A plate containing frozen kidney cell aggregates, a kit for pharmacokinetic examination using the plate, a kit for toxicity examination, and a kit for drug development

Abstract

An object of the present disclosure is to provide a method of inhibiting deterioration of a cell state when freezing a cryopreservation solution containing cell aggregates of kidney cells. The present disclosure provides a plate having a plurality of wells, characterized in that a liquid containing cell aggregates of kidney cells is housed in a frozen state in the wells, and the liquid in a frozen state contains 7.5 to 40 mass% of a saccharide based on the mass of the liquid in a frozen state.

Description

Technical Field

[0001] This disclosure relates to plates containing frozen kidney cell aggregates, pharmacokinetic assay kits using these plates, toxicity assay kits, and drug development kits. Background Technology

[0002] When a drug is administered to a living organism, it is absorbed into the body and excreted from the blood into the urine via the proximal renal tubules. Therefore, drug nephrotoxicity frequently causes kidney damage. In drug development research, investigating pharmacokinetics in the kidneys is crucial for clarifying drug action. Based on this, there is a desire to develop a drug development aid that can utilize renal cells to evaluate pharmacokinetics and toxicity. Furthermore, such a drug development aid would also be useful for developing therapeutics for kidney-related diseases such as diabetes, renal cell carcinoma, and hyperuricemia.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 2021-191305 Summary of the Invention

[0006] The problem that the invention aims to solve

[0007] Here, renal cell aggregates (e.g., Patent Document 1) are proposed as a useful technique for confirming pharmacokinetics and drug development. Moreover, for drugs that are test subjects, cell aggregates of multiple samples (typically plates with multiple wells and cell aggregates arranged in each well) are typically used to confirm and evaluate pharmacokinetics, toxicity, and efficacy.

[0008] Here, from the viewpoint of reducing the load on cell aggregates during transportation and improving operability, the inventors investigated a method of loading cryopreservation solution containing renal cell aggregates into each well of a plate and then freezing it together with the plate. During this process, the inventors observed that when using conventional cryopreservation solutions for freezing, the renal cell aggregates exhibit cell deterioration. Therefore, the objective of this disclosure is to provide a method for inhibiting cell deterioration when freezing cryopreservation solutions containing renal cell aggregates.

[0009] Methods for solving problems

[0010] One aspect of this disclosure is a plate having multiple holes, characterized in that...

[0011] The aforementioned pore contains a liquid containing kidney cell aggregates in a frozen state.

[0012] The above-mentioned liquid in a frozen state contains 7.5 to 40% by mass of sugars, based on the mass of the above-mentioned liquid in a frozen state.

[0013] Here, the above-mentioned liquid in a frozen state may further contain 1 to 30% by mass of dimethyl sulfoxide based on the mass of the above-mentioned liquid.

[0014] In addition, the volume of the liquid in the above-mentioned hole that is in a frozen state can be 5% to 80% of the hole volume.

[0015] In addition, the number of cells in the above-mentioned renal cell aggregates can be more than 100 cells and less than 10,000 cells.

[0016] Alternatively, it could be a pharmacokinetic testing kit containing the aforementioned plate.

[0017] Alternatively, it could be a toxicity testing kit containing the aforementioned plates.

[0018] Alternatively, it could be a kit for developing innovative drugs that includes the aforementioned plates.

[0019] One aspect of this disclosure is a plate having multiple holes, characterized in that...

[0020] The aforementioned pore contains a liquid containing kidney cell aggregates in a frozen state.

[0021] The viscosity of the above liquid is 1 mPa·s to 100 mPa·s.

[0022] Here, the liquid in the frozen state may further contain 1 to 30% by mass of dimethyl sulfoxide based on the mass of the liquid in the frozen state.

[0023] In addition, the volume of the liquid in the above-mentioned hole that is in a frozen state can be 5% to 80% of the hole volume.

[0024] In addition, the number of cells in the above-mentioned renal cell aggregates can be more than 100 cells and less than 10,000 cells.

[0025] Alternatively, it could be a pharmacokinetic testing kit containing the aforementioned plate.

[0026] Alternatively, it could be a toxicity testing kit containing the aforementioned plates.

[0027] Alternatively, it could be a kit for developing innovative drugs that includes the aforementioned plates.

[0028] Invention Effects

[0029] According to this disclosure, it is possible to inhibit the deterioration of cell state when freezing cryopreservation solutions containing cell aggregates of kidney cells. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the plate used in this method.

[0031] Figure 2 This is a diagram (photograph) showing an evaluation standard example of the state of cells after thawing.

[0032] Figure 3 This is a graph showing the difference in survival rates due to different DMSO concentrations when the sugar concentration is constant.

[0033] Figure 4 This is a graph showing the state (score) of thawed cell aggregates under varying cryopreservation volume and sugar concentration.

[0034] Figure 5 The figure shows the results of measuring cell viability after adding various amounts of culture medium to a frozen liquid containing cell aggregates (sugar concentration: 10% by mass) and thawing the frozen state.

[0035] Figure 6 This is a graph showing the survival rate of cell aggregates under the same conditions (DMSO concentration: 10% by mass / sugar concentration: 10% by mass) with varying cell number and cryopreservation volume (relative values ​​based on 100 μl).

[0036] Figure 7 This is a graph showing the differences in survival rates after long-term cryopreservation under varying sugar concentrations.

[0037] Figure 8 This is a graph showing the differences in the state (score) of cell aggregates after long-term cryopreservation under varying sugar concentrations. Detailed Implementation

[0038] <<Plate containing kidney cell aggregates (before freezing)>>

[0039] The plate of this method has multiple wells. Furthermore, each well contains a cryopreservation medium and an aggregate of one or more kidney cells located within that cryopreservation medium. For example, Figure 1 This is a culture container for a well plate, which is an example of this method. For example... Figure 1 As shown on the left (top and bottom), the culture vessel has a well plate (96 wells, V-bottom shown in the figure) and a well plate cap. Additionally, as described later, as... Figure 1As shown on the right, the cryopreservation medium and condensate are contained within the wells to a degree that does not completely fill them, and an air layer exists between the upper surface of the cryopreservation medium and the well plate cap. The following details each element. The surface of the culture vessel can be covered by a covering member (e.g., a membrane, such as a sheet membrane), in which case an air layer exists between the upper surface of the cryopreservation medium and the covering member. Preferably, the water vapor transmission rate of the covering member (measured according to JIS Z0208 at a temperature of 40°C and a humidity of 90% RH) is 500 g / m³. 2 / 24hr or more, preferably 2000g / m 2 / 24hr or more, the optimal value is 4000g / m 2 / 24hr or more. It should be noted that the upper limit for water vapor transmission rate is, for example, 100,000 g / m³. 2 / less than 24hr, 50000g / m 2 / less than 24hr, 25000g / m 2 / less than 24hr, 10000g / m 2 / 24hr or less. Additionally, the preferred covering member has a pore size of 10μm to 50μm. It should be noted that the covering member can be used to cover all the pores within the plate in one piece, or it can be divided into multiple pore groups and covered according to each pore group, or it can be covered individually.

[0040] <Kidney cell aggregates>

[0041] The kidney cells used in this method can be cultured regardless of their source. Kidney cells are preferably derived from mammals, particularly primates such as humans and monkeys. However, depending on the purpose, kidney cells can be derived from normal kidneys or diseased kidneys. Examples of kidney cells include those that constitute the epithelium, cortex, proximal tubules, distal tubules, collecting ducts, and glomeruli; specifically, examples include proximal tubular epithelial cells (RPTEC) and mesangial cells. Kidney cells can be primary cells or stem cell-derived kidney cells such as iPS cells or ES cells. Furthermore, kidney cells can be immortalized kidney cells, lineage cells (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 with genes introduced to express specific transport proteins or other proteins. More specifically, examples of kidney cells include, for instance, 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 induced from differentiation of human iPS cells or human ES cells. For use in drug development studies, proximal tubular epithelial cells, particularly those derived from normal human kidneys, are preferred.

[0042] Here, a cell "agglomerate" is a clump-like collection of several or more cells. It is also called an agglomerate or spheroid. The number of cells constituting the agglomerate is, for example, 5 or more, 25 or more, 50 or more, preferably 100 or more, more preferably 125 or more, further preferably 200 or more, and particularly preferably 500 or more. The number of cells constituting the agglomerate is, for example, 4000 or less, preferably 10000 or less, more preferably 2000 or less, or 1000 or less. If the number of cells constituting the agglomerate is within this range, almost all the cells in the culture medium remain viable, achieving higher homogeneity among the multiple agglomerates within the plate.

[0043] (morphological parameters)

[0044] As morphological parameters, we can cite the diameter, volume, cross-sectional area, perimeter, density, roundness, and aspect ratio of an aggregate. The following sections will detail each parameter.

[0045] The diameter of the condensate is preferably 100 μm or more and 800 μm or less. It should be noted that the diameter of the condensate is defined as its maximum width. That is, the diameter of the condensate is the length of the longest straight line connecting two points on the outer edge of the condensate. The diameter of the condensate can be measured, for example, using a photograph taken with a phase-contrast microscope based on a known method. A BZ-X710 (Keyence Corporation) phase-contrast microscope can be used, and analysis software can be used for diameter measurement.

[0046] The volume of the aggregate is preferably, for example, 0.001 mm. 3 Above, 0.300mm 3 The volume of the condensate can be calculated from the measured diameter, since the condensate is approximately spherical.

[0047] The cross-sectional area of ​​the condensate is preferably 40,000 μm. 2 Above and 100,000 μm 2 Below, 45000μm is more preferred. 2 Above and 90000μm 2 Below. Here, the cross-sectional area (and also the perimeter, compactness, roundness, and aspect ratio described below) is calculated based on known methods using CQ1 (confocal imaging cytometer, Yokogawa electrophysiology) to identify an image {slice image (cross-sectional view)} of an aggregate.

[0048] The perimeter of the condensate is preferably 600 μm or more and 1800 μm or less, more preferably 800 μm or more and 1600 μm or less.

[0049] In addition, the compactness of the aggregate is preferably 1.0 or higher and 3.0 or lower, more preferably 1.2 or higher and 2.5 or lower.

[0050] In addition, the roundness 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.

[0051] 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.

[0052] (Coefficient of variation of ATP)

[0053] 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%.

[0054] (Coefficient of variation of morphological parameters)

[0055] The coefficient of variation of the cross-sectional area of ​​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 of 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 of 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 roundness of 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 of the multiple cell aggregates is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less.

[0056] <board>

[0057] Plates containing renal cell aggregates are not particularly limited in design as long as each well can hold one or more aggregates; any type of plate can be used. Examples of culture containers include 6-well plates, 24-well plates, 48-well plates, 96-well plates, 384-well plates, and various sizes of dishes. Furthermore, the shape of the bottom is not particularly limited; flat bottoms, V-shaped bottoms, and U-shaped bottoms are available. For example, the well volume of a 96-well plate is typically 280–310 mm³. 3 (For example, the PrimeSurface 96V manufactured by Sumitomo Bakelite is approximately 310mm thick.) 3 The EZ BindShut SP 96-hole plate made by AGC Techno Glass is approximately 280mm in diameter. 3 ).

[0058] <Liquid>

[0059] The medium constituting the liquid (cryopreservation medium) within each well is not particularly limited; it can be the culture medium used for preparing aggregates, fresh culture medium, or a liquid other than a culture medium (e.g., physiological saline, buffer solution, etc.). Furthermore, this liquid preferably contains one or more cryoprotectant components that mitigate damage caused by intracellular ice crystals, such as dimethyl sulfoxide (DMSO), mammalian serum, dextran, glycogen, methylcellulose or carboxymethylcellulose, polyethylene glycol, polyvinylpyrrolidone, glucose, sucrose, etc. Alternatively, the medium can be a culture medium or other liquid. It can also be a solution appropriately formulated with two or more of these cryoprotectants. Therefore, commercially available cryopreservation media such as CELLBANKER 1plus (Zenoaq Resource), CELLBANKER 1 (Zenoaq Resource), and CELLBANKER 2 (Zenoaq Resource) can be used. In addition, the liquid may also contain sugars, salts, buffers, serum, vitamins, amino acids, electrolytes, antibiotics, and growth factors (compounds, proteins).

[0060] Here, the liquid is used as a reference, with the preferred lower limit being 7.5% by mass, 9% by mass, or 10% by mass of sugars, and the preferred upper limit being 40% by mass, 30% by mass, or 20% by mass of sugars. If the sugar content is within this range, the dehydrating effect of the cryoprotectant (e.g., DMSO), which causes water to be expelled from the cell, works antagonistically with the water-retaining effect of the sugars. If dehydration leads to complete loss of intracellular water, it will damage the cell; conversely, if the cell is rich in water, the water will crystallize during freezing, damaging the cell. Therefore, the balance between dehydration and water retention becomes important. The term "sugar" is not particularly limited, and examples 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 astragalus gum), neutral polysaccharides (e.g., dextran, tamarind gum, guar gum, locust bean gum, and pullulan), and basic polysaccharides (e.g., chitosan). Sugars having one or more consecutive glucose backbones are preferred. Furthermore, the sugar can be one or more types. Additionally, known methods can be considered for the quantification of these sugars, such as methods using the phenol-sulfuric acid method or high-performance liquid chromatography (HPLC).

[0061] Furthermore, as mentioned above, the liquid preferably contains a cryoprotectant. Dimethyl sulfoxide (DMSO) is particularly preferred as this cryoprotectant. DMSO is known to have low cytotoxicity and high cell permeability among organic solvents. Because it can remove intracellular water and inhibit ice crystal formation during freezing, DMSO has long been widely used, along with glycerol, as a cryoprotectant for dispersed cells. However, it is known that DMSO is cytotoxic in dispersed cells and affects differentiation ability in undifferentiated cells; therefore, the use of DMSO substitutes, zwitterions, etc., is encouraged. However, the renal cell aggregates targeted by this liquid differ from dispersed cells. Due to the formation of aggregates, strong cell adhesion develops, and renal cells express polar aquaporins specific to them. Therefore, it is considered that solvents with low permeability, such as substitutes, and polar zwitterions cannot penetrate into the cells and will not function as cryoprotectants. Thus, DMSO, with its high intracellular permeability and non-polarity, is most likely to penetrate into the renal cell aggregates, dehydrating them and thereby improving cryoprotection. Furthermore, the sugars used simultaneously are known to have water-retaining capabilities. By inhibiting ice crystal formation intracellularly through dehydration using DMSO and moderately retaining water extracellularly, cytotoxicity caused by dehydration is suppressed, excessive dehydration during cell thawing is inhibited, and damage to cells is reduced. Moreover, 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.

[0062] Furthermore, the viscosity of the liquid is preferably within a given range. If the viscosity is within this range, during the freezing of kidney cell aggregates, ice crystal formation can be moderately suppressed within the cell aggregates through dehydration by DMSO, and excessive dehydration can be suppressed outside the aggregates by the viscosity generated by sugars, thereby inhibiting cell damage. During thawing, water retention from the extracellular space is achieved through the viscosity generated by sugars, thereby suppressing cell damage. If the viscosity is low, water retention is insufficient, and the cells are excessively affected by the dehydration effect of DMSO, resulting in damage to cell morphology and viability. If the viscosity is high during thawing, the extracellular water content is high, making it susceptible to damage from ice crystal formation during freezing, thus causing morphological collapse of the extracellular space during thawing. From the viewpoint of protecting cells from water depletion caused by DMSO dehydration during cell thawing, it is preferable that the liquid has a viscosity within the given range. 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 determined by the method described below.

[0063] (Determination Method)

[0064] Viscosity was measured using a TVB-10M digital viscometer manufactured by Toki Sangyo Co., Ltd. After powering on the digital viscometer and allowing it to preheat for 10 minutes, samples with a sugar content up to 10% by mass were measured at 60 rpm using the M1 rotor for low viscosity. For samples containing 10% or more sugar, the THM1 rotor was used at 60 rpm; for samples containing 30% or more sugar, the THM1 rotor was used at 30 rpm; and for samples containing 40% or more sugar, the THM1 rotor was used at 12 rpm. After confirming the stability and contact properties (contact ≤ 20%) of each rotor used, measurements were performed for 3 minutes at room temperature (18°C).

[0065] <Quantity of liquid>

[0066] There is no particular limitation on the volume ratio of liquid to volume in each well; however, the preferred lower limit is 5%, 10%, and 15%, and the preferred upper limit is 80%, 60%, and 40%. Within this range, a better cell state can be maintained while ensuring high cell viability.

[0067] <Number of holes>

[0068] The number of aggregates within the well is one or more. Here, when there are multiple aggregates, it is preferable to have two or more but less than 1000. For example, in a 6-well plate, it is envisioned that they be arranged in a configuration of 500 to 1000 per well.

[0069] <<Methods for Manufacturing Cell Aggregates>>

[0070] Kidney cells can be cultured using a suitable culture medium and culture vessel, following conventional methods, such as at 37°C and 5% CO2. The culture can be static, shaking, or stirred. It can also be adherent, however, it is preferred that at least a portion of the culture be performed in a non-adherent state (e.g., suspension culture). Kidney cells can form aggregates by culturing them in a non-adherent state in a culture vessel. The term "non-adherent state" refers to a state where all or most of the cells are not attached to the surface of the culture vessel, including: a state where all or most of the cells are detached from the surface of the culture vessel; and a state where, even when in contact with the surface of the culture vessel, the cells can easily detach from the surface without the use of instruments, enzymes, etc., through coating of the culture vessel, convection of the culture medium, etc.

[0071] For example, in some cases, renal cell aggregates form within 24 hours of the start of renal cell culture. Furthermore, by culturing renal cells in an aggregate state for a portion of the time, the physiological function of renal cells that has been reduced due to dedifferentiation can be restored. The period for culturing renal cells in a non-adherent state in the culture vessel is generally preferably 120 hours or more. This allows for the acquisition of cultured renal cells with a higher expression of physiological function. During culture, the culture medium is preferably changed regularly. For example, the culture medium should be changed every 2 days.

[0072] As a culture medium, any known culture medium may be appropriately used. For example, in the case of culturing proximal renal tubular epithelial cells, commercially available renal tubular cell culture medium may be used. Examples of preferred culture media include REGM (registered trademark) (LONZA), EpiCM (registered trademark) (ScienCell), and Keratinocyte SFM (registered trademark) (Thermo Fisher Scientific).

[0073] In addition, conventionally known materials and additives useful for cell culture can be appropriately used. For example, collagen I (type I collagen) can be added to the culture medium. Collagen I has the function of promoting adhesion between kidney cells. Therefore, by culturing kidney cells in a culture medium containing collagen I, aggregate formation can be promoted. Collagen I is preferably full-length collagen I, but it can also be the α1 chain or α2 chain that constitutes collagen I, or more specifically, collagen peptides obtained by fragmenting the chains. Furthermore, the source of collagen I is not particularly limited and can be from humans or other animals.

[0074] Any culture container can be used. Here, to promote aggregate formation, it is preferable to perform a cell non-adhesion (or low-adhesion) treatment or to use a cell non-adhesion (or low-adhesion) material. Examples of cell non-adhesion (or low-adhesion) treatments include cell non-adhesion hydrogel coating, MPC (2-methacryloyloxyethyl phosphorylcholine) coating, Proteo Save (registered trademark) SS coating, and mirror polishing. Examples of cell non-adhesion (or low-adhesion) materials include glass, and polymers 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 the cell non-adhesion (or low-adhesion) treatment can be transferred to another culture container for use. In this case, culture containers that have undergone cell non-adhesion (or low-adhesion) treatment or cell adhesion treatment can be used. In addition, there are no particular limitations on the shape of the bottom of the culture container used after transfer; examples include flat bottom, V-bottom, and U-bottom.

[0075] When forming a large number of agglomerates, high-density spherical plates or dishes can be used. Alternatively, culture vessels such as spinnerets can be used as needed to form agglomerates. For example, culture vessels from the ELPLASIA (registered trademark) series (Corning Corporation) and the EZSPHERE (registered trademark) series (AGC Techno Glass Corporation) are preferred. These culture vessels are available in 6-well plates, 24-well plates, 96-well plates, 384-well plates, and various sizes of dishes; the number of agglomerates that can be formed varies depending on the bottom area of ​​the vessel. For example, when using 96-well plates (V-bottom), 96-well plates (U-bottom), and 384-well plates (U-bottom) that have undergone low-adhesion treatment, one agglomerate is formed in each well.

[0076] Aggregates prepared using high-density spherical plates or dishes can be recovered and then cultured in suspension with shaking. When performing suspension with shaking culture, it is preferable to place the aggregates in a dish or plate that has undergone cell non-adhesion (or low-adhesion) treatment on a shaker. Reciprocating shakers and rotary shakers can be used as shakers.

[0077] <<Freezing Method>>

[0078] A method for manufacturing a plate in a frozen state includes, for example, a step of freezing a kidney cell aggregate while substantially maintaining the aggregate's aggregated state, a step of adding the frozen aggregate and liquid into a well, and a step of freezing the liquid in the well. The freezing step of the aggregate will be described in detail below.

[0079] The freezing of condensates is performed by cooling them to a low temperature at which cells can be frozen until the cells are frozen. Here, the freezing of kidney cells is preferably carried out while the rate of temperature decrease of the cells to be frozen is adjusted to a given range. The rate of temperature decrease can be adjusted using commercially available cryopreservation containers, programmable freezers capable of setting freezing conditions for cells and tissues, etc. The rate of temperature decrease of the cells during the freezing process can be set while minimizing damage to the cells during freezing. For example, the rate of temperature decrease during slow freezing can be in the range of about 0.2°C to about 3°C ​​per minute, preferably about 1°C per minute.

[0080] Cooling of renal cells can be performed using conventional freezing methods with a cryostat or liquid nitrogen. For example, in the case of cryopreservation of proximal tubular epithelial cells, an ultra-low temperature cryostat that can preferably be set to -80°C to -150°C, more preferably -100°C to -150°C, can be used. When storing in a liquid nitrogen container, storage can be performed at a temperature of -196°C in the liquid phase and -150°C to -196°C in the gas layer.

[0081] The phrase "substantially maintaining the aggregated state" in the freezing process means that no actions are taken to intentionally destroy or disperse the aggregates contained in the recovered kidney cells (such as trypsin treatment), thus preserving the morphology of the aggregates. Furthermore, "substantially" means that the reduction in the amount of aggregates after freezing is within a non-problematic range compared to the amount of aggregates contained in the recovered kidney cells before freezing; it does not necessarily mean that the dispersion of the cells constituting the aggregates will not occur at all during the freezing process.

[0082] It should be noted that, as long as it does not significantly adversely affect the maintenance of the morphology of the aggregates, any step may be included between the recovery and freezing steps, such as other steps that are beneficial to cell cryopreservation. For example, it may include the step of adding a suitable liquid (the aforementioned cryopreservation medium) to the recovered kidney cells.

[0083] <Thawing>

[0084] There are no particular limitations on the thawing conditions of the cryopreservation solution; for example, it can be carried out in an incubator heated to 37°C. Furthermore, after thawing, it is preferable to add culture medium to maintain the viability of the kidney cell aggregates. The method of adding the culture medium can be any of the following: (Step A) adding fresh culture medium to the thawed cryopreservation solution; (Step B) adding fresh culture medium while the solution is frozen; or (Step C) replacing the thawed cryopreservation solution with fresh culture medium. However, regarding (Step A) and (Step B), since fresh culture medium is added to the wells, 100% cell survival rate can be achieved. On the other hand, regarding (Step C), due to the viscosity of the cryopreservation solution, there is a risk of cell loss during replacement.

[0085] <<Applications>>

[0086] Plates containing cell aggregates prepared in this manner can be provided as drug evaluation systems or cell products. As drug evaluation systems, examples include systems for evaluating pharmacokinetics and nephrotoxicity in kidney cells. Furthermore, such kidney cells can also be used as tools for mechanism analysis and therapeutic drug discovery in kidney-related diseases such as diabetes, kidney cancer, and hyperuricemia.

[0087] This disclosure is not limited to the above-described embodiments. Various design changes and modifications can be made based on the knowledge of those skilled in the art, and such modified embodiments are also included within the scope of this disclosure.

[0088] Example

[0089] Manufacturing Example

[0090] <The Manufacturing of Cell Conglomerates>

[0091] Human proximal renal tubular epithelial cells obtained from LONZA (Clonetics, registered trademark, catalog number CC-2553, RPTEC - Proximal Renal Tubular Epithelial Cells) were used as kidney cells. Frozen vials stored in liquid nitrogen were thawed by immersion in a 37°C incubator. After thawing, the cell suspension in the frozen vials was mixed with the recommended culture medium (REGM, registered trademark, LONZA) and cultured in petri dishes. Cells were cultured at 37°C and 5% CO2, with the medium changed every two days. Cells were recovered before reaching confluence and seeded and cultured in 96-well V-type plates (PrimeSurface, registered trademark, Sumitomo Bakelite) treated with low cell adhesion to reach a given number of cells per well (1000 unless otherwise specified) to form aggregates. Aggregates were cultured with the medium changed every two days. It should be noted that the term "convergence" refers to a state in which the proportion of the area occupied by the cells relative to the entire culture surface of the culture container is approximately 100%, that is, a state in which the cells have proliferated to the point of filling the entire culture surface without any gaps.

[0092] <Manufacturing of Frozen Plates Containing Cell Aggregates>

[0093] In each well of a 96-well V-type plate {PrimeSurface (registered trademark) 96V plate, Sumitomo Bakelite, Inc.}, a given amount (in a given proportion relative to the well volume) of cryopreservation solution containing a given concentration of DMSO and a given concentration of sugar (dextran in this example) was added, followed by the addition of the cell aggregates (cryocysts) prepared above. It should be noted that the viscosity of the cryopreservation solution in this example is shown in Table 1. At this time, the specific gravity of the cell aggregates relative to the culture medium was 1.00 to 1.07. Furthermore, the volume ratio of the contents (liquid + aggregates) to the volume of the well was 48%. In addition, the volume ratio of the aggregates to the liquid in the well was 0.013%. Subsequently, the plates were cryopreserved by placing them in a cryostat that could be set to -80°C.

[0094]

[0095] <Thawing of Frozen Plates>

[0096] Remove the plate from the cryostat that can be set to -80°C, and let it stand in an incubator heated to 37°C for about 1 to 3 minutes. While the edges of the plate are slightly thawed and the cryopreservation solution is not completely thawed, add 100 μl of fresh culture medium heated to 37°C in a constant temperature bath to each well and thaw it.

[0097] <Survival rate determination>

[0098] After thawing, ATP levels in 96 cell aggregates that had been subjected to a 2-3 day culture medium change and then left to stand for one week were determined using a luminescence assay with the CellTiter-Glo (registered trademark) 3D Cell Viability Assay (Promega). Specifically, the cell aggregates were recovered along with the culture medium, and an equal volume of CellTiter-Glo 3D Reagent was added. The mixture was incubated at room temperature for 30 minutes. After thorough mixing, the luminescence values ​​were measured using a microplate reader (Perkin Elmer). It should be noted that ATP levels were also measured in the unfrozen cell aggregates as a control group.

[0099] <Determination of Cell State>

[0100] After thawing, the cell aggregates (96 aggregates) were observed and photographed within the wells using a phase-contrast microscope (BZ-X710, Keyence) after the culture medium was changed every 2-3 days and the cells were left to stand for one week. The images of the cell aggregates are shown below. Figure 2 The cells are categorized into A, B, and C based on their degree of cell breakdown, and scored according to the evaluation criteria shown in Table 2. The total score for each group frozen in the same cryopreservation solution is calculated and then converted to a maximum score of 100. For example, in a group of 10 cells, with 4 cells in group A, 3 cells in group B, and 3 cells in group C, the total score for this group is 4 × 1 + 3 × 0.5 + 3 × 0 = 5.5. Converted to a maximum score of 100, the score is 100 × (5.5 ÷ 10) = 55.

[0101] [Table 2]

[0102]

[0103] ≪Results≫

[0104] <The Effect of DMSO Concentration>

[0105] Figure 3 This is a graph showing the difference in survival rates due to different DMSO concentrations at a constant sugar concentration (relative values ​​based on cell aggregates before freezing (control group)). From Figure 3 It can be seen that high cell aggregate survival can be confirmed regardless of the DMSO concentration. In particular, extremely high cell aggregate survival rates can be confirmed at DMSO concentrations of 1.0–30% by mass (the data shown in the figure are 2.5–20% by mass).

[0106] <The Influence of Cryopreservation Liquid Volume>

[0107] Figure 4 This graph shows the state (score) of thawed cell aggregates under varying cryopreservation solution volume and sugar concentration. As can be seen, examples with higher sugar concentrations consistently show higher scores regardless of cryopreservation solution volume, compared to examples with 0% and 7% sugar. It should be noted that... Figure 3 The volume of cryopreservation solution in the solution is 40 μl to 100 μl, which is equivalent to 15 to 40% of the volume of cryopreservation solution relative to the pore volume.

[0108] <Effect of Culture Medium Addition Amount>

[0109] Figure 5 This graph shows the results of measuring cell viability after adding various amounts of culture medium to a frozen liquid containing cell aggregates (sugar concentration: 10% by mass) and thawing the liquid (relative values ​​compared to the cell aggregates before freezing (control group)). The graph shows that almost all cell aggregates survived regardless of the amount of culture medium added.

[0110] <The Influence of Cell Number on Cell Aggregates>

[0111] Figure 6 This graph shows the cell aggregate survival rate (relative values ​​based on 100 μl) under the same conditions (DMSO concentration: 10% by mass / sugar concentration: 10% by mass) with varying cell numbers and cryopreservation solution volumes. The graph shows that cell aggregates are sufficiently viable regardless of cell number or cryopreservation solution volume; however, higher survival rates are observed when the cryopreservation solution volume exceeds 100 μl (cryopreservation solution volume ratio relative to well volume: 40%).

[0112] <Summary>

[0113] For the frozen plate in the embodiment, even after thawing, it can maintain a high survival rate, good cell state, and preferred morphological parameters.

[0114] Long-term cryopreservation experiment

[0115] Next, to evaluate the effect of long-term cryopreservation on the cell aggregates, we conducted the following experiment. The culture medium was aspirated from each well of a 96-well V-plate containing the cell aggregates prepared in the above-described manufacturing example, and 100 μl (40% of the well volume) of a cryopreservation solution (viscosity: 4.26 mPa·s) containing 10% by mass DMSO and 10% by mass sugar (dextran) was added to each well. It should be noted that the specific gravity of the cell aggregates relative to the culture medium was 1.00–1.07. Furthermore, the volume ratio of the well contents (liquid + aggregates) to the volume was 48%. Additionally, the volume ratio of the aggregates to the liquid in the well was 0.013%. Subsequently, the plates were cryopreserved by placing them statically in a cryostat capable of being set to -120°C. The same treatment as above was performed, except that cryopreservation was performed using a cryopreservation solution containing 10% by mass DMSO and 7% by mass sugar (dextran), and the results were compared. After 1 month, 6 months, and 9 months of cryopreservation, the plates were removed from the cryogenic freezer and thawed using the methods described above. Subsequently, the ATP content was measured using the methods described in the "Determination of Viability" section above. Additionally, the state of cell aggregates was determined using the methods described in the "Determination of Cell State" section above.

[0116] The results are expressed as Figure 7 , Figure 8 middle. Figure 7 This is a graph showing the differences in survival rates after long-term cryopreservation under varying sugar concentrations (relative values ​​based on pre-freezing cell aggregates (control group)). (By...) Figure 7 It can be seen that when the sugar concentration in the cryopreservation solution is less than 7.5% by mass, the survival rate of cell aggregates tends to decrease as the cryopreservation period increases. However, when the sugar concentration in the cryopreservation solution is above 7.5% by mass, even if the cryopreservation period is more than 9 months, the survival rate of cell aggregates can be confirmed to be extremely high. Figure 8 This is a graph showing the differences in the state (score) of cell aggregates after long-term cryopreservation under varying sugar concentrations. Figure 8 It can be seen that when the sugar concentration in the cryopreservation solution is less than 7.5% by mass, the score shows a decreasing trend as the cryopreservation period increases. However, when the sugar concentration in the cryopreservation solution is 7.5% by mass or higher, it can be confirmed that the score is extremely high under any cryopreservation period conditions.

[0117] Cross-referencing of related applications

[0118] 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.

Claims

1. A plate having a plurality of holes, characterized in that, The pore contains a liquid containing kidney cell aggregates in a frozen state. The liquid in its frozen state contains 7.5% to 40% by mass of sugars based on the mass of the liquid in its 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 according to claim 1, wherein, The volume of the frozen liquid in the pore is 5% to 80% of the pore volume.

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

5. A pharmacokinetic assay kit comprising the plate according to 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 reagent kit for developing innovative drugs, comprising the plate according to any one of claims 1 to 4.

8. A plate having a plurality of holes, characterized in that, The pore contains a liquid containing kidney cell aggregates in a frozen state. The viscosity of the liquid is 1 mPa·s to 100 mPa·s.

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

10. The plate according to claim 8, wherein, The volume of the frozen liquid in the pore is 5% to 80% of the pore volume.

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

12. A pharmacokinetic assay 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 reagent kit for developing innovative drugs, comprising the plate according to any one of claims 8 to 11.

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