed after thawing the freezing plate, and the same results were obtained.

[0087] [Table 1]

[0088]

[0089] [Table 2]

[0090]

[0091] Industrial Applicability

[0092] The present invention relates to a plate containing renal cell aggregates, a pharmacokinetic examination kit, a toxicity examination kit, and a pharmaceutical development kit using the same, and can be applied, for example, in the industry for developing therapeutic drugs for treating kidney-related diseases.

Claims

1. A plate having a plurality of holes, characterized in that, the holes accommodate aggregates of renal cells and liquid, the openings of the holes are covered by a covering member, the ratio of the total volume of the aggregates and the liquid to the total volume of the holes is 35% or more and 75% or less.

2. The plate according to claim 1, wherein, the specific gravity of the aggregates relative to the liquid is 1.00 or more and 1.20 or less.

3. The plate according to claim 1, wherein, The water vapor transmission rate of the covering member is 500 g / m 2 / 24 hr or more.

4. The plate according to claim 1, wherein, the aggregates and the liquid are frozen.

5. A kit for pharmacokinetic examination, comprising the plate according to any one of claims 1 to 4.

6. A kit for toxicity examination, comprising the plate according to any one of claims 1 to 4.

7. A kit for pharmaceutical development, comprising the plate according to any one of claims 1 to 4.

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