Plate containing frozen renal cell aggregates, and pharmacokinetics test kit, toxicity test kit, and drug discovery and development kit using same

Abstract

The present disclosure addresses the problem of providing a means for suppressing deterioration of the cell state when freezing a cryopreservation liquid containing cell aggregates of renal cells. The present disclosure provides a plate having a plurality of wells, the plate being characterized in that a liquid containing renal cell aggregates is accommodated in a frozen state in the wells, and the liquid in a frozen state contains 7.5-40 mass% of saccharides relative to the mass of the liquid in a frozen state.

Description

Plate containing frozen renal cell aggregates, pharmacokinetic test kit, toxicity test kit and drug discovery development kit using the same

[0001] The present disclosure relates to a plate containing frozen renal cell aggregates, and a pharmacokinetic testing kit, a toxicity testing kit, and a drug discovery and development kit using the same.

[0002] Drugs administered to the body are absorbed into the body and then excreted from the blood into the urine via the proximal tubules of the kidney. Therefore, nephrotoxicity of drugs often leads to kidney damage. In drug discovery research, it is extremely important to investigate pharmacokinetics in the kidney to clarify the effects of drugs. Therefore, there is a need for the development of a drug discovery support device that can evaluate pharmacokinetics and toxicity using renal cells. Furthermore, such a drug discovery support device would also be useful in developing drugs for treating kidney-related diseases (e.g., diabetes, kidney cancer, hyperuricemia, etc.).

[0003] Patent Publication No. 2021-191305

[0004] Renal cell aggregates have been proposed as a useful tool for confirming pharmacokinetics and for drug discovery (see, for example, Patent Document 1). Typically, the pharmacokinetics, toxicity, and efficacy of a drug to be tested are confirmed and evaluated using multiple samples of cell aggregates (typically, a plate having multiple wells, with a cell aggregate placed in each well).

[0005] Here, from the viewpoints of reducing the load on cell aggregates during transportation and improving handling, the present inventors investigated a method of placing a cryopreservation solution containing cell aggregates of renal cells into each well of a plate and then freezing the plate as a whole. During this study, the present inventors discovered that freezing using conventional cryopreservation solutions can sometimes cause deterioration in the cellular state of renal cell aggregates. Therefore, an object of the present disclosure is to provide a means for suppressing deterioration in the cellular state when freezing a cryopreservation solution containing cell aggregates of renal cells.

[0006] One aspect of the present disclosure is a plate having a plurality of wells, wherein the wells contain a liquid containing renal cell aggregates in a frozen state, and the frozen liquid contains 7.5 to 40 mass% of sugars based on the mass of the frozen liquid. Here, the frozen liquid may further contain 1 to 30 mass% of dimethyl sulfoxide based on the mass of the liquid. The volume of the frozen liquid in the wells may be 5 to 80% of the well volume. The number of cells in the renal cell aggregates may be 100 cells or more and 10,000 cells or less. The present disclosure may also be a pharmacokinetic testing kit including the plate. The present disclosure may also be a toxicity testing kit including the plate. The present disclosure may also be a drug discovery and development kit including the plate. One aspect of the present disclosure is a plate having a plurality of wells, wherein the wells contain a liquid containing renal cell aggregates in a frozen state, and the viscosity of the liquid is 1 mPa·s to 100 mPa·s. Here, the frozen liquid may further contain 1 to 30% by mass of dimethyl sulfoxide based on the mass of the frozen liquid. The volume of the frozen liquid in the well may be 5 to 80% of the well volume. The number of cells in the renal cell aggregates may be 100 to 10,000 cells. The plate may also be a pharmacokinetic testing kit. The plate may also be a toxicity testing kit. The plate may also be a drug discovery and development kit.

[0007] According to the present disclosure, when a cryopreservation solution containing cell aggregates of kidney cells is frozen, it is possible to suppress deterioration of the cell condition.

[0008] FIG. 1 is a schematic diagram of a plate according to the present embodiment. FIG. 2 is a diagram (photograph) showing an example of evaluation criteria for cell state after thawing. FIG. 3 is a diagram showing the difference in viability depending on the DMSO concentration when the sugar concentration is constant. FIG. 4 is a diagram showing the state (score) of cell aggregates after thawing when the volume of cryopreservation solution and the sugar concentration are changed. FIG. 5 is a diagram showing the results of adding various amounts of medium to a liquid containing frozen cell aggregates (sugar concentration: 10% by mass), thawing the frozen state, and then measuring the cell viability. FIG. 6 is a diagram showing the viability of cell aggregates when the number of cells in the cell aggregates and the volume of cryopreservation solution are changed under the same conditions (DMSO concentration: 10% by mass / sugar concentration: 10% by mass) (relative values ​​based on 100 μl). FIG. 7 is a diagram showing the difference in viability after long-term cryopreservation when the sugar concentration is changed. FIG. 8 is a diagram showing the difference in the state (score) of cell aggregates after long-term cryopreservation when the sugar concentration is changed.

[0009] <<Plate Containing Renal Cell Aggregates (Before Freezing)>> The plate according to this embodiment has multiple wells. Each well contains a cryopreservation medium and one or more renal cell aggregates contained within the cryopreservation medium. For example, FIG. 1 shows a culture vessel including a well plate according to one example of this embodiment. As shown on the left (top and bottom) of FIG. 1 , the culture vessel includes a well plate (a 96-well, V-bottom well is illustrated) and a well plate lid. As will be described later, as shown on the right of FIG. 1 , the cryopreservation medium and aggregates are contained within the wells to the extent that they do not fill the wells, and an air space exists between the top surface of the cryopreservation medium and the well plate lid. Each element is described in detail below. The surface of the culture vessel may be covered with a covering member (e.g., a film, e.g., a sheet-like film). In this case, an air space exists between the top surface of the cryopreservation medium and the covering member. A suitable covering member has a water vapor permeability of 500 g / m (measured in accordance with JIS Z0208 at a temperature of 40°C and a humidity of 90% RH). 2 / 24hr or more is preferred, and 2000g / m 2 / 24hr or more is more preferable, and 4000g / m 2The upper limit of the water vapor permeability is, for example, 100,000 g / m 2 / 24hr or less, 50000g / m 2 / 24hr or less, 25000g / m 2 / 24hr or less, 10000g / m 2 / 24 hr or less. A suitable covering member has a pore size of 10 μm to 50 μm. This covering member may cover all wells in the plate with one piece, or may be divided into multiple well groups and each well group may be covered, or each well may be covered individually.

[0010] <Kidney Cell Aggregates> The kidney cells used in this embodiment may be cultureable and may be derived from any source. Kidney cells are preferably derived from mammals, and preferably from primates such as humans or monkeys. Furthermore, kidney cells may be derived from normal kidneys or diseased kidneys, depending on the purpose. Examples of kidney cells include cells constituting the epithelium, cortex, proximal tubules, distal tubules, collecting ducts, glomeruli, etc., specifically, proximal renal tubule epithelial cells (RPTEC), mesangial cells, etc. Kidney cells may be primary cells or kidney cells derived from stem cells such as iPS cells or ES cells. Kidney cells may also be immortalized kidney cells, established cell lines (e.g., HK-2 cells), cells derived from other animal species (e.g., MDCK cells, LLC-PK1 cells, JTC-12 cells), or cells expressing specific proteins such as transporters, which have been gene-transduced into kidney cells to express the proteins. More specifically, examples of renal cells include human proximal tubule epithelial cells, human distal tubule epithelial cells, and human collecting duct epithelial cells collected and isolated from the kidney, as well as proximal tubule epithelial cells, distal tubule epithelial cells, and collecting duct epithelial cells induced to differentiate from human iPS cells or human ES cells. For use in drug discovery research, proximal tubule epithelial cells, particularly proximal tubule epithelial cells derived from normal human kidneys, are preferred.

[0011] Here, the term "aggregate" of cells refers to a mass-like collection of several or more cells. It is also called an aggregate or a 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, even more preferably 200, and particularly preferably 500 or more. The number of cells constituting the aggregate is, for example, 4,000 or less, preferably 10,000 or less, more preferably 2,000 or less, or 1,000 or less. When the number of cells constituting the aggregate is within this range, almost all cells remain viable in the culture medium, allowing for greater homogeneity among multiple aggregates within a plate.

[0012] (Morphological parameters) Examples of morphological parameters include diameter, volume, cross-sectional area, perimeter, compactness, circularity, and aspect ratio of the aggregate. Each parameter will be described in detail below.

[0013] The diameter of the aggregate is preferably, for example, 100 μm or more and 800 μm or less. The diameter of the aggregate is defined as the maximum width of the aggregate. In other words, the diameter of the aggregate is the length of the longest line connecting two points on the outer edge of the aggregate. The diameter of the aggregate can be measured, for example, based on a well-known method using a photograph 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.

[0014] The volume of the aggregate is, for example, 0.001 mm ３ More than 0.300 mm ３ The following is preferred: The volume of the agglomerates can be calculated from the measured diameter, since the agglomerates are approximately spherical.

[0015] The cross-sectional area of ​​the aggregate is preferably 40,000 μm ２ More than 100000 μm ２ More preferably, 45,000 μm or less ２ More than 90000μm ２ Here, the cross-sectional area (as well as the perimeter, compactness, circularity, and aspect ratio, which will be described below) is calculated based on a well-known method by recognizing an image (slice image (cross-sectional view)) of one aggregate using a CQ1 (confocal image cytometer, manufactured by Yokogawa Electric Corporation).

[0016] 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.

[0017] 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.

[0018] The circularity of the aggregate is preferably 0.3 or more and 1.0 or less, and more preferably 0.4 or more and 1.0 or less.

[0019] 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.

[0020] (ATP Coefficient of Variation) The ATP coefficient of variation among a plurality of cell aggregates is preferably 20% or less, more preferably 15% or less, and most preferably 10% or less. The lower limit is 0%.

[0021] (Coefficient of variation of morphological parameters) The coefficient of variation of the cross-sectional area among the multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less. The coefficient of variation of the perimeter among the multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less. The coefficient of variation of the compactness among the multiple cell aggregates is preferably 25% or less, more preferably 20% or less, and even more preferably 15% or less. The coefficient of variation of the circularity among the multiple cell aggregates is preferably 25% or less, more preferably 20% or less, and even more preferably 15% or less. The coefficient of variation of the aspect ratio among the multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less.

[0022] <Plate> The plate containing the renal cell aggregates is not particularly limited as long as it has multiple wells capable of accommodating one or more aggregates per well, and any plate can be used. These culture vessels include, for example, 6-well plates, 24-well plates, 48-well plates, 96-well plates, 384-well plates, and dishes of various sizes. The shape of the bottom is also not particularly limited, and includes flat, V-bottom, U-bottom, and other types. For example, the well volume of a 96-well plate is generally 280 to 310 mm ３ (For example, PrimeSurface 96V manufactured by Sumitomo Bakelite is about 310 mm ３ The EZ BindShut SP 96-well plate manufactured by AGC Technoglass is approximately 280 mm ３ ).

[0023] <Liquid> The medium constituting the liquid (cryopreservation medium) present in each well is not particularly limited and may be the culture medium used when the aggregates were prepared, fresh culture medium, or a liquid other than culture medium (e.g., physiological saline, buffer solution, etc.). The liquid preferably contains one or more cryoprotectant components that reduce damage caused by intracellular ice crystals, such as dimethyl sulfoxide (DMSO), mammalian serum, dextran, glycogen, methylcellulose or carboxymethylcellulose, polyethylene glycol, polyvinylpyrrolidone, glucose, sucrose, etc. The medium may also be a liquid such as a culture medium. It may also be a solution containing an appropriate blend of two or more of these cryoprotectants. Therefore, commercially available cryopreservation media, such as CELLBANKER® 1plus (Zenoac Resources), CELLBANKER® 1 (Zenoac Resources), and CELLBANKER® 2 (Zenoac Resources), may be used. Additionally, the liquid may contain sugars, salts, buffers, serum, vitamins, amino acids, electrolytes, antibiotics, growth factors (compounds, proteins).

[0024] Here, the liquid preferably contains saccharides in amounts of 7.5%, 9%, or 10% by mass, with preferred lower limits and 40%, 30%, or 20% by mass, based on the mass of the liquid. When the saccharide content is within this range, the dehydrating action of the cryoprotectant (e.g., DMSO) to expel water outside the cells works in opposition to the water-retaining action of the sugars that retain water inside the cells. If water is completely removed from the cells due to dehydration, the cells will be damaged. Conversely, if there is an abundance of water, the water will turn into ice crystals during freezing, damaging the cells. Therefore, a balance between dehydration and water retention is important. The sugars are not particularly limited, and examples thereof include monosaccharides such as glucose, fructose, galactose, mannose, ribose, and deoxyribose; disaccharides such as sucrose, maltose, and lactose; and polysaccharides such as acidic polysaccharides (e.g., carrageenan, pectin, gum arabic, xanthan gum, gellan gum, agar, and tragacanth gum), neutral polysaccharides (e.g., dextran, tamarind seed gum, guar gum, locust bean gum, and pullulan), and basic polysaccharides (e.g., chitosan). Sugars having one or multiple consecutive glucose backbones are preferred. The sugars may be of one or more types. These sugars may be quantified by well-known methods, such as the phenol-sulfuric acid method and high-performance liquid chromatography (HPLC) method.

[0025] As mentioned above, the liquid preferably contains a cryoprotectant. Dimethyl sulfoxide (DMSO) is particularly suitable as a cryoprotectant. DMSO is known to have relatively low cytotoxicity among organic solvents and high cell penetration. Because it dehydrates intracellular water and suppresses ice crystal formation during freezing, DMSO has long been widely used, along with glycerol, as a cryoprotectant for dispersed cells. However, due to its known cytotoxicity and its effect on the differentiation potential of undifferentiated cells, the use of DMSO substitutes or zwitterions has been proposed. However, renal cell aggregates, which are the target of this liquid, differ from dispersed cells in that they exhibit strong intercellular adhesion due to their aggregate formation and express polar aquaporins specific to renal cells. Therefore, solvents with low penetration such as substitutes or polar zwitterions are thought to be unable to penetrate cells and therefore do not function as cryoprotectants. Therefore, DMSO, which has high intracellular penetration and is non-polar, penetrates renal cell aggregates the most, thereby dehydrating the aggregates and enhancing their cryoprotective ability. Furthermore, the sugars used at the same time are known to have water-retaining properties, and by suppressing the formation of ice crystals inside the cells through dehydration with DMSO and maintaining appropriate levels of water outside the cells with sugars, cytotoxicity caused by dehydration is suppressed from the outside of the cells, excessive dehydration during cell thawing is suppressed, and damage to the cells is attenuated. Furthermore, based on the mass of the liquid, the lower limit of the DMSO concentration in the liquid is preferably 1% by mass, 2.5% by mass, or 5% by mass. The upper limit of the DMSO concentration in the liquid is preferably 30% by mass. The DMSO concentration in the liquid is more preferably 2.5 to 20% by mass, and particularly preferably 5 to 10% by mass.

[0026] Furthermore, it is preferable that the viscosity of the liquid be within a predetermined range. When the viscosity is within the predetermined range, during freezing of renal cell aggregates, dehydration by DMSO moderately suppresses ice crystal formation within the cell aggregates, while the viscosity of sugars prevents excessive dehydration outside the aggregates, thereby minimizing damage to the cells. During thawing, the viscosity of sugars prevents cell damage by retaining water from the extracellular space. If the viscosity is low, water retention is insufficient, resulting in excessive exposure to the dehydrating effect of DMSO, resulting in reduced cell morphology and viability. If the viscosity is high during thawing, the extracellular water content is high, making the extracellular space susceptible to damage from ice crystal formation during freezing, resulting in the collapse of the extracellular morphology upon thawing. Having the viscosity of the liquid within the predetermined range is preferable from the viewpoint of protecting cells from depletion of water due to dehydration by DMSO upon thawing. Specifically, the lower limit of the viscosity of the liquid is preferably 1 mPa·s, 1.5 mPa·s, 2.0 mPa·s, 2.5 mPa·s, 3.0 mPa·s, 3.5 mPa·s, 4.0 mPa·s, 4.5 mPa·s, or 5.0 mPa·s. The upper limit of the viscosity of the liquid is preferably 100 mPa·s, 90 mPa·s, 80 mPa·s, 70 mPa·s, 60 mPa·s, or 50 mPa·s. Here, the viscosity in this specification is a value measured by the following method. (Measurement Method) Viscosity was measured using a digital viscometer, manufacturer: Toki Sangyo Co., Ltd., model: TVB-10M. The digital viscometer was turned on and warmed up for 10 minutes, after which measurements were taken at 60 rpm using a low-viscosity M1 rotor for samples containing up to 10% by weight of sugar. For samples containing 10% by weight or more of sugar, a THM1 rotor was used and measurements were taken at 60 rpm. For samples containing 30% by weight or more of sugar, a THM1 rotor was used and measurements were taken at 30 rpm. For samples containing 40% by weight or more of sugar, a THM1 rotor was used and measurements were taken at 12 rpm. After confirming the stability and contact of each rotor used (contact was 20% or less), measurements were taken at room temperature (18 ° C) for 3 minutes.

[0027] <Liquid Amount> The volume ratio of the liquid to the volume of each well is not particularly limited, but suitable lower limits are 5%, 10%, and 15%, and suitable upper limits are 80%, 60%, and 40%. Within these ranges, it is possible to maintain a good cell condition while ensuring a high cell survival rate.

[0028] <Number in Well> The number of aggregates in a well may be one or more. Here, when there are more than one, the number is preferably 2 to 1000. For example, in a 6-well plate, an embodiment in which 500 to 1000 aggregates are arranged per well is envisioned.

[0029] <<Method for Producing Cell Aggregates>> Renal cells are cultured in a culture vessel and in a medium suitable for the cells to be cultured, according to a conventional method, for example, at 37°C and 5% CO ２ The culture can be carried out under these conditions. The culture may be static culture, shaking culture, or agitation culture. The culture may be adherent culture, but it is preferable to culture in a non-adherent state (e.g., suspension culture) for at least a portion of the period. Renal cells can form aggregates by culturing them in a non-adherent state to a culture vessel. The term "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 present away from the surface of the culture vessel, and a state in which, even if the cells are in contact with the surface of the culture vessel, they can easily detach from the surface of the culture vessel due to the coating of the culture vessel or convection of the medium without the use of tools, enzymes, etc.

[0030] For example, in some cases, renal cell aggregates are formed within 24 hours after the start of renal cell culture. By culturing renal cells in an aggregate state for a certain period of time, it is possible to restore the physiological function of renal cells that has been reduced due to dedifferentiation. The period for culturing renal cells in a non-adherent state to a culture vessel is generally 120 hours or longer. This allows for the production of cultured renal cells in a state where physiological function is more highly expressed. It is preferable to periodically change the medium during the culture period. For example, the medium is changed every two days.

[0031] Any known medium can be used as appropriate. For example, in the case of culturing proximal tubular epithelial cells, commercially available renal tubular cell culture media can be used, and preferred examples of the medium include REGM (registered trademark) (LONZA), EpiCM (registered trademark) (ScienCell), and Keratinocyte SFM (registered trademark) (Thermo Fisher Scientific).

[0032] 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 renal cells to each other. Therefore, culturing renal cells in a culture medium containing collagen I promotes the formation of aggregates. While full-length collagen I is preferred, it may also be the α1 chain or α2 chain that constitutes collagen I, or collagen peptides obtained by fragmenting each chain. Furthermore, the source of collagen I is not particularly limited, and it may be derived from humans or other animals.

[0033] Any culture vessel can be used. To promote aggregate formation, it is preferable that the vessel be subjected to a non- (low) cell-adhesive treatment or be made of a non- (low) cell-adhesive material. Examples of non- (low) cell-adhesive treatments include coating the vessel surface with a non-cell-adhesive hydrogel, coating with MPC (2-methacryloyloxyethyl phosphorylcholine), coating with ProteoSave (registered trademark) SS, and mirror polishing. Examples of non- (low) cell-adhesive materials include glass and polymeric materials such as low-density polyethylene, medium-density polyethylene, polyvinyl chloride, polyethylene-vinyl acetate copolymer, poly(ethylene-ethyl acrylate) copolymer, poly(ethylene-methacrylate) copolymer, poly(ethylene vinyl acetate) copolymer, and mixtures of two or more of these polymers. Aggregates cultured using a non- (low) cell-adhesive treatment can be transferred to another culture vessel for use. In this case, a culture vessel that has been subjected to a non- (low) cell-adhesive treatment or a cell-adhesive treatment can be used. The shape of the well bottom of the culture vessel that has been moved is not particularly limited, and examples thereof include a flat bottom, a V-bottom, and a U-bottom.

[0034] When forming a large amount of aggregates, a high-density spheroid-forming plate or dish can be used. Furthermore, if necessary, aggregates may be formed using a culture vessel such as a spinner flask. For example, it is preferable to use the ELPLASIA® series of culture vessels (Corning) or the EZSPHERE® series of culture vessels (AGC Technoglass). These culture vessels include 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 formed varies 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 subjected to low-adhesion treatment, one aggregate is formed per well.

[0035] Aggregates prepared using a high-density spheroid preparation plate or dish can be recovered and cultured in suspension with shaking. When performing suspension with shaking, it is preferable to culture the aggregates by placing a dish or plate that has been treated to prevent (or reduce) cell adhesion on a shaker. Reciprocating shakers and rotary shakers can be used as the shaker.

[0036] <<Freezing Method>> A method for producing a plate in a frozen state includes, for example, the steps of producing aggregates of renal cells and then freezing the aggregates while substantially maintaining the aggregated state of the aggregates, placing the frozen aggregates and a liquid into wells, and freezing the liquid in the wells. The step of freezing the aggregates is described in detail below.

[0037] The freezing of the aggregates is carried out by cooling them at a low temperature at which the cells can be frozen until they are frozen. Here, the freezing of renal cells is preferably carried out while adjusting the temperature drop rate of the cells to be frozen within a predetermined range. The temperature drop rate can be adjusted by using commercially available cryopreservation containers, programmable freezers that can set the freezing conditions for cells and tissues, or the like. The temperature drop rate of the cells during freezing can be set so as to minimize damage to the cells during freezing. For example, in the case of slow freezing, the temperature drop rate can be in the range of about 0.2°C to about 3°C ​​per minute, with a rate of about 1°C per minute being preferred.

[0038] Renal cells can be cooled using a general freezing method such as a freezer or liquid nitrogen. For example, when freezing aggregates of proximal tubular epithelial cells, an ultra-low temperature freezer that can be set to a temperature of preferably -80°C to -150°C, more preferably -100°C to -150°C, can be used. When storing in a liquid nitrogen storage vessel, the cells can be stored at a temperature of -196°C in the liquid phase and at a temperature of -150°C to -196°C in the gas phase.

[0039] "Substantially maintaining the aggregated state of the aggregates" in the freezing step means that the morphology of the aggregates is not damaged by not intentionally destroying or dispersing the aggregates contained in the collected renal cells (e.g., trypsin treatment). Furthermore, "substantially" means that the reduction in the amount of aggregates after freezing is within an insignificant range compared to the amount of aggregates contained in the collected renal cells before freezing, and does not necessarily mean that the cells that make up the aggregates are not dispersed at all during freezing.

[0040] Any step, such as adding a suitable liquid (the aforementioned cryopreservation medium) to the collected kidney cells, may be included between the collection and freezing steps, as long as it does not significantly adversely affect the maintenance of the aggregate morphology.

[0041] <Thawing> The conditions for thawing the cryopreservation solution are not particularly limited, and thawing can be carried out, for example, in an incubator heated to 37°C. After thawing, it is preferable to add a medium to maintain the viability of the renal cell aggregates. The medium addition method may be any of the following: (Step A) adding new fresh medium to the thawed cryopreservation solution; (Step B) adding new fresh medium in a frozen state; or (Step C) replacing the thawed cryopreservation solution with new fresh medium. However, of these, (Step A) and (Step B) involve adding new fresh medium to the well, which allows for a 100% cell survival rate. On the other hand, (Step C) carries the risk of losing cells during medium replacement due to the viscosity of the frozen medium.

[0042] <<Applications>> The plate containing the cell aggregates according to this embodiment can be provided as a drug evaluation system or a cell product. Examples of drug evaluation systems include systems for evaluating pharmacokinetics in renal cells and nephrotoxicity. Furthermore, such renal cells can be used to analyze the mechanisms of kidney-related diseases such as diabetes, kidney cancer, and hyperuricemia, and as a tool for discovering therapeutic drugs.

[0043] The present disclosure is not limited to the above-described embodiments, and various modifications such as design changes may be made based on the knowledge of those skilled in the art, and embodiments with such modifications are also included in the scope of the present disclosure.

[0044] <<Production Example>> <Production of Cell Aggregates>> Human proximal tubular epithelial cells (Clonetics (registered trademark), catalog number CC-2553, RPTEC - renal proximal tubular epithelial cells) obtained from LONZA were used as kidney cells. A frozen vial stored in a liquid nitrogen storage vessel was immersed in a 37°C incubator and thawed. After thawing, the cell suspension in the frozen vial was mixed with the recommended medium (REGM (registered trademark), LONZA) and cultured in a culture dish. The cells were incubated at 37°C and 5% CO ２ The cells were cultured under these conditions, with the medium being changed every two days. The cells were harvested before they reached confluence and seeded onto a 96-well V-bottom plate (PrimeSurface® Plate 96V, Sumitomo Bakelite Co., Ltd.) treated with low cell adhesion to form aggregates at a predetermined number of cells per well (1,000 cells unless otherwise specified). The aggregates were cultured with the medium being changed every two days. "Confluent" refers to a state in which the ratio of the cell area to the entire culture surface of the culture vessel is approximately 100%, i.e., cells have proliferated over the entire culture surface without any gaps.

[0045] <Preparation of a Freezing Plate Containing Cell Aggregates> A predetermined amount (predetermined ratio relative to the well volume) of a cryopreservation solution containing a predetermined concentration of DMSO and a predetermined concentration of sugar (dextran in this example) was placed into each well of a 96-well V-bottom plate (PrimeSurface® Plate 96V, Sumitomo Bakelite Co., Ltd.), and then the cell aggregates (frozen bodies) prepared above were added. The viscosity of the cryopreservation solution according to the examples is shown in Table 1. The specific gravity of the cell aggregates relative to the medium was 1.00 to 1.07. The volume ratio of the contents (liquid + aggregates) to the well volume was 48%. The volume ratio of aggregates to liquid in the well was 0.013%. The plate was then frozen and stored in an ultra-low temperature freezer capable of being set to -80°C.

[0046]

[0047] <Thawing of frozen plate> The plate was removed from the ultra-low temperature freezer, which can be set to a temperature of -80°C, and left to stand in an incubator heated to 37°C for 1 to 3 minutes. Once the periphery of the plate had thawed slightly and the cryopreservation solution had not completely thawed, 100 μl of fresh medium, which had been heated to 37°C in a thermostatic bath, was added to each well to thaw the plate.

[0048] <Measurement of Viability> After thawing, the cell aggregates (96 cells) were left to stand for one week with repeated medium changes every 2-3 days, and the ATP level was measured by luminescence using the CellTiter-Glo (registered trademark) 3D Cell Viability Assay (Promega). Specifically, the aggregates were collected together with the medium, and an amount of CellTiter-Glo 3D Reagent equal to the volume of the medium was added. This mixture was incubated at room temperature for 30 minutes. After thorough mixing, the luminescence value was measured using a microplate reader (Perkin Elmer). The ATP level was similarly measured for the cell aggregates before freezing and used as a control.

[0049] <Measurement of Cell Condition> After thawing, the cell aggregate groups (96 cells) were allowed to stand for one week, with the medium replaced every two to three days. The state of each aggregate in the well was observed and photographed using a phase-contrast microscope BZ-X710 (Keyence). The photographed images of the cell aggregates were classified into A, B, and C based on the cell disintegration shown in Figure 2, and evaluated by assigning a score based on the evaluation criteria shown in Table 2. The total score for each group frozen in the same cryopreservation solution was calculated and converted to a score out of 100. For example, if one group contains 10 cells and includes 4 cells of type A, 3 cells of type B, and 3 cells of type C, the total score for that group is 4 x 1 + 3 x 0.5 + 3 x 0 = 5.5. Converted to a score out of 100, the score is 100 x (5.5 ÷ 10) = 55 points.

[0050]

[0051] <Results> <Effect of DMSO concentration> Figure 3 shows the difference in viability depending on the DMSO concentration when sugar is at a constant concentration (relative values ​​based on the cell aggregates before freezing (control)). As can be seen from Figure 3, high viability of cell aggregates was confirmed regardless of the DMSO concentration. In particular, an extremely high viability of cell aggregates was confirmed at DMSO concentrations of 1.0 to 30% by mass (2.5 to 20% by mass in the data shown in the figure).

[0052] <Effect of cryopreservation solution volume> Figure 4 shows the state (score) of cell aggregates after thawing when the volume of cryopreservation solution and sugar concentration are varied. As can be seen, compared to the examples with sugar concentrations of 0% and 7% by mass, the examples with higher sugar concentrations show higher scores regardless of the volume of cryopreservation solution. In Figure 3, the volume of cryopreservation solution ranging from 40 μl to 100 μl corresponds to a volume of cryopreservation solution of 15% to 40% of the well volume.

[0053] <Effect of amount of culture medium added> Figure 5 shows the results of adding various amounts of culture medium to a liquid containing frozen cell aggregates (sugar concentration: 10% by mass), thawing the frozen state, and then measuring the cell viability (relative values ​​based on the cell aggregates before freezing (control)). As can be seen from the figure, it can be confirmed that almost all of the cell aggregates survive, regardless of the amount of culture medium added.

[0054] <Effect of cell number in cell aggregates> Figure 6 is a diagram showing the viability of cell aggregates (relative values ​​based on 100 μl) when the number of cells in the cell aggregates and the amount of cryopreservation solution were changed under the same conditions (DMSO concentration: 10% by mass / sugar concentration: 10% by mass). As can be seen from the figure, it was confirmed that the cell aggregates were sufficiently viable regardless of the number of cells in the cell aggregates and the amount of cryopreservation solution, and that a higher viability was achieved when the amount of cryopreservation solution was higher than 100 μl (volume ratio of cryopreservation solution to well volume: 40%).

[0055] <Summary> The freezing plates according to the examples were able to maintain a high survival rate, good cell condition, and favorable morphological parameters even after thawing.

[0056] <Long-Term Cryopreservation Test> Next, to evaluate the effects of long-term cryopreservation on aggregates, we conducted the following test. The medium was aspirated from each well of a 96-well V-bottom plate containing the cell aggregates prepared in the above Preparation Example, and 100 μl (40% of the well volume) of a cryopreservation solution (viscosity: 4.26 mPas) containing 10% by mass of DMSO and 10% by mass of sugar (dextran) was added to each well. The specific gravity of the cell aggregates relative to the medium was 1.00 to 1.07. The volume ratio of the contents (liquid + aggregates) to the well volume was 48%. The volume ratio of aggregates to liquid in the well was 0.013%. The plate was then cryopreserved by placing it in an ultra-low temperature freezer capable of being set to a temperature of -120°C. The same procedures as above were performed, except that cryopreservation was performed using a cryopreservation solution containing 10% by mass of DMSO and 7% by mass of sugar (dextran). Comparisons were also made. After 1 month, 6 months, and 9 months of cryopreservation, the plates were removed from the ultra-low temperature freezer and thawed as described above. The ATP level was then measured as described above in <Measurement of Viability>. The state of the cell aggregates was also measured as described above in <Measurement of Cell State>.

[0057] The results are shown in Figures 7 and 8. Figure 7 shows the difference in viability after long-term cryopreservation when the sugar concentration was changed (relative values ​​based on the cell aggregates (control) before freezing). As can be seen from Figure 7, when the sugar concentration in the cryopreservation solution was less than 7.5% by mass, the viability of the cell aggregates tended to decrease as the cryopreservation period increased, whereas when the sugar concentration in the cryopreservation solution was 7.5% by mass or higher, extremely high viability of the cell aggregates was confirmed even for cryopreservation periods of 9 months or longer. Figure 8 shows the difference in the state (score) of the cell aggregates after long-term cryopreservation when the sugar concentration was changed. As can be seen from Figure 8, when the sugar concentration in the cryopreservation solution was less than 7.5% by mass, the score tended to decrease as the cryopreservation period increased, whereas when the sugar concentration in the cryopreservation solution was 7.5% by mass or higher, extremely high scores were confirmed for all cryopreservation periods. CROSS-REFERENCE TO RELATED APPLICATIONS

[0058] This application claims priority based on Japanese Patent Application No. 2024-072475, filed with the Japan Patent Office on April 26, 2024, the entire disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A plate having a plurality of wells, wherein a liquid containing renal cell aggregates is contained in a frozen state in the wells, and the liquid in the frozen state contains 7.5 to 40% by mass of sugars based on the mass of the liquid in the frozen state.

2. The plate according to claim 1, wherein the liquid in a frozen state further contains 1 to 30% by mass of dimethyl sulfoxide based on the mass of the liquid.

3. The plate of claim 1, wherein the volume of the liquid in the well in a frozen state is 5 to 80% of the well volume.

4. The plate according to claim 1, wherein the number of cells in the renal cell aggregate is 100 to 10,000 cells.

5. A pharmacokinetic test kit comprising the plate described in any one of claims 1 to 4.

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

7. A kit for drug discovery and development, comprising the plate described in any one of claims 1 to 4.

8. A plate having a plurality of wells, wherein a liquid containing renal cell aggregates is contained in a frozen state in the wells, and the viscosity of the liquid is 1 mPa·s to 100 mPa·s.

9. The plate according to claim 8, wherein the liquid in a frozen state further contains 1 to 30% by mass of dimethyl sulfoxide based on the mass of the liquid in a frozen state.

10. The plate of claim 8, wherein the volume of the liquid in the well in a frozen state is 5 to 80% of the well volume.

11. The plate according to claim 8, wherein the number of cells in the renal cell aggregate is 100 to 10,000 cells.

12. A pharmacokinetic test kit comprising the plate according to any one of claims 8 to 11.

13. A toxicity testing kit comprising the plate according to any one of claims 8 to 11.

14. A kit for drug discovery and development, comprising the plate described in any one of claims 8 to 11.

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