Solution for immersing frozen-thawed three-dimensional cell culture for transplantation, solution production method, frozen-thawed product of three-dimensional cell culture for transplantation, production method for obtaining frozen-thawed product, method for using solution, and kit for transplantation

Abstract

The present invention provides a solution for immersing a three-dimensional cell culture for transplantation, the solution comprising: one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, and the like; and an aqueous solvent, wherein said solution satisfies at least one among the following (a)-(c), and has an osmotic pressure of 150-1,200 mOsm / L. (a) When calcium salts are contained, the content thereof is 5-700 ppm in terms of calcium ions. (b) When magnesium salts are contained, the content thereof is 5-1,000 ppm in terms of magnesium ions. (c) When other divalent metal salts are contained, the content thereof is 0.01-100 ppm in terms of metal ions.

Description

A solution for immersing frozen and thawed three-dimensional cell cultures for transplantation, a method for producing the solution, frozen and thawed three-dimensional cell cultures for transplantation, a method for producing the frozen and thawed products, a method for using the solution, and a transplantation kit.

[0001] This invention relates to a solution, etc., for improving the cell viability or metabolic activity rate of three-dimensional cell cultures for transplantation after freeze-thawing.

[0002] In the medical field, including regenerative medicine, there has been rapid progress in recent years in cell transplantation technology, which involves creating three-dimensional cell cultures, such as cell sheets formed from cells in a sheet-like structure, and transplanting them to the affected area for the repair and improvement of damaged tissue. In such cell transplantation, it is necessary to maintain good cell viability and preserve the three-dimensional cell culture for a long period of time after culturing and proliferating the cells until the cells are transplanted to the patient. One method for preserving such three-dimensional cell cultures is to cryopreserve them in the presence of a cryopreservation solution containing cryoprotective agents, etc. (Patent Document 1). However, there is a problem that the cell viability decreases after thawing cryopreserved cells, so a method to improve the cell viability after thawing is needed. As such a method, Patent Document 2 discloses a method to improve cell viability by diluting a cell suspension obtained by thawing cryopreserved cells with a buffer containing human-derived albumin and optionally inorganic salts, amino acids, vitamins, etc.

[0003] Japanese Patent Publication No. 2022-8269, International Publication No. 2021 / 065971

[0004] As mentioned above, there is a problem in that the cell viability of three-dimensional cell cultures for transplantation decreases after freezing and thawing, and there is still a need for suitable methods to improve cell viability after thawing.

[0005] The inventors of the present invention diligently studied methods for improving the cell viability or metabolic activity rate of three-dimensional cell cultures for transplantation after cryopreservation. As a result, they discovered that immersing the three-dimensional cell cultures for transplantation in a solution containing a specific concentration of divalent metal salt and having a specific osmotic pressure after freezing and thawing improves the cell viability or metabolic activity rate, leading to the completion of the present invention.

[0006] In other words, the present invention relates to the following:

[0007] The following are examples of reference forms. 1. A solution for immersing a frozen-thawed three-dimensional cell culture for transplantation, wherein the solution comprises one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and an aqueous solvent, satisfying at least one of the following (a) to (c), and having an osmotic pressure of 150 mOsm / L or more and 1200 mOsm / L or less. (a) If the calcium salt is included, the content of the calcium salt is 5 ppm or more and 700 ppm or less in terms of calcium ions, based on the total weight of the solution. (b) If the magnesium salt is included, the content of the magnesium salt is 5 ppm or more and 1000 ppm or less in terms of magnesium ions, based on the total weight of the solution. (c) If the divalent metal salt other than the calcium salt and the magnesium salt is included, the content of the other divalent metal salt is 0.01 ppm or more and 100 ppm or less in terms of metal ions, based on the total weight of the solution. 2. The solution according to 1, wherein the aqueous solvent is physiological saline. 3. The solution according to 1, wherein the aqueous solvent is buffer. 4. 1. to 3. A solution according to any one of the above, wherein at least one of the culture medium essential amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine is not contained, or its concentration (mg / L) in the solution is less than or equal to the specified values ​​shown in (i) to (xi) below.(i) L-histidine ≤ 20 mg / L (ii) L-isoleucine ≤ 40 mg / L (iii) L-leucine ≤ 40 mg / L (iv) L-lysine ≤ 50 mg / L (v) L-methionine ≤ 10 mg / L (vi) L-phenylalanine ≤ 20 mg / L (vii) L-threonine ≤ 7 mg / L (viiii) L-valine ≤ 40 mg / L (ix) L-arginine ≤ 40 mg / L (x) L-cystine ≤ 20 mg / L (xi) L-tyrosine ≤ 10 mg / L 5. 4. A solution as described in (1), wherein the solution contains two or more amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine, and the total concentration of two or more essential amino acids belonging to the group contained in the solution is 300 mg / L or less. 6. A solution as described in (4) or (5), wherein the essential amino acid of the culture medium contains at least one of (ii) L-isoleucine at 40 mg / L or less, (iii) L-leucine at 40 mg / L or less, (iv) L-lysine at 50 mg / L or less, and (ix) L-arginine at 40 mg / L or less. 7. A solution as described in any one of (1) to (6), which is a non-processing solution for cells. 8. (1) to (7) A solution according to any one of the above, wherein the solution substantially does not contain animal-derived components. 9. A solution according to any one of 1 to 8, wherein the solution substantially does not contain cryoprotective agents. 10. A solution according to any one of 1 to 9, wherein the three-dimensional cell culture is a cell sheet, a spheroid, an organoid, or a combination thereof. 11. A solution according to 10, wherein the cell sheet is on a scaffold or culture carrier substrate. 12. A solution according to any one of 1 to 11, wherein the cells in the three-dimensional cell culture are cells derived from mammals. 13. A solution according to 12, wherein the cells derived from mammals are selected from the group consisting of living tissue cells, mesenchymal stem cells, and pluripotent stem cells, at least one of these.14. A method for producing a solution according to any one of 1 to 13, comprising a compounding step of obtaining the solution by adding one or more inorganic salt powders selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts to an aqueous solvent, or by adding a solution obtained by dissolving the inorganic salt powder in a liquid to an aqueous solvent. 15. A method for producing a solution according to 14, wherein in the compounding step, 50% by volume or more of basal culture medium is not added to 100% by volume of the solution. 16. A frozen-thawed three-dimensional cell culture for transplantation comprising a solution according to any one of 1 to 13, and a three-dimensional cell culture immersed in the solution. 17. 16. A frozen-thawed product of a three-dimensional cell culture for transplantation as described in 17, wherein a cryoprotective agent is mixed in the solution. 18. A frozen-thawed product of a three-dimensional cell culture for transplantation as described in 17, wherein the concentration of the cryoprotective agent is 7 w / v% or more based on the volume of the solution. 19. A frozen-thawed product of a three-dimensional cell culture for transplantation as described in 17, wherein the concentration of the cryoprotective agent is less than 0.5 w / v% based on the volume of the solution. 20. A frozen-thawed product of a three-dimensional cell culture for transplantation as described in 16, wherein the three-dimensional cell culture is a cell sheet, and the cell sheet is on a scaffold or culture carrier substrate. 21. A method for producing a frozen-thawed three-dimensional cell culture for transplantation, comprising: (a) a step of preparing a frozen three-dimensional cell culture for transplantation; and (b) a step of thawing the three-dimensional cell culture and immersing the thawed three-dimensional cell culture in any one of the solutions described in 1. to 13.22. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation as described in 21, comprising: (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container; and (b) thawing the three-dimensional cell culture and the cryopreservation solution, removing the thawed cryopreservation solution from the container, then adding a solution described in any one of 1 to 13 to the container, and immersing the three-dimensional cell culture in the solution. 23. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation as described in 21, comprising: (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container; and (b) thawing the three-dimensional cell culture and the cryopreservation solution, removing the thawed three-dimensional cell culture from the container, and then immersing it in a solution described in any one of 1 to 13 in another container. 24. 21 to 23. A method for producing a frozen-thawed three-dimensional cell culture for transplantation according to any one of the above, wherein in step (b), the three-dimensional cell culture is immersed in the solution for at least 30 seconds. 25. A method for producing a frozen-thawed three-dimensional cell culture for transplantation according to any one of the above, wherein in step (b), the three-dimensional cell culture is thawed at 0 to 45°C. 26. A method for producing a frozen-thawed three-dimensional cell culture for transplantation according to any one of the above, wherein the three-dimensional cell culture is a cell sheet, and the cell sheet is on a scaffold or culture carrier substrate. 27. A method for using the solution according to any one of the above, wherein the three-dimensional cell culture is a cell sheet, and the cell sheet is on a scaffold or culture carrier substrate. 28. A transplantation kit comprising a frozen three-dimensional cell culture for transplantation and the solution according to any one of the above,

[0008] According to the present invention, by immersing a frozen-thawed three-dimensional cell culture for transplantation in a solution containing a specific concentration of a divalent metal salt and having a specific osmotic pressure, a good cell viability rate or metabolic activity rate can be achieved for the cells in the frozen-thawed three-dimensional cell culture for transplantation. Furthermore, the preparation of the above solution is very simple, requiring only a simple operation of immersing the three-dimensional cell culture for transplantation in the solution after thawing, thus reducing implementation costs. Moreover, it is optional to include cytotoxic animal-derived components in the solution, which would be more preferable from a therapeutic standpoint.

[0009] (A) This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in solutions of the present invention having various calcium salt concentrations. (B) This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in solutions of the present invention having various magnesium salt concentrations. This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in solutions of the present invention having various osmotic pressures. This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in phosphate-buffered saline (PBS(-)), cryopreservation solution, and solutions of the present invention over various time periods. This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in solutions of the present invention, basal media without animal-derived components, and basal media containing animal-derived components. This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in solutions of the present invention or various control solutions or liquids. This figure shows the metabolic activity rate of cells when three-dimensional cultures for transplantation, after freeze-thawing, are immersed in solutions of the present invention containing various concentrations of DMSO.

[0010] In one embodiment of the present invention, a solution for immersing a freeze-thawed three-dimensional cell culture for transplantation is provided, the solution comprising one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and an aqueous solvent, satisfying at least one of the following (a) to (c), and having an osmotic pressure of 150 mOsm / L or more and 1200 mOsm / L or less (hereinafter referred to as "the solution of the present invention"). (a) If calcium salts are included, the calcium salt content shall be 5 ppm or more and 700 ppm or less in terms of calcium ions, based on the total weight of the solution. (b) If magnesium salts are included, the magnesium salt content shall be 5 ppm or more and 1000 ppm or less in terms of magnesium ions, based on the total weight of the solution. (c) If other divalent metal salts other than calcium salts and magnesium salts are included, the content of other divalent metal salts shall be 0.01 ppm or more and 100 ppm or less in terms of metal ions, based on the total weight of the solution. By immersing the three-dimensional cell cultures for transplantation in the solution of the present invention during and / or after thawing, the viability of the cells after thawing can be improved. The detailed mechanism is not clear, but in an environment with low osmotic pressure, such as distilled water, water flows excessively into the cells, making it impossible to maintain cell morphology. On the other hand, in a high osmotic pressure environment, water flows out of the cells, resulting in a dehydrated state. Therefore, it is presumed that the solution used to immerse the three-dimensional cell culture reduces stress on the cells by maintaining an appropriate osmotic pressure environment, and contributes to improving cell viability after thawing through the action of divalent metal salts.

[0011] As used herein, "freezing" means a solid state in which ice crystals are not formed inside and outside the cells, or preferably a small amount of ice crystals are formed inside and outside the cells. Also, as used herein, "freezing treatment" refers to a treatment for freezing cells. Typically, it refers to placing a three-dimensional cell culture for transplantation in a cryopreservation solution in an environment below the freezing temperature of the cryopreservation solution to freeze the cells present in the three-dimensional cell culture for transplantation. Also, "cryopreservation" means storing the frozen cells for a certain period (including either a long period or a short period). Therefore, as used herein, "freezing" is used as a broad term representing the state in which cells are frozen and includes "cryopreservation".

[0012] As used herein, "thawing" refers to thawing frozen cells. Typically, it is to place a three-dimensional cell culture for transplantation frozen together with a cryopreservation solution in an environment above the freezing temperature of the cryopreservation solution to thaw the cells in the three-dimensional cell culture for transplantation. In practice, when the frozen cryopreservation solution is thawed and in a liquid state (this confirmation may be done visually), it is considered that the cells in the three-dimensional cell culture for transplantation are thawed. In this specification, the term "thawing" can be used interchangeably with the term "defrosting", and includes not only the case where all of the cryopreservation solution is in a liquid state after thawing, but also the case where a part of the cryopreservation solution is in a liquid state.

[0013] As used herein, "immersing" means a state in which the solution of the present invention is in contact with the three-dimensional cell culture for transplantation. In addition to the form of immersing or dipping all or part of the three-dimensional cell culture for transplantation in a solution containing the solution of the present invention, it may also be achieved in a form such that the solution of the present invention is added to the cryopreservation solution remaining after thawing the three-dimensional cell culture for transplantation to dilute the cryopreservation solution, or it may be achieved in a form such that the solution of the present invention is poured into the cryopreservation solution of the three-dimensional cell culture for transplantation and washed away.

[0014] The calcium salts, magnesium salts, and other divalent metal salts contained in the solution are at least partially or almost completely dissociated in water, and calcium ions (Ca ２＋ ), magnesium ions (Mg ２＋), and exist in ionic state as other divalent metal ions. The content of calcium salt in terms of calcium ion conversion means the concentration of calcium ions. Similarly, for the content of magnesium salt and the content of divalent metal salt, when converted to ions, they respectively mean the concentration of magnesium ions and the concentration of divalent metal ions. Hereinafter, the content of calcium salt, the content of magnesium salt, and the content of other divalent metal salts may also be referred to as calcium ion concentration, magnesium ion concentration, and other divalent metal ion concentration, respectively. "Content based on the total weight of the solution" means a value obtained by converting the ratio of the components contained in the total amount in terms of mass with the total mass (100% by mass) of the solution as the reference. The calcium ion concentration, magnesium ion concentration, and other divalent metal ion concentration in the solution may be calculated from the raw material ratio, and if it is in the state before freezing or after freeze-thawing, it can be measured by ion chromatography. In addition, when calculating from the raw material ratio, the calcium ion concentration, magnesium ion concentration, and other divalent metal ion concentration may be calculated on the premise that all the salts present in the solution are dissociated into ions. For example, taking calcium salt as an example, when both calcium phosphate (Ca ３ (PO ４ ) ２ ) and calcium chloride (CaCl ２ ) are contained, it is assumed that calcium ions (Ca ２＋ ) and phosphate ions (PO ４ ３－ ) are completely dissociated, and further calcium ions (Ca ２＋ ) and chloride ions (Cl － ) are completely dissociated, and the calcium ions (Ca ２＋The one with the adjusted concentration is defined as the concentration of the calcium salt contained in the solution of the present invention. Also, the "calcium salt" may be a calcium salt such as hydrochloric acid, nitric acid, sulfuric acid, etc., and for example, calcium chloride or the like is added. The "magnesium salt" may be a magnesium salt such as hydrochloric acid, nitric acid, sulfuric acid, etc., and for example, magnesium sulfate or the like is added. For other divalent metal salts, divalent metal salts of anions such as hydrochloric acid, nitric acid, sulfuric acid, ammonia, etc. and other cations such as copper ions, manganese ions, iron ions, molybdenum ions, nickel ions, cobalt ions, etc. can be used.

[0015] <Ion Chromatography Analysis> An example of the measurement method by ion chromatography for calcium ions, magnesium ions, and ions of other divalent metal salts in a solution is as follows. Regarding calcium ions and magnesium ions in the cryopreservation solution, they are measured by ion chromatography (ICS2100, manufactured by Thermo Fisher Scientific). The measurement sample is one from which proteins have been removed by ultrafiltration, and is measured in the matrix illumination (MI) mode. Ion chromatography is carried out with the following parameters. Separation column: Ion Pac CS16 (inner diameter 4 mm × 250 mm) Guard column: Ion Pac CG16 (inner diameter 4 mm × 250 mm) Top column: TCC-LP1 (inner diameter 4 mm × 35 mm) Suppressor: CDRS600 (external mode) Column temperature: 35 °C Detector: Electrical conductivity Eluent flow rate: 1.0 mL / min Eluent: 30 mM methanesulfonic acid aqueous solution MI conditions: Eluent; water, MI flow rate; 1 mL / min, MI liquid feeding time; -1 to 0 min Sample introduction volume: 25 μL Calibration curve: Prepared by arbitrarily diluting a cation mixed standard solution II (07197-96, manufactured by Kanto Chemical Co., Inc.) with ultrapure water

[0016] The calcium salt concentrations of 5 to 700 ppm mentioned above include any values ​​within that numerical range, in increments of 0.1 ppm or 1 ppm. Therefore, the lower limit of the calcium salt concentration is, for example, based on the total weight of the solution, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 The values ​​may be selected from 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500. The lower limit of the calcium salt concentration is more preferably 50 ppm or higher. Furthermore, the upper limit of the calcium salt concentration is set such that the lower limit < upper limit, for example, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, You may choose from 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 ppm. Therefore, the concentrations of calcium salts are, for example, 5-700, 5-650, 5-600, 5-550, 5-500, 5-450, 5-400, 5-350, 5-300, 5-250, 5-200, 5-150, 5-100 ppm; 10-700, 10-650, 10-600, 10-550, 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100 ppm;20~700、20~650、20~600、20~550、20~500、20~450、20~400、20~350、20~300、20~250、20~200、20~150、20~100ppm;30~700、30~650、30~600、30~550、30~500、30~450、30~400、30~350、30~300、30~250、30~200、30~150、30~100ppm;40~700、40~650、40~600、40~550、40~500、40~450、40~400、40~350、40~300、40~250、40~200、40~150、40~100ppm;50~700、50~650、50~600、50~550、50~500、50~450、50~400、50~350、50~300、50~250、50~200、50~150、50~100ppm;60~700、60~650、60~600、60~550、60~500、60~450、60~400、60~350、60~300、60~250、60~200、60~150、60~100ppm;70~700、70~650、70~600、70~550、70~500、70~450、70~400、70~350、70~300、70~250、70~200、70~150、70~100ppm;80~700、80~650、80~600、80~550、80~500、80~450、80~400、80~350、80~300、80~250、80~200、80~150、80~100ppm;90~700、90~650、90~600、90~550、90~500、90~450、90~400、90~350、90~300、90~250、90~200、90~150、90~100ppm;100~700、100~650、100~600、100~550、100~500、100~450、100~400、100~350、100~300、100~250、100~200、100~150ppm;150~700、150~650、150~600、150~550、150~500、150~450、150~400、150~350、150~300、150~250、150~200ppm;Alternatively, 200-700, 200-650, 200-600, 200-550, 200-500, 200-450, 200-400, 200-350, 200-300, or 200-250 ppm may be used.

[0017] Furthermore, the above magnesium salt concentrations of 5 to 1000 ppm include any value within that numerical range, in increments of 0.1 ppm or 1 ppm. Therefore, the lower limit of the magnesium salt concentration is, for example, based on the total weight of the solution, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 6 The magnesium salt concentration may be selected from 6, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, or 600 ppm. The lower limit of the magnesium salt concentration is preferably 10 ppm or higher, and more preferably 50 ppm or higher. The upper limit of the magnesium salt concentration is set such that the lower limit < upper limit, for example, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 5 The concentrations may be selected from 30, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 ppm. Therefore, the magnesium salt concentration can be, for example, 5-1000, 5-950, 5-900,5~850、5~800、5~750、5~700、5~650、5~600、5~550、5~500、5~450、5~400、5~350、5~300、5~250、5~200、5~150、5~100ppm;10~1000、10~950、10~900、10~850、10~800、10~750、10~700、10~650、10~600、10~550、10~500、10~450、10~400、10~350、10~300、10~250、10~200、10~150、10~100ppm;20~1000、20~950、20~900、20~850、20~800、20~750、20~700、20~650、20~600、20~550、20~500、20~450、20~400、20~350、20~300、20~250、20~200、20~150、20~100ppm;30~1000、30~950、30~900、30~850、30~800、30~750、30~700、30~650、30~600、30~550、30~500、30~450、30~400、30~350、30~300、30~250、30~200、30~150、30~100ppm;40~1000、40~950、40~900、40~850、40~800、40~750、40~700、40~650、40~600、40~550、40~500、40~450、40~400、40~350、40~300、40~250、40~200、40~150、40~100ppm;50~1000、50~950、50~900、50~850、50~800、50~750、50~700、50~650、50~600、50~550、50~500、50~450、50~400、50~350、50~300、50~250、50~200、50~150、50~100ppm;60~1000、60~950、60~900、60~850、60~800、60~750、60~700、60~650、60~600、60~550、60~500、60~450、60~400、60~350、60~300、60~250、60~200、60~150、60~100ppm;70~1000、70~950、70~900、70~850、70~800、70~750、70~700、70~650、70~600、70~550、70~500、70-450, 70-400, 70-350, 70-300, 70-250, 70-200, 70-150, 70-100ppm; 80-1000, 80-950, 80-900, 80-850, 80-800, 80-750, 80-700, 80-650, 80-600, 80-550, 80-500, 80-450, 80-400, 80-350, 80-300, 80-250, 80-200, 80-150, 80-1 00ppm; 90-1000, 90-950, 90-900, 90-850, 90-800, 90-750, 90-700, 90-650, 90-600, 90-550, 90-500, 90-450, 90-4 00, 90-350, 90-300, 90-250, 90-200, 90-150, 90-100ppm; 100-1000, 100-950, 100-900, 100-850, 100-800, 100-750 , 100-700, 100-650, 100-600, 100-550, 100-500, 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-15 0ppm; 150-1000, 150-950, 150-900, 150-850, 150-800, 150-750, 150-700, 150-650, 150-600, 150-550, 150-500, 1 50-450, 150-400, 150-350, 150-300, 150-250, 150-200 ppm; or 200-1000, 200-950, 200-900, 200-850, 200-800, 200-750, 200-700, 200-650, 200-600, 200-550, 200-500, 200-450, 200-400, 200-350, 200-300, 200-250 ppm.

[0018] The concentrations of other divalent metal ions may be one concentration selected from the group consisting of copper ions, manganese ions, zinc ions, iron ions, molybdenum ions, nickel ions, and cobalt ions, or the sum of two or more concentrations.

[0019] The copper ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm increments within these numerical ranges. Alternatively, the lower limit of the copper ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the copper ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0020] The manganese ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm within these numerical ranges. Alternatively, the lower limit of the manganese ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the manganese ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0021] The zinc ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm increments within these numerical ranges. Alternatively, the lower limit of the zinc ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the zinc ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0022] The iron(II) ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm within these numerical ranges. Alternatively, the lower limit of the iron(II) ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the iron(II) ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0023] The molybdenum(II) ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm increments within these numerical ranges. Alternatively, the lower limit of the molybdenum(II) ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the molybdenum(II) ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0024] The nickel ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm increments within these numerical ranges. Alternatively, the lower limit of the nickel ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the nickel ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0025] The cobalt(II) ion concentration, based on the total weight of the solution, is 0.01 ppm or higher, 0.02 ppm or higher, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, and 0.02 to 5 ppm, and also includes concentration ranges between any values ​​of 0.01 ppm, 0.1 ppm, or 1 ppm increments within these numerical ranges. Alternatively, the lower limit of the cobalt(II) ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any values ​​between these values ​​in increments of 0.01 ppm or 0.1 ppm) based on the total weight of the solution, and the upper limit of the cobalt(II) ion concentration may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ppm (including any values ​​between these values ​​in increments of 0.1 ppm or 1 ppm).

[0026] The solution may further contain calcium salts, magnesium salts, and inorganic salts other than the divalent metal ions mentioned above. Examples of inorganic salts include metal salts, which include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (excluding calcium salts and magnesium salts). The metal salts may be divalent or trivalent metal salts. Specifically, they can be used as metal salts of hydrochloric acid, nitric acid, sulfuric acid, etc., and examples include potassium chloride and sodium chloride. These can be added separately from the metal salts contained in the cell culture medium. The type and concentration of the metal salt can be used as appropriate to be advantageous for cell survival, and although there are no limitations on the concentration, it can be used in the range of 0.01 to 10000 ppm, preferably 1 to 1000 ppm.

[0027] The solution of the present invention has an osmotic pressure of 200 to 1000 mOsm / L, and the osmotic pressure value includes any value within that numerical range in increments of 1 mOsm / L or 10 mOsm / L. Therefore, the lower limit of the osmotic pressure is, for example, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520. , 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 may be selected, and the upper limit of osmotic pressure shall be set on the condition that the lower limit < the upper limit, for example ba, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, You may choose from 670, 680, 690, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 mOsm / L.Therefore, the osmotic pressure is, for example, 200-1000, 200-950, 200-900, 200-850, 200-800, 200-750, 200-700, 200-650, 200-600, 200-550, 200-500, 200-450, 200-400, 200-350, 200-300 mOsm / L; 250-1000, 250-95 0, 250-900, 250-850, 250-800, 250-750, 250-700, 250-650, 250-600, 250-550, 250-500, 250- 450, 250-400, 250-350, 250-300mOsm / L; 300-1000, 300-950, 300-900, 300-850, 300-800, 300- 750, 300-700, 300-650, 300-600, 300-550, 300-500, 300-450, 300-400, 300-350mOsm / L; 350- 1000, 350-950, 350-900, 350-850, 350-800, 350-750, 350-700, 350-650, 350-600, 350-550, 35 The osmotic pressure may be 0-500, 350-450, 350-400 mOsm / L; or 400-1100, 400-1050, 400-1000, 400-950, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, or 400-450 mOsm / L. The lower limit of the osmotic pressure is preferably 250 mOsm / L or higher, more preferably 300 mOsm / L or higher, and the upper limit of the osmotic pressure is more preferably less than 800 mOsm / L, and even more preferably less than 500 mOsm / L.

[0028] The osmotic pressure of a solution can be measured using an osmometer at room temperature (25°C). If the solution does not contain organic solvents such as DMSO, the osmotic pressure may be calculated from the ion concentration in the solution.

[0029] Methods for achieving the desired osmotic pressure described above are known in the fields of organic and biochemistry and can typically be achieved by adding inorganic salts. Such inorganic salts include metal salts, such as alkali metal salts (sodium salts, potassium salts, etc.) or alkaline earth metal salts. The metal salts may be divalent or trivalent. Specifically, they are used as metal salts of hydrochloric acid, nitric acid, etc., such as sodium chloride and potassium chloride.

[0030] In one embodiment, the solution may contain physiological saline as an aqueous solvent, or it may contain a buffer solution. That is, the method for producing the solution (solution production method) may include a compounding step of obtaining the solution by adding one or more inorganic salt powders selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts to an aqueous solvent, or by adding a solution obtained by dissolving the inorganic salt powder in a liquid such as water or a buffer solution to an aqueous solvent. Furthermore, the solution production method of this embodiment can be configured so that no basal culture medium is added in the compounding step. Specifically, in the compounding step, for example, 50% or more, 40% or more, 30% or more, 20% or more, 10% or more, or 5% or more of basal culture medium per 100% by volume of the solution may not be added. In this way, by including a step of adding inorganic salt powder or its solution according to the desired metal ion concentration, it becomes possible to produce the solution stably and efficiently.

[0031] In one embodiment, the solution of the present invention can be prepared using physiological saline or a buffer solution. Physiological saline is an aqueous solution of sodium chloride (NaCl) having approximately the same osmotic pressure as human blood or tissue fluid. A buffer solution is a solution that maintains the pH within a certain range in an aqueous solution and has a buffering effect against acids or bases added from the outside. Known buffer solutions include, but are not limited to, carbonate buffers (sodium carbonate, sodium bicarbonate), phosphate-buffered physiological saline (phosphoric acid, sodium phosphate, potassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, dihydrogen phosphate, trisodium phosphate, dipotassium dihydrogen phosphate, tripotassium phosphate), Tris, Good's buffers (HEPES, MES, PIPES, etc.), citrate buffer (trisodium citrate), acetate buffer (sodium acetate, potassium acetate), borate buffer (sodium borate, sodium tetraborate), tartrate buffer (sodium tartrate), amino acid buffers (histidine, taurine, aspartic acid), etc. Combinations of two or more buffer solutions can also be used.

[0032] In one embodiment, the solution may not contain at least one of the culture medium essential amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine, or its concentration (mg / L) in the solution may be less than or equal to the specified values ​​shown in (i) to (xi) below. (i) L-histidine less than 20 mg / L (ii) L-isoleucine less than 40 mg / L (iii) L-leucine less than 40 mg / L (iv) L-lysine less than 50 mg / L (v) L-methionine less than 10 mg / L (vi) L-phenylalanine less than 20 mg / L (vii) L-threonine less than 7 mg / L (viiii) L-valine less than 40 mg / L (ix) L-arginine less than 40 mg / L (x) L-cystine less than 20 mg / L (xi) L-tyrosine less than 10 mg / L

[0033] Furthermore, the solution may have a form that does not contain the essential amino acids of the culture medium (hereinafter referred to as Form I). In Form I, the solution may be composed of a composition that does not contain at least one of the following 11 essential amino acids of the culture medium used in the cell culture medium: L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine. Here, "does not contain" means that the content of the amino acid is below the detection limit. The solution may also be composed of a composition that does not contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the above 11, and it may contain the essential amino acids of the culture medium that are not specified as not to be contained.

[0034] Furthermore, the solution may have an embodiment (hereinafter referred to as Embodiment II) in which at least one of the culture medium essential amino acids listed in (i) to (xi) above is present in the solution at a concentration (mg / L) below a specified value. In Embodiment II, at least one of the 11 culture medium essential amino acids contained in the solution may be present at a concentration below the specified value shown in (i) to (xi) above. Here, "containing a culture medium essential amino acid at a concentration below the specified value" means that the concentration of the amino acid is present above the detection limit and below the specified value. The numerical value of the specified value for each culture medium essential amino acid is set individually according to the type of amino acid. Furthermore, the solution may contain any number of amino acids from the 11 culture medium essential amino acids shown in (i) to (xi) above, ranging from two to eleven, and may contain two, three, four, five, six, seven, eight, nine, ten, or eleven of the culture medium essential amino acids shown in (i) to (xi) above.

[0035] If the solution contains (i) L-histidine at a concentration of 20 mg / L or less, the concentration of L-histidine is 20 mg / L or less, preferably 10 mg / L or less, and more preferably 5 mg / L or less. If the solution contains (ii) L-isoleucine at a concentration of 40 mg / L or less, the concentration of L-isoleucine is 40 mg / L or less, preferably 20 mg / L or less, and more preferably 15 mg / L or less. If the solution contains (iii) L-leucine at a concentration of 40 mg / L or less, the concentration of L-leucine is 40 mg / L or less, preferably 20 mg / L or less, and more preferably 15 mg / L or less. If the solution contains (iv) L-lysine at a concentration of 50 mg / L or less, the concentration of L-lysine is 50 mg / L or less, preferably 20 mg / L or less, and more preferably 15 mg / L or less. If the solution contains (v) L-methionine at a concentration of 10 mg / L or less, the concentration of L-methionine is 10 mg / L or less, preferably 8 mg / L or less, and more preferably 6 mg / L or less. If the solution contains (vi) L-phenylalanine at a concentration of 20 mg / L or less, the concentration of L-phenylalanine is 20 mg / L or less, preferably 15 mg / L or less, and more preferably 10 mg / L or less. If the solution contains (vii) L-threonine at a concentration of 7 mg / L or less, the concentration of L-threonine is 7 mg / L or less, preferably 5 mg / L or less, and more preferably 3 mg / L or less. If the solution contains (viii) L-valine at a concentration of 40 mg / L or less, the concentration of L-valine is 40 mg / L or less, preferably 20 mg / L or less, and more preferably 10 mg / L or less. If the solution contains (ix) L-arginine at a concentration of 40 mg / L or less, the concentration of L-arginine is 40 mg / L or less, preferably 35 mg / L or less, and more preferably 30 mg / L or less. If the solution contains (x) L-cystine at a concentration of 20 mg / L or less, the concentration of L-cystine is 20 mg / L or less, preferably 10 mg / L or less, and more preferably 5 mg / L or less. If the solution contains (xi) L-tyrosine at a concentration of 10 mg / L or less, the concentration of L-tyrosine is 10 mg / L or less, preferably 8 mg / L or less, and more preferably 5 mg / L or less.The essential amino acids in the culture medium contained in the solution of this embodiment may be added as amino acids themselves or in the form of amino acid salts. Regardless of the form of addition, it is sufficient that the concentration calculated by converting to amino acids falls within the aforementioned numerical range.

[0036] In embodiment II, the solution may contain at least one of the following: (ii) L-isoleucine at a concentration of 40 mg / L or less, (iii) L-leucine at a concentration of 40 mg / L or less, (iv) L-lysine at a concentration of 50 mg / L or less, and (ix) L-arginine at a concentration of 40 mg / L or less. Specifically, the solution may contain two of the following: (ii) and (iii), (ii) and (iv), (ii) and (ix), (iii) and (iv), (iii) and (ix), (iv) and (ix), (ii), (iii) and (ix), (iii), (iv) and (ix), or four of the following: (ii), (iii), (iv) and (ix).

[0037] In embodiment II, the solution may contain two or more solutions selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine, with the total concentration of these solutions being, for example, 300 mg / L or less. Furthermore, the total concentration of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 types selected from the group may be, for example, 300 mg / L or less, 250 mg / L or less, 200 mg / L or less, 190 mg / L or less, 180 mg / L or less, 170 mg / L or less, 150 mg / L or less, 120 mg / L or less, 110 mg / L or less, 100 mg / L or less, 90 mg / L or less, 80 mg / L or less, 70 mg / L or less, 60 mg / L or less, 50 mg / L or less, 40 mg / L or less, 30 mg / L or less, 20 mg / L or less, 10 mg / L or less, or 5 mg / L or less. The total concentration of the four components (ii), (iii), (iv), and (ix) may be 300 mg / L or less, 200 mg / L or less, 150 mg / L or less, 120 mg / L or less, 110 mg / L or less, 100 mg / L or less, 90 mg / L or less, 80 mg / L or less, 70 mg / L or less, 60 mg / L or less, 50 mg / L or less, 40 mg / L or less, 30 mg / L or less, 20 mg / L or less, 10 mg / L or less, and 5 mg / L or less.

[0038] The concentration of essential amino acids in a culture medium contained in a solution can be measured according to known methods for quantifying the amount of free amino acids in a solution. Specifically, based on the "AAA (AminoAcidAnalysis) method" provided by Agilent, the concentration of various amino acids can be measured with high sensitivity and accuracy by pretreatment combining OPA derivatization and FMOC derivatization, separation and detection by liquid chromatography (LC), and quantification by a fluorescence detector. In this measurement method, the solution is first thawed and homogenized, then diluted with buffer if the DMSO concentration is high, and filtered through a 0.22 μm filter to obtain the sample. For the initial analysis, it is desirable to perform an LC scan in an underivatized state to check for matrix interference. Subsequently, OPA derivatization reagent is added to the sample to which borate buffer has been added as pretreatment, and then FMOC derivatization reagent is added to form derivatives covering all types of amino acids, including primary and secondary amines. These derivatives are separated under LC conditions using an appropriate mobile phase and detected and quantified by a fluorescence detector. For detection, by using an excitation wavelength of 340 nm / fluorescence wavelength of 450 nm for OPA derivatives and an excitation wavelength of 266 nm / fluorescence wavelength of 305 nm for FMOC derivatives, it is possible to detect the peaks corresponding to each amino acid with high sensitivity. Calibration curves are created using amino acid standard solutions of known concentrations (e.g., 10, 25, 100, 250 μM) and obtained by performing linear regression processing based on the correlation with each peak area. In particular, for tryptophan, regression processing at the FMOC detection window is desirable to avoid fluorescence interference. Based on the concentration measurements obtained in this way, it is possible to objectively determine whether the concentration of essential amino acids in the culture medium (e.g., L-histidine, L-leucine, L-arginine, etc.) contained in the solution is below a predetermined specified value.

[0039] Here, the essential amino acids used in cell culture media such as basal media are generally the 11 mentioned above plus L-tryptophan, totaling 12 types. However, there are several problems in quantitative measurement of L-tryptophan, such as its susceptibility to oxidation and photodegradation, its poor reaction with derivatization reagents, and its high background noise. For this reason, in the solution according to this embodiment, by defining the solution after excluding L-tryptophan from its composition in either Embodiment I, which does not contain essential amino acids, or Embodiment II, which contains essential amino acids at a concentration below a specified value, stability in terms of both measurement reproducibility and product quality control can be improved, and Embodiments I and II, which are practically suitable, can be realized. Note that by employing the following L-tryptophan quantitative means, the solution may have a composition that does not contain L-tryptophan in Embodiment I, or a composition that contains L-tryptophan at concentrations below a specified value of 2.0 mg / L or less, 1.5 mg / L or less, or 1.0 mg / L or less in Embodiment II. L-tryptophan can be quantitatively measured using the Tryptophan Assay Kit (Cell Biolab, product number MET-5177). This kit utilizes hydrogen peroxide, produced when tryptophan is oxidized by tryptophan oxidase, as a fluorescence reaction. The fluorescence intensity of this reaction is detected by a fluorescence plate reader, allowing for highly accurate measurement of the tryptophan concentration in a sample. Calibration curves are created using tryptophan standard solutions of known concentrations (e.g., 6.25, 12.5, 25, 50, 100 μM) and obtained by performing linear regression based on the correlation with fluorescence intensity.

[0040] The solution according to this embodiment may be configured to contain other amino acids besides the above-mentioned essential amino acids for the culture medium, as needed. However, amino acids that are not classified as essential amino acids for the culture medium but are often included in cell culture media as important components in cell culture (so-called conditionally essential amino acids), such as L-serine, may be configured to be excluded depending on the specific amino acid, or their concentration in the solution may be controlled to be below a predetermined threshold. Examples of such thresholds include being set to a specified value of 50 mg / L or less, 40 mg / L or less, 30 mg / L or less, 20 mg / L or less, 15 mg / L or less, or 10 mg / L or less. In addition, the solution according to this embodiment may be configured to contain at least one of carbohydrates (sugars) and vitamins as other components included in the basal culture medium besides the above-mentioned amino acids and inorganic salts.

[0041] The solution according to this embodiment has a configuration that includes either Embodiment I, which does not contain essential amino acids in the culture medium, or Embodiment II, in which the amount of essential amino acids added to the culture medium is below a specified value. Therefore, even when the solution is used to immerse a frozen and thawed three-dimensional cell culture for transplantation, it can be used within the scope that does not fall under "processing of cells and tissues" or "manufacturing" as defined in relevant guidelines and laws, and a solution suitable for so-called non-processing of cells can be provided. In other words, this solution can be used to immerse a frozen and thawed three-dimensional cell culture for transplantation without processing the cells and tissues, and a method of using the solution that is not considered processing of cells and tissues can be provided.

[0042] Here, in the Ministry of Health, Labour and Welfare's Pharmaceutical and Food Safety Bureau Director's Notification No. 0907-3 dated September 7, 2012, "Guidelines on Ensuring the Quality and Safety of Human Allogeneic Somatic Stem Cell Processed Pharmaceuticals, etc.," "processing of cells and tissues" is defined as follows: "The act of artificially proliferating or differentiating cells and tissues, establishing cell lines, treating cells with drugs for the purpose of cell activation, modifying biological properties, combining with non-cellular components, or genetically engineering modifications, etc., for the purpose of treating diseases or repairing or reconstructing tissues." It should be noted that "separation of tissues, shredding of tissues, separation of cells, isolation of specific cells, treatment with antibiotics, washing, sterilization with gamma rays, etc., freezing, thawing, etc." are not considered processing. Furthermore, the "Act on Ensuring the Safety of Regenerative Medicine, etc.," which applies to the field of regenerative medicine, also provides the following definitions: "Cell processed product": Cells of humans or animals that have been cultured or otherwise processed. "Specific cell processed product": Cell processed products used in regenerative medicine other than regenerative medicine products. "Manufacturing": The act of culturing or otherwise processing specific cell processed products. "Specific cell processing product manufacturing facilities": These are facilities that manufacture these products and require permission from the Minister of Health, Labour and Welfare. As described above, the guidelines and laws clearly define "cell and tissue culture operations" as acts that constitute processing or manufacturing. However, if a solution for processing cells contains approximately 50-70% basal culture medium, considering that its main component is basal culture medium for cell culture, the operation of immersing a frozen-thawed three-dimensional cell culture for transplantation with the solution for processing the cells may be considered to constitute "cell and tissue culture operations." Therefore, there was a need to develop a solution for processing cells that is suitable for so-called non-processing applications, which is composed in a way that does not fall under "processing of cells and tissues" or "manufacturing" as defined in the guidelines and laws, even if it is used to immerse a frozen-thawed three-dimensional cell culture for transplantation. The inventors of this invention investigated the composition of a solution for processing cells suitable for non-processing applications (hereinafter referred to as "solution for non-processing cells"). In doing so, they focused on "essential amino acids for cell culture" contained in basal culture medium, and attempted to construct a composition that would not be considered processing by excluding these amino acids.However, while it is possible to remove essential amino acids from the culture medium by reducing the proportion of basal culture medium in the cell treatment solution or by not using basal culture medium at all, this also removes other components contained in the basal culture medium, especially inorganic salts. As a result, the overall function of the cell treatment solution is reduced, and it has been confirmed that this leads to a decrease in cell viability and metabolic activity after freezing and thawing.

[0043] Furthermore, biological raw materials are raw materials derived from humans and other living organisms (excluding plants) used in pharmaceuticals, quasi-drugs, cosmetics, medical devices, and regenerative medicine products. When these are used in the manufacture of pharmaceuticals, etc., necessary measures to ensure the quality, efficacy, and safety of pharmaceuticals, etc. are stipulated in Japan by the "Standards for Biological Raw Materials" (Ministry of Health, Labour and Welfare Notification No. 37 / established February 28, 2018). The solution according to this embodiment can be provided as a product to which the Standards for Biological Raw Materials do not apply. This eliminates the need to excessively tighten quality control standards compared to products to which the Standards for Biological Raw Materials apply. Specific examples of such products include solutions used in the field of regenerative medicine, but they can also be applied to medical devices, research reagents, etc. Hereinafter, examples of reference embodiments of methods using the solution of this embodiment are noted: ・An ex vivo cell treatment method in which the solution is brought into contact with the cells during the process of processing frozen and thawed cells. ・The use of a solution for immersing frozen and thawed cells in a non-therapeutic and ex vivo manner. - A non-therapeutic, ex vivo method for treating frozen-thawed cells, comprising the step of bringing a solution into contact with the cells. - A method for treating frozen-thawed cells ex vivo, comprising the step of bringing a solution into contact with the cells (excluding methods for treating human or animal bodies). However, the cells are a three-dimensional cell culture having interconnected structures between cells.

[0044] The solution according to this embodiment is a product used to immerse a three-dimensional cell culture having interconnected cell structures after freezing and thawing. In the freezing of single cells, it is known that the extracellular osmotic pressure increases during the freezing process, resulting in water leakage from the cells, dehydration, and shrinkage. On the other hand, in the freezing of three-dimensional cell structures such as cell sheets, dehydration and shrinkage occur similarly to single cells, and it has also been reported that intercellular junctions and the extracellular matrix are further destroyed by ice crystal formation. Although the detailed mechanism of this phenomenon is not yet clear, it is thought that when cells are strained due to dehydration and shrinkage associated with freezing, single cells have a high degree of freedom for deformation of the cell membrane because there are no physical constraints from surrounding cells. In contrast, in a three-dimensional cell culture where cells are densely interconnected, each cell is structurally constrained by other adjacent cells, resulting in a low degree of freedom for individual cell membranes and a structure where strain tends to concentrate. As a result, it is inferred that during the freezing process, the structure of the cell membrane and intercellular junctions is prone to breakdown, and membrane damage is likely to occur.

[0045] Regarding the cell membrane repair mechanism, when the cell membrane is locally damaged, calcium ions (Ca) are released from the site of the rupture. ２＋ ) flows into the cell, and its high concentration of Ca ２＋ It is known that a signal activates the process by which intracellular vesicles fuse with the cell membrane (exocytosis). ２＋It is believed that cell membrane repair is promoted by the supply of new membrane components to the damaged site through cell-dependent exocytosis. However, the entire mechanism of cell membrane repair described above is still not clearly understood, and its behavior is particularly complex in cell populations with a three-dimensional structure. For example, in a single cell, since the entire cell is in contact with the solution, calcium ions can easily penetrate from the outside to any damaged site of the membrane. On the other hand, when areas such as intercellular junctions or the extracellular matrix in a three-dimensional cell culture are damaged, it is assumed that calcium ions and the solution cannot easily penetrate locally due to the structure in which these areas are covered by surrounding cells. As a result of diligent research by the inventors, we have found that the cell viability after the first freeze-thaw test is improved by using a solution prepared with a high concentration of calcium ions. Although the detailed mechanism of action is not yet clear, it is believed that during the immersion process after freezing cells, external calcium ions flow into the cell from the peripheral region of the cell membrane that has suffered mechanical stress or freeze damage, and Ca ２＋ It is presumed that this induces calcium-dependent exocytosis. At this time, it is thought that by using a high concentration of calcium ions, a similar repair mechanism is more easily induced even in cell-to-cell linkage structures that are normally difficult for the solution to come into contact with, and as a result, it is inferred that the cell viability after freeze-thawing in three-dimensional cell cultures will improve. Furthermore, the inventors have found that by including a high concentration of magnesium ions and other divalent metal ions, an improvement in metabolic activity rate after a second freeze-thaw is obtained. In addition, it has been confirmed that by using a combination of high concentrations of calcium ions and magnesium ions, it is possible to further improve cell viability through a further improvement in metabolic activity.

[0046] Furthermore, in this embodiment, a solution can be used to provide a freeze-thawed three-dimensional cell culture for transplantation, a method for producing the freeze-thawed product, a method for using the solution, and a transplantation kit.

[0047] An example of a frozen-thawed three-dimensional cell culture for transplantation in this embodiment includes a solution and a three-dimensional cell culture immersed in the solution. The solution in the frozen-thawed material contains one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and an aqueous solvent, satisfying at least one of the following (a) to (c), and having an osmotic pressure of 150 mOsm / L or more and 1200 mOsm / L or less. (a) If the calcium salt is included, the calcium salt content is 5 ppm or more and 700 ppm or less in terms of calcium ions, based on the total weight of the solution. (b) If the magnesium salt is included, the magnesium salt content is 5 ppm or more and 1000 ppm or less in terms of magnesium ions, based on the total weight of the solution. (c) If other divalent metal salts other than the calcium salt and magnesium salt are included, the content of the other divalent metal salts is 0.01 ppm or more and 100 ppm or less in terms of metal ions, based on the total weight of the solution.

[0048] In the above-mentioned frozen-thawed product, the solution may contain a cryoprotectant. The concentration of the cryoprotectant in the above-mentioned frozen-thawed product may be 7 w / v% or more based on the volume of the solution. Alternatively, the concentration of the cryoprotectant in the above-mentioned frozen-thawed product may be less than 0.5 w / v% based on the volume of the solution.

[0049] An example of a manufacturing method for obtaining a frozen and thawed three-dimensional cell culture for transplantation according to this embodiment includes: (a) a step of preparing a frozen three-dimensional cell culture for transplantation; and (b) a step of thawing the three-dimensional cell culture and immersing the thawed three-dimensional cell culture in a solution. The solution used in the step of immersing the thawed three-dimensional cell culture in the solution comprises one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and an aqueous solvent, satisfying at least one of the following (a) to (c), and having an osmotic pressure of 150 mOsm / L or more and 1200 mOsm / L or less. (a) If the calcium salt is included, the calcium salt content is 5 ppm or more and 700 ppm or less in terms of calcium ions, based on the total weight of the solution. (b) If the magnesium salt is included, the magnesium salt content is 5 ppm or more and 1000 ppm or less in terms of magnesium ions, based on the total weight of the solution. (c) If other divalent metal salts other than the calcium salt and magnesium salt are included, the content of the other divalent metal salts is 0.01 ppm or more and 100 ppm or less in terms of metal ions, based on the total weight of the solution.

[0050] One example of how to use the solution in this embodiment is to immerse a frozen and thawed three-dimensional cell culture for transplantation in the solution.

[0051] An example of a transplantation kit of this embodiment includes a frozen three-dimensional cell culture for transplantation and a solution. The solution in the transplantation kit contains one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and an aqueous solvent, satisfying at least one of the following (a) to (c), and having an osmotic pressure of 150 mOsm / L or more and 1200 mOsm / L or less. (a) If the calcium salt is included, the calcium salt content is 5 ppm or more and 700 ppm or less in terms of calcium ions, based on the total weight of the solution. (b) If the magnesium salt is included, the magnesium salt content is 5 ppm or more and 1000 ppm or less in terms of magnesium ions, based on the total weight of the solution. (c) If other divalent metal salts other than the calcium salt and magnesium salt are included, the content of the other divalent metal salts is 0.01 ppm or more and 100 ppm or less in terms of metal ions, based on the total weight of the solution.

[0052] In one embodiment, the solution of the present invention is free of or substantially free of cryoprotective agents. A "cryoprotective agent" is a substance used to reduce damage to cells caused by freezing and thawing during cell cryopreservation. "Substantially free of cryoprotective agents" means that cryoprotective agents are not intentionally added, and is distinct from contamination of the solution with cryoprotective agents contained in cryopreservation solutions, where trace amounts remain because the cryopreservation solution used for freezing three-dimensional cell cultures is not completely removed after thawing. The solution of the present invention, which does not contain cryoprotective agents known to be biotoxic (especially cell-penetrating cryoprotective agents such as DMSO), is advantageous from a therapeutic standpoint. Therefore, considering that contamination with cryoprotective agents as described above may occur, from a clinical standpoint, it is preferable that the concentration of cryoprotective agents (especially cell-penetrating cryoprotective agents such as DMSO) contained in the solution of the present invention, specifically in the solution containing the buffer, be less than 0.5 w / v% based on the volume of the solution of the present invention.

[0053] In another embodiment, the solution of the present invention may contain a cryoprotective agent. "Containing" a cryoprotective agent typically refers to a case where, after thawing, some or all of the cryopreservation solution containing the cryoprotective agent used during freezing is left intact, and the solution of the present invention is added to the remaining cryopreservation solution, resulting in the solution of the present invention containing a cryoprotective agent. However, intentionally adding a cryoprotective agent to the solution of the present invention is not excluded. Examples of cryoprotective agents include cell-impermeable cryoprotective agents and cell-permeable cryoprotective agents. Specific examples of cell-impermeable cryoprotective agents include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeable cryoprotective agents include dimethyl sulfoxide (DMSO), polyol, glycerin (glycerol), propylene glycol, ethylene glycol, and propanediol. Furthermore, these cryoprotective agents may be formulated individually or in combination of two or more. These cryoprotective agents can be appropriately selected from known ones depending on the type of cell, the composition of the cryopreservation solution, etc. The content of the cryoprotective agent can be appropriately changed depending on the type of component used. The upper limit of the cryoprotective agent content may be selected from 20 w / v%, 15 w / v%, 10 w / v%, 9 w / v%, 8 w / v%, 7 w / v%, 6 w / v%, 5 w / v%, 4 w / v%, and 3 w / v%, based on the weight and volume of the solution of the present invention containing the cryoprotective agent, and the lower limit of the cryoprotective agent content may be selected from 5 w / v%, 4 w / v%, 3 w / v%, 2.5 w / v%, 2 w / v%, 1.5 w / v%, 1 w / v%, and 0.5 w / v%, provided that the upper limit > lower limit.

[0054] In one embodiment, the solution of the present invention can be prepared using a cryopreservation solution. Cryopreservation solutions are generally prepared by adding a cryoprotectant to a buffer solution. Numerous cryopreservation solutions are commercially available and can be used. For example, commercially available cryopreservation solutions containing DMSO include Stem Cell Banker® GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), BanBanKer® hRM (GC Lymphotek), BanBanKer® (GC Lymphotek), and iStock® (GC Lymphotek). In addition, commercially available cryopreservation solutions that do not contain DMSO include, for example, Stem Cell Banker (registered trademark) DMSO-free GMP grade (Nippon Zenyaku Kogyo Co., Ltd.), Banbanker (registered trademark) DMSO-free (GC Lymphotec Co., Ltd.), Cryoscarless (registered trademark) DMSO-free (Bioverde Co., Ltd.), Stem Cell Keep (Bioverde Co., Ltd.), CryoNovo (registered trademark) X12 (Akron BioProducts LCC Co., Ltd.), and CryoNovo (registered trademark). Examples include P24 (Akron BioProducts LCC), DMSO-free cryopreservation solution for human ES / iPS cells (ReproCELL), CellReservoirOne (Nacalai Tesque), TheliKeep (registered trademark: BioVerde), Cellvation (registered trademark: ProtidePharmaceuticals), ReproCryoRM (ReproCELL), SOFOROCryo (SARAYA), etc.

[0055] In addition to the above-mentioned substances or components, the solution of the present invention may further contain any other substances or components known in the art to improve cell viability, such as amino acids, sugars, vitamins, inorganic salts, and / or antioxidants. The "amino acids" may be natural or unnatural amino acids, such as glutamic acid, glutamine, arginine, cystine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, cysteine, proline, and hydroxyproline. Examples of "sugars" include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides. Examples of "vitamins" include sodium L-ascorbate, L-ascorbic acid diphosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12. Examples of inorganic salts include metal salts, which include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), and salts of copper, zinc, and iron. Metal salts may be divalent or trivalent metal salts. Specifically, these are used as metal salts such as hydrochloride, carbonate, nitrate, and sulfate, including potassium chloride, sodium chloride, calcium chloride, sodium sulfate, magnesium sulfate, magnesium carbonate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate. Examples of "antioxidants" include ascorbic acid and glutathione. Furthermore, the solution of the present invention may substantially not contain at least one or more of the following: amino acids, sugars, vitamins, or antioxidants. Specifically, the concentrations of amino acids, sugars, vitamins, and antioxidants in the solution of the present invention may be, for example, 0.5% w / v or less.0.5% w / v or less also includes a configuration in which at least one of these four components is not present in the solution, or a configuration in which at least one of the four components is at a concentration below the detection limit.

[0056] The solution of the present invention may contain animal-derived components, but it is preferable that it does not contain animal-derived components. "Substantially free of animal-derived components" means that animal-derived components are not intentionally added, and is distinct from contamination of the cryopreservation solution with animal-derived components as a result of trace amounts remaining after thawing, when the cryopreservation solution used for freezing three-dimensional cell cultures is not completely removed. When animal-derived components are not included, it is possible to avoid changes in the properties of cells caused by components that are not originally necessary for cell preservation, such as various cytokines and growth factors contained in serum, or the influence of components in the basal culture medium of unknown origin, and is useful from the viewpoint of being able to be safely applied to living organisms in clinical use. Therefore, considering that contamination of cryoprotective agents as described above may occur, from a clinical standpoint, it is preferable that the concentration of animal-derived components in the cryopreservation solution used for freezing be 0.5% by weight or less, based on the weight of the solution of the present invention. Examples of animal-derived components include albumin, serum (calf serum, neonatal calf serum, fetal bovine serum, horse serum, etc.), plasma, and basal culture medium.

[0057] In another aspect of the present invention, the solution of the present invention may be provided in a container or packaging such as a cold-resistant container, and the container or packaging may be accompanied by instructions for use of the solution of the present invention. Furthermore, in one aspect of the present invention, a kit may be provided comprising frozen three-dimensional cell cultures for transplantation and the solution of the present invention.

[0058] One embodiment of the present invention provides a three-dimensional cell culture for transplantation that has been frozen and thawed, and is immersed in the solution of the present invention described above.

[0059] A "three-dimensional cell culture for transplantation" is a three-dimensional cell culture used for transplantation to treat a disease or disorder or to improve its symptoms. The recipient of the transplantation is preferably a mammal, such as a human or a domestic pet (dog, cat, etc.), with humans being particularly preferred.

[0060] A "three-dimensional cell culture" is an aggregate of cultured cells that has a three-dimensional structure due to physical and / or functional linkage between cells via adhesion molecules or the extracellular matrix. A "three-dimensional cell culture" includes, but is not limited to, three-dimensional cell sheets, spheroids, organoids, etc. The shape of a three-dimensional cell culture can be, but is not limited to, three-dimensional sheets, organ-like, spherical, tissue-like, hollow, or block-like. Typically, adherent cells (cells that adhere to and proliferate on a culture substrate, such as fibroblasts) easily form three-dimensional cell aggregates through physical and / or functional linkage, as in a cell sheet. However, even suspension cells (i.e., cells that float and proliferate in a culture medium, such as hematopoietic cells or blood cells) can be considered a three-dimensional cell culture if they form a three-dimensional cell aggregate through physical and / or functional linkage, as in a spheroid.

[0061] The method for producing a three-dimensional cell culture can be carried out by methods known to those skilled in the art and is not particularly limited. Examples of methods for producing a three-dimensional cell culture include those taught in the literature such as Japanese Patent Publication No. 2012-120696, Japanese Patent Publication No. 2017-176025, and Japanese Patent Publication No. 2015-149905. The density of cells to be cultured in the culture of a three-dimensional cell culture is not particularly limited as long as it is suitable for the cells to be cultured, the culture vessel (e.g., a multi-well cell culture plate), or the intended use of the cultured cells, but for example, 5 × 10 ２ cells / cm ２ The above 1 x 10 ９ cells / cm ２ The following applies. More preferably, the lower limit is 1 × 10⁻⁶. ３ cells / cm ２ More preferably 5 x 10 ３ cells / cm ２ In particular, 5 x 10 ４ cells / cm ２ That concludes the explanation. On the other hand, a more preferable upper limit is 1 × 10⁻⁶. ８ cells / cm ２ More preferably 5 × 10 ７ cells / cm ２The following is particularly preferable: 1 x 10 ７ cells / cm ２ The following applies: Furthermore, the culture medium used for culturing three-dimensional cell cultures for transplantation is not particularly limited and includes, for example, basic media such as AIMV medium, HFDM-1 medium, DMEM, EMEM, α-MEM, IMDM, GMEM, Ham's F-10 medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 medium, Williams' medium E, Brinster's BMOC-3 medium, and E8 medium. These basic culture media may be used individually or in combination of two or more types.

[0062] A three-dimensional cell culture may be cultured in three dimensions using a scaffold. For example, adherent cells can adhere to and proliferate on a scaffold having a mesh structure or the like. The scaffold material used is a material that can adhere or hold the cells to be cultured and enhance intercellular interactions during culture. The scaffold material can be appropriately selected from known materials depending on the cells to be cultured, but examples include collagen, polyester, polyisoprene, polybutadiene, polyurethane, polyurea, polytetrafluoroethylene, polyethylene oxide, polyethylene glycol, polycaprolactone, silk fibroin, polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), and Examples of scaffolding materials include polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), fibroin, cellulose, regenerated cellulose, cyclic olefin polymers, acrylate resins, methacrylate resins and their derivatives and copolymers. Scaffolding materials may be used individually or in combination of two or more materials. Their shape is not particularly limited and examples include films, fibers, and nonwoven fabrics. Furthermore, to improve cell adhesion and propagation, an extracellular matrix (ECM) with high affinity for cells may be used. The extracellular matrix (ECM) is a noncellular component present in all tissues and organs, and is mainly composed of two types of macromolecules: fibrous proteins and proteoglycans. The main fibrous proteins in the ECM are collagen, elastin, fibronectin, and laminin.On the other hand, three-dimensional cell cultures may be cultured without using a scaffold (scaffold-free). Scaffold-free three-dimensional cell cultures, such as scaffold-free spheroids obtained by culturing suspension cells, have advantages such as reducing the risk of inflammatory reactions and infections caused by foreign body contamination when using a scaffold.

[0063] A "cell sheet" is a sheet structure in which cells are physically and / or functionally connected to each other via adhesion molecules or an extracellular matrix. The method for manufacturing a cell sheet can be carried out by methods known to those skilled in the art and is not particularly limited. Examples of methods for manufacturing cell sheets include those taught in literature such as International Publication No. 2016 / 068217, Japanese Patent Publication No. 2019-000038, Japanese Patent Publication No. 2011-006490, and International Publication No. 2015 / 068505. A cell sheet may be a monolayer structure composed of one cell layer, or a laminated structure composed of two or more cell layers (two, three, four, five, etc.). The thickness of the cell sheet can be appropriately set to exhibit a high viability of cells in the cell sheet and excellent shape retention advantageous for cell transplantation, but may be, for example, 0.001 mm to 2.0 mm.

[0064] When using cell sheets as a three-dimensional cell culture medium, it is preferable to form the cell sheets on a culture carrier substrate. Forming the cell sheets on a culture carrier substrate reduces the possibility of tearing when removing the cell sheets from the culture vessel or cryopreservation container, and allows for easy removal. The culture carrier substrate is used both as a cell sheet scaffold for culturing cells and forming cell sheets, and as a cell sheet support for transporting the cultured cell sheets. It is a substrate that is peeled off from the cell sheets after they have been attached to the transplant site. The area of ​​the culture carrier substrate is not particularly limited, but it is desirable that it be smaller than the culture vessel and the same as or larger than the cell sheet, for example, 0.3 cm². ２ ~1000cm ２The following applies. Furthermore, the thickness of the culture carrier substrate is preferably 5 μm to 250 μm or less. The culture surface of the culture carrier substrate is preferably subjected to hydrophilic treatment, such as UV ozone treatment or plasma treatment, in order to improve cell adhesion. Materials that constitute the culture carrier substrate include polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), fibroin, cellulose, regenerated cellulose, cyclic olefin polymer, gelatin, collagen, and the like. Polyether ether ketone (PEEK), polyethylene terephthalate (PET), and polybutylene terephthalate (PBT) are particularly preferred. The culture carrier substrate before being used for culturing cell sheets can be a packaged product enclosed in packaging material.

[0065] An example of a culture carrier substrate may consist of a film containing at least one of these materials as its main component. It is preferably made of a transparent material with a specific gravity greater than 1.0 and excellent durability, mechanical strength, and processability. Preferably, it is a polyetheretherketone (PEEK) film, polyethylene terephthalate (PET) film, or polystyrene, more preferably a polyetheretherketone (PEEK) film. The culture carrier substrate is preferably a resin film containing one or more resin layers made of the above-mentioned resin material. The two or more resin layers may contain different types of materials. The culture carrier substrate may also consist of a resin substrate containing resin layers made of the above-mentioned material, or a laminate substrate containing a resin layer made of the above-mentioned material and a resin layer and / or an inorganic layer made of other materials. However, the resin layer does not need to include layers formed by chemical vapor deposition such as parylene or a wet coating layer. Furthermore, the inorganic layer in the laminate substrate preferably does not contain glass, but may contain metal foil. Additionally, it is preferable that the culture carrier substrate does not contain nonwoven fabric or fiber substrate on the culture surface side. When considering all aspects from the viewpoints of low coefficient of thermal expansion, solvent resistance, heat resistance, impact resistance, and sterilization resistance, culture carrier substrates containing PEEK are preferable to those containing PET. Furthermore, it is preferable that the culture carrier substrate is composed of a material with a specific gravity higher than that of the culture medium 30.

[0066] The lower limit of the surface roughness Ra of the culture surface of the culture carrier substrate is 0.3 nm or more, preferably 0.5 nm or more, and the upper limit is 100 nm or less, preferably 10 nm or less, more preferably 2 nm or less. Here, surface roughness Ra refers to the arithmetic mean roughness in a square region with sides of 100 nm, using surface shape data measured by an atomic force microscope (AFM). The cell culture film substrate is such that when the culture surface of the culture carrier substrate is measured with a laser microscope, there are three or fewer holes in a 100 μm square area, with a diameter of 1 μm or more and 100 μm or less, and a depth of 0.5 μm or more and 100 μm or less. The diameter of the holes is, for example, 1 μm or more and 100 μm or less, preferably 2 μm or more and 50 μm or less, more preferably 3 μm or more and 30 μm or less. The depth of the holes is, for example, 0.5 μm or more and 100 μm or less, preferably 1 μm or more and 50 μm or less, more preferably 2 μm or more and 20 μm or less. The ranges of hole diameter and hole depth include, for example, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 0.5 μm or more and 100 μm or less, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 1 μm or more and 50 μm or less, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 2 μm or more and 20 μm or less, a hole diameter of 2 μm or more and 50 μm or less and a hole depth of 0.5 μm or more and 100 μm or less, and the hole diameter The holes are 2 μm to 50 μm in diameter and 1 μm to 50 μm in depth, 2 μm to 50 μm in diameter and 2 μm to 20 μm in depth, 3 μm to 30 μm in diameter and 0.5 μm to 100 μm in depth, 3 μm to 30 μm in diameter and 1 μm to 50 μm in depth, and 3 μm to 30 μm in diameter and 2 μm to 20 μm in depth. The upper limit of the porosity of the culture carrier substrate is, for example, 15% or less, preferably 10% or less, and more preferably 5% or less. On the other hand, the lower limit of the porosity of the culture carrier substrate is not particularly limited, but may be 0% or more. By having three or fewer holes on the culture surface and / or by keeping the porosity of the culture carrier substrate below the upper limit, the adhesion force to the cell sheet can be made appropriate. The presence or absence of pores may be measured on the surface of the resin layer formed on the culture surface side of the culture carrier substrate. The porosity is calculated from the theoretical density and the measured density.Specifically, the porosity is calculated using the formula: porosity = {1 - (actual density / theoretical density)} × 100.

[0067] Furthermore, the culture surface of the culture carrier substrate may be configured so as not to contain temperature-responsive polymers. This suppresses a decrease in adhesion between the cell sheet and the culture carrier substrate in low-temperature environments such as cryopreservation processes. Temperature-responsive polymers are materials that exhibit cell adhesion at the temperature at which cells are cultured, and then exhibit cell non-adhesion by changing the temperature, allowing for easy detachment of the cell sheet. It is preferable that the temperature range in which the temperature-responsive polymer exhibits cell adhesion is 10°C to 45°C, particularly 33°C to 40°C, as this allows for stable cell culture. It is also preferable that the temperature range in which the temperature-responsive polymer exhibits cell non-adhesion is 1°C to 36°C, particularly 4°C to 32°C, as this reduces damage to the detachment of the cell sheet. Examples of temperature-responsive polymers that can be used as materials for temperature-responsive polymers include poly-N-isopropylacrylamide (PNIPAAm), poly-N-n-propylacrylamide, poly-N-n-propylmethacrylamide, poly-N-ethoxyethylacrylamide, poly-N-tetrahydrofurfurylacrylamide, poly-N-tetrahydrofurfurylmethacrylamide, and poly-N,N-diethylacrylamide, among which PNIPAAm, poly-N-n-propylmethacrylamide, and poly-N,N-diethylacrylamide are preferred. The bottom surface of the culture vessel may also be constructed so as not to contain temperature-responsive polymers.

[0068] The culture carrier substrate before being used for cell sheet culture can be packaged in packaging material. By using a sealed package, the culture surface can be stored and distributed without contamination. This package may also be sterilized. Sterilization methods include electron beam sterilization, gamma ray sterilization, EOG sterilization, and autoclaving.

[0069] An example of a method for manufacturing a cell sheet-attached culture carrier substrate may include, for example, a culture step in which multiple cells, a culture medium (culture solution), and a culture carrier substrate are introduced into the inside of a culture vessel (for example, each well of a cell culture multiwell plate), and the cell sheet is cultured on the culture carrier substrate; and a removal step in which the cell sheet-attached culture carrier substrate is removed from the culture solution. After the cell sheet is attached to a target site such as a wound or affected area, the culture carrier substrate may be peeled off from the cell sheet. Furthermore, the cell sheet may be used for treatment, prevention, etc., such as cell transplantation therapy, after being peeled off from the cell sheet-attached culture carrier substrate. In other words, the method of using the cell sheet-attached culture carrier substrate may be either to attach the cell sheet-attached culture carrier substrate to the affected area and then peel off the culture carrier substrate, or to peel off the cell sheet from the cell sheet-attached culture carrier substrate and then attach it to the affected area.

[0070] A "spheroid" generally refers to a mass (typically ball-shaped) formed by the aggregation of cells, or a simple cluster (aggregate) of cells. Spheroids are typically formed by the aggregation of thousands of cells into a spherical structure, and three-dimensional cultures can maintain high levels of functional expression for longer periods compared to two-dimensional cell cultures. Spheroids may consist of, for example, 1,000 or more, 10,000 or more, or 100,000 or more cells, and the preparation and culture of spheroids can be carried out using known methods, such as the method taught in Scientific Reports 6(1):31063, "Microfabric Vessels for Embryoid Body Formation and Rapid Difference of Pluripotet Stem Cells", DOI:10.1038 / srep31063. It is preferable to culture spheroids in a three-dimensional culture that proliferates them three-dimensionally while maintaining their shape in vivo.

[0071] An "organoid" refers to a collection of cells that have been given the function of an organ. Actual organs cannot be cultured because it is difficult to continuously supply nutrients to them, but organoids can be cultured because they are smaller and simpler than actual organs. For example, organoids can be passaged from human organ stem cells, or organ stem cells can be differentiated to obtain hollow structures composed of multiple types of organ-specific cells. The difference between organoids and spheroids is that spheroids are typically clumps of simple types of cells cultured in three dimensions, with cells filling the entire structure, while organoids are typically more complex tissues composed of multiple cells to have a specific function as an organ, and they have a hollow structure. The preparation and cultivation of organoids can be carried out using known methods, such as those taught in StemCellsInternational, Volume 2021, Article ID: 9929461, "Development of Human International Organoid Model for In Vitro Study Gut Inframation and Fibrosis", doi: 10.1155 / 2021 / 9929461.

[0072] The "cells" contained in the three-dimensional cell culture for transplantation may be any cells used for transplantation. While not limited to these, the cells may include, for example, clinically useful cells for treating or preventing symptoms related to cell, tissue, or organ defects and dysfunction, or cultureable cells used in non-clinical trials, etc., that have been isolated from a living organism. Examples include living tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissues, pluripotent stem cells capable of differentiating into various living tissues, and differentiated stem cells or progenitor cells. The cells used for cell transplantation may be autologous cells, allogeneic non-autologous cells, or xenologous cells. Furthermore, preferred cells are human autologous cells from the viewpoint of clinical application and safety, and human non-autologous cells from the viewpoint of clinical application and productivity.

[0073] Specific examples of living tissue cells include, for example, fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiomyocytes, myoblasts, osteocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, renal cells, gingival cells, periosteal cells, skin cells, and endothelial cells. Specific examples of mesenchymal stem cells include, for example, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells. Specific examples of pluripotent stem cells include, for example, induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured individually or in combination of two or more types. These cells may be appropriately selected from known types depending on the intended use of the cells.

[0074] The origin of the cells is not particularly limited, but examples include mammals, birds, amphibians, fish, insects, plants, and microorganisms. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens.

[0075] The three-dimensional cell cultures for transplantation immersed in the solution of the present invention described above may be provided in a container such as a cold-resistant container or a culture vessel (e.g., a multi-well cell culture plate). In the case of a multi-well cell culture plate, the three-dimensional cell cultures for transplantation are actually placed in each well. The term "container" includes any container capable of containing the three-dimensional cell cultures for transplantation described above, and also includes kits, etc. The container may also be accompanied by or come with instructions for use regarding the three-dimensional cell cultures for transplantation. Therefore, in one embodiment of the present invention, a container containing three-dimensional cell cultures for transplantation immersed in the solution of the present invention described above is provided. In another embodiment, a kit is provided containing frozen three-dimensional cell cultures for transplantation and the solution of the present invention.

[0076] One embodiment of the present invention provides a method for obtaining a three-dimensional cell culture for transplantation immersed in a solution, comprising the steps of (a) preparing a frozen three-dimensional cell culture for transplantation, and (b) thawing the three-dimensional cell culture for transplantation and immersing the thawed three-dimensional cell culture for transplantation in the solution of the present invention. Furthermore, another embodiment of the present invention provides a method for improving the viability or metabolic activity of cells in a three-dimensional cell culture for transplantation after freezing and thawing, comprising the steps of (a) and (b) above. This method is performed to improve the viability or metabolic activity of cells in a three-dimensional cell culture for transplantation after freezing and thawing. The steps of preparing, freezing, and thawing the three-dimensional cell culture for transplantation, and the step of immersing it in the solution of the present invention are described below.

[0077] First, a three-dimensional cell culture for transplantation is prepared. The three-dimensional cell culture for transplantation can be obtained by culturing cells for transplantation, which have been molded into a desired three-dimensional shape such as a sheet, sphere, or hollow, in a culture medium containing the above-mentioned culture solution (see, for example, publicly available literature on the production of cell sheets, spheroids, and organoids as exemplified above). In this case, a scaffold may be used as described above to form the three-dimensional cell culture on the scaffold. Next, a cryopreservation solution to be used for cryopreservation (for example, the commercially available cryopreservation solution mentioned above) is prepared. Then, after cell culture, the cryopreservation solution is added to the culture vessel containing the three-dimensional cell culture for transplantation, thereby placing or immersing the three-dimensional cell culture in the cryopreservation solution, or placing the three-dimensional cell culture in contact with the cryopreservation solution in any manner. If the cells were cultured in a state of adhesion to the culture vessel, such as a cell sheet, the cryopreservation solution may be added to the culture vessel while the cultured cells are still attached to the culture vessel without being detached from it. Alternatively, to facilitate the detachment of three-dimensional cell cultures, such as cell sheets, from the culture vessel after thawing, the three-dimensional cell cultures may be detached from the culture vessel and then placed back into the culture vessel. Methods for detachment may include enzymatic treatment using trypsin or other enzymes, or physical methods such as direct use of forceps.

[0078] One method for placing or immersing cultured cells in cryopreservation solution is to replace the culture medium in the culture vessel with cryopreservation solution and then place or immerse the cultured cells in the cryopreservation solution. The method for replacing the culture medium in the culture vessel with cryopreservation solution is not particularly limited, and known means can be used. Specific examples of replacement methods include, once the cultured cells have reached the desired state, using a pipette to remove the culture medium from the culture vessel and add the cryopreservation solution to the culture vessel; removing a portion of the culture medium from the culture vessel and adding the cryopreservation solution; or adding the cryopreservation solution to the culture medium in the culture vessel. Alternatively, a separate culture vessel may be prepared from the one used for culturing the three-dimensional cell culture, the cultured three-dimensional cell culture may be detached and transferred to the separate culture vessel, and then the three-dimensional cell culture may be immersed in cryopreservation solution in that separate culture vessel.

[0079] The freezing process involves freezing three-dimensional cell cultures for transplantation, typically by freezing them together with a cryopreservation solution. Typically, this involves freezing the three-dimensional cell cultures for transplantation, which are placed in or immersed in a cryopreservation solution at or above the coagulation temperature, or by placing the three-dimensional cell cultures, which are in contact with the cryopreservation solution in any manner, under conditions below the coagulation temperature of the cryopreservation solution. The freezing method is not particularly limited, and known freezing methods can be used. For example, a method of rapidly cooling a culture vessel containing the cryopreservation solution and the three-dimensional cell cultures for transplantation, which are under conditions above the coagulation temperature, to the freezing temperature is one example.

[0080] The freezing temperature is any temperature below the solidification temperature of the cryopreservation solution, and is not particularly limited as long as it can freeze the cultured cells and the cryopreservation solution. The solidification temperature of the cryopreservation solution is not limited, but may be -30°C to 0°C, -25°C to 0°C, -20°C to 0°C, -20°C to -5°C, or -15°C to -5°C, preferably -15°C to -5°C. For example, the lower limit of the freezing temperature may be selected from -220°C or higher, -196°C or higher, -180°C or higher, -165°C or higher, or -150°C or higher. On the other hand, the upper limit of the freezing temperature may be selected from -120°C or lower, -100°C or lower, -80°C or lower, -50°C or lower, -30°C or lower, -25°C or lower, or -20°C or lower, for example, the freezing temperature is -196°C to -25°C.

[0081] The freezing method is not particularly limited, and known freezing methods can be used. Examples include contact of a coolant with the liquid or gas phase, or the use of a cooling device. Examples of coolants used in the freezing process include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice. Examples of cooling devices used in the freezing process include rapid freezing devices and cryogenic refrigeration devices. As for the cooling device, a freezing device that does not contact the heat transfer means and freezes the culture vessel by blowing cold air onto the culture vessel from multiple directions, preferably all directions, rather than one direction, is preferable from the viewpoint of freezing the cells at a uniform temperature and increasing the viability of the cells after thawing, compared to a cooling device that freezes the culture vessel and cells by contacting the heat transfer means with the culture vessel. Furthermore, the storage time after freezing can be appropriately selected depending on the intended use, but examples include 30 minutes, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 10 hours or more, 20 hours or more, 1 day or more, 5 days or more, 10 days or more, 1 month or more, 6 months or more, 1 year or more, or 5 years or more. Three-dimensional cell cultures for transplantation, frozen together with the cryopreservation solution, can be stored and transported in the form of a container or package containing these.

[0082] The thawing method is not particularly limited, and any known thawing method can be used. Methods of freezing and thawing include simply placing the container containing the cell culture and cryopreservation solution in an environment above the freezing point of the cryopreservation solution, or using, for example, a water bath, bead bath, incubator, or hot plate. The thawing temperature or the ambient temperature in contact with the frozen material during thawing is not particularly limited, as long as it is above the freezing point of the cryopreservation solution and 45°C or lower. A thawing temperature higher than 45°C is undesirable because it may cause thermal damage to the cells. Under the condition that the thawing temperature is above the condensation temperature of the cell preservation solution, the upper limit of the thawing temperature may be selected from 45°C, 40°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, or 25°C, and the lower limit of the thawing temperature may be selected from 0°C, 5°C, 10°C, 15°C, or 20°C. Alternatively, under the condition that the thawing temperature is above the condensation temperature of the cell preservation solution, the range is 0 to 45°C (including any value within these ranges at 1°C intervals), more preferably 10 to 35°C or 15 to 30°C, and even more preferably 15 to 25°C or 20 to 25°C. Furthermore, the time required to thaw the frozen material is not particularly limited as long as no damage to the cells occurs due to thawing. Typically, thawing can be done without damaging the cells in 10 seconds to 60 minutes. The lower limit of the time required to thaw a frozen product may be 10 seconds or more, preferably 20 seconds or more, more preferably 30 seconds or more, even more preferably 1 minute or more, and may be 2 minutes or more. The upper limit may be 60 minutes or less, preferably 50 minutes or less, more preferably 40 minutes or less, and even more preferably 30 minutes or less. For example, the time required to thaw is 10 seconds to 60 minutes, 10 seconds to 50 minutes, 10 seconds to 40 minutes, 10 seconds to 30 minutes, 20 seconds to 60 minutes, 20 seconds to 50 minutes, 20 seconds to 40 minutes, 20 seconds to 30 minutes, 30 seconds to 60 minutes, 30 seconds to 50 minutes, 30 seconds to 40 minutes, 30 seconds to 30 minutes, 1 minute to 60 minutes, 1 minute to 50 minutes, 1 minute to 40 minutes, or 1 minute to 30 minutes.

[0083] In one embodiment of the step of immersing in the solution of the present invention in step (b) above, first, the three-dimensional cell culture for transplantation and the cryopreservation solution are thawed, and typically, the supernatant of the cryopreservation solution, which has become a solution, is absorbed and removed. After removing the thawed cryopreservation solution, the solution of the present invention is added to the container and the three-dimensional cell culture for transplantation is immersed in the solution of the present invention. Therefore, one embodiment of the present invention provides a method for obtaining a three-dimensional cell culture for transplantation immersed in a solution, comprising the steps of (a) freezing the three-dimensional cell culture for transplantation together with the cryopreservation solution in a container, and (b) thawing the three-dimensional cell culture for transplantation and the cryopreservation solution, removing the thawed cryopreservation solution from the container, then adding the solution of the present invention to the container and immersing the three-dimensional cell culture for transplantation in the solution. In this case, after the removal of the cryopreservation solution, some of the cryopreservation solution may remain, and cryoprotective agents and / or animal-derived components contained in the remaining cryopreservation solution may be mixed into the solution. To prevent contamination, it is preferable to remove all or substantially all of the cryopreservation solution. For example, it is preferable that 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the cryopreservation solution is removed. Therefore, considering that such contamination of cryoprotective agents may occur, from a clinical standpoint, it is preferable that the concentrations of cryoprotective agents and animal-derived components in the cryopreservation solution used for freezing be low. Specifically, it is preferable that the amount of cryoprotective agent in the cryopreservation solution be less than 0.5 w / v% based on the volume of the solution of the present invention, and that the amount of animal-derived components in the cryopreservation solution be 0.5% by weight or less based on the weight of the solution of the present invention.

[0084] Alternatively, in one embodiment of the step of immersing in the solution of the present invention in step (b) above, the frozen and thawed three-dimensional cell cultures in the container may be removed from the container and transferred to another container containing the solution of the present invention for immersion. Accordingly, in one embodiment of the present invention, a method is provided for obtaining three-dimensional cell cultures for transplantation immersed in a solution, comprising the steps of (a) freezing the three-dimensional cell cultures for transplantation together with the cryopreservation solution in a container, and (b) thawing the three-dimensional cell cultures for transplantation and the cryopreservation solution, removing the three-dimensional cell cultures for transplantation from the container, and then immersing them in the solution of the present invention in another container. It should be noted that the cryopreservation solution may adhere to the three-dimensional cell cultures for transplantation removed in step (b), and when placed in the solution of the present invention in another container, the cryoprotective agent and animal-derived components contained in the attached cryopreservation solution may be mixed in. Accordingly, considering that contamination with cryoprotective agents may occur as described above, from a clinical standpoint, it is preferable that the concentration of cryoprotective agents and animal-derived components contained in the cryopreservation solution used for freezing be low, and the preferred concentrations are the same as those for contamination occurring in the same container as described above.

[0085] Alternatively, in one embodiment of the step of immersing in the solution of the present invention in step (b) above, after thawing the three-dimensional cell culture for transplantation, all or part of the cryopreservation solution may be left behind, and the necessary inorganic salts may be added to the remaining cryopreservation solution to adjust the concentrations of calcium salts and magnesium salts in the remaining cryopreservation solution and the osmotic pressure of the remaining cryopreservation solution to the predetermined concentrations and osmotic pressures. In this specification, such an adjusted cryopreservation solution may be considered synonymous with "the solution of the present invention". Accordingly, in one aspect of the present invention, a method is provided for obtaining a three-dimensional cell culture for transplantation immersed in a solution, comprising the steps of: a) freezing the three-dimensional cell culture for transplantation together with a cryopreservation solution in a container; and b) thawing the three-dimensional cell culture for transplantation and the cryopreservation solution, leaving some or all of the thawed cryopreservation solution, and adjusting the calcium salt concentration and / or magnesium salt concentration in the remaining cryopreservation solution and the osmotic pressure of the remaining cryopreservation solution so that the remaining cryopreservation solution contains 5 to 700 ppm of calcium salt and / or 5 to 1000 ppm of magnesium salt, and has an osmotic pressure of 150 to 1200 mOsm / L, based on the total weight of the remaining cryopreservation solution. As described above, from a clinical standpoint, it is preferable that the concentrations of cryoprotective agents and animal-derived components contained in the cryopreservation solution used for freezing be low, and the preferred concentrations thereof are the same as in the case of contamination described above.

[0086] Alternatively, in one embodiment of the step of immersing in the solution of the present invention in step (b) above, after thawing, all or part of the cryopreservation solution may be left behind and the remaining cryopreservation solution may be diluted with any liquid (for example, the solution of the present invention, PBS, or physiological saline) (in this case, the liquid may be added to the container while partially thawing the cryopreservation solution is removed by aspirate), and then the necessary inorganic salts may be added to the diluted cryopreservation solution to adjust it so that the above-mentioned predetermined calcium salt and / or magnesium salt concentrations and predetermined osmotic pressure are achieved. In this specification, such an adjusted diluted cryopreservation solution may also be considered synonymous with "the solution of the present invention". Accordingly, in one aspect of the present invention, a method is provided for obtaining a three-dimensional cell culture for transplantation immersed in a solution, comprising the steps of (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container, and (b) thawing the three-dimensional cell culture and the cryopreservation solution, leaving some or all of the thawed cryopreservation solution, diluting the remaining cryopreservation solution with any liquid, and adjusting the calcium salt concentration and / or magnesium salt concentration in the diluted cryopreservation solution and the osmotic pressure of the diluted cryopreservation solution, based on the total weight of the diluted cryopreservation solution (i.e., the above liquid and the cryopreservation solution combined), so that the diluted cryopreservation solution contains 5 to 700 ppm of calcium salt and / or 5 to 1000 ppm of magnesium salt and has an osmotic pressure of 150 to 1200 mOsm / L. From a clinical standpoint, it is preferable to use a low concentration of cryoprotective agents and animal-derived components in the cryopreservation solution for freezing, similar to the concentration in the case of the above-mentioned contamination, or to adjust the amount of liquid used for dilution to lower the concentration of cryoprotective agents and animal-derived components in the diluent.

[0087] The immersion time in the solution of the present invention described above may be at least 30 seconds, for example, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or more, 1 day or more, 2 days or more, 5 days or more, 10 days or more, or longer. Preferably it is 3 minutes or more, more preferably 5 minutes or more. Furthermore, as shown in Example 3 described later, when a three-dimensional cell culture for transplantation is immersed directly in the cryopreservation solution without removing the thawed cryopreservation solution, the cell viability tends to decrease over time, whereas when immersed in the solution of the present invention, it has been observed that a high metabolic activity rate is maintained even after, for example, 2 hours.

[0088] Alternatively, after immersing the three-dimensional cell culture for transplantation in the solution of the present invention, the three-dimensional cell culture for transplantation may be removed from the culture vessel or the like and washed with physiological saline solution or the like.

[0089] By immersing a three-dimensional cell culture for transplantation in the solution of the present invention, a three-dimensional cell culture for transplantation with improved cell viability or metabolic activity after thawing can be obtained. Here, the "metabolic activity rate" when immersed in the solution of the present invention is defined as the value obtained by dividing the metabolic activity C1 of the three-dimensional cell culture for transplantation obtained by immersing the three-dimensional cell culture for transplantation in the solution of the present invention by the metabolic activity C0 obtained by immersing the three-dimensional cell culture in a control solution (a solution that does not contain the predetermined concentrations of calcium and magnesium salts specified for the solution of the present invention; typical examples of a control solution that does not contain the predetermined concentrations of calcium and magnesium salts include phosphate-buffered saline or saline). Specifically in this specification, metabolic activity is measured using tetrazolium salt WST-8 (CellCountReagentSF, manufactured by Nacalai Tesque, product number 07553-44), which is a reagent for measuring the number of viable cells. This is a cell counting method that uses intracellular reductase activity as an indicator, and utilizes the fact that when reduced in the presence of an electron carrier, a highly water-soluble orange formazan dye is produced. Those skilled in the art will understand that the "metabolic activity rate" when the solution of the present invention is used compared to a control solution is an indicator for determining whether the "cell viability" (defined as the ratio of the number of viable cells to the reference value (C0 in this specification)) has improved. The specific procedure for measuring "metabolic activity" is as follows: After immersing the above-mentioned three-dimensional culture medium for transplantation in the solution of the present invention, first remove the solution of the present invention, then add a 2% serum-containing medium, and set the temperature to 37°C and 5% CO2. ２ The cells are re-cultured in the specified environment for 24 hours. After re-culture, the WST-8 viable cell count reagent is diluted 20-fold with 2% serum-containing medium (reagent / medium = 1 / 19 volume ratio) to prepare the test solution. Then, a certain amount of the above test solution is added to a container containing the three-dimensional cell culture, and the cells are incubated at 37°C and 5% CO2. ２Incubate in the specified environment for 1 hour. After incubation, add the supernatant of the test solution to a multi-well cell culture plate, then measure this supernatant with a plate reader and determine the metabolic activity using the following formula: Metabolic activity C = {Absorbance of the sample (450 nm) - Absorbance of the sample (630 nm)} - {Absorbance of the blank (450 nm) - Absorbance of the blank (630 nm)}. The test solution is used as the blank. In the examples described later, the above measurement method is used in all cases.

[0090] In one embodiment of the present invention, the metabolic activity rate (C1 / C0) obtained by dividing the metabolic activity C1 when using the solution of the present invention obtained by the above measurement by the metabolic activity C0 when using physiological saline is 1.04 or higher, preferably 1.08 or higher, 1.1 or higher, 1.15 or higher, or 1.2 or higher, more preferably 1.25 or higher, 1.3 or higher, 1.35 or higher, even more preferably 1.4 or higher, 1.45 or higher, or 1.5 or higher, even more preferably 1.55 or higher, 1.6 or higher, or 1.65 or higher, even more preferably 1.7 or higher, 1.75 or higher, or 1.8 or higher, even more preferably 1.85 or higher, 1.9 or higher, or 1.95 or higher, most preferably 2.0 or higher, 2.05 or higher, or 2.1 or higher. Furthermore, in one embodiment of the present invention, the metabolic activity rate (C1 / C0) obtained by dividing the metabolic activity C1 when using the solution of the present invention obtained by the above measurement by the metabolic activity C0 when using phosphate-buffered saline is 1.05 or higher, preferably 1.09 or higher, 1.1 or higher, 1.15 or higher, or 1.2 or higher, more preferably 1.25 or higher, 1.3 or higher, 1.35 or higher, even more preferably 1.4 or higher, 1.45 or higher, or 1.5 or higher, even more preferably 1.55 or higher, 1.6 or higher, or 1.65 or higher, even more preferably 1.7 or higher, 1.75 or higher, or 1.8 or higher, even more preferably 1.85 or higher, 1.9 or higher, or 1.95 or higher, most preferably 2.0 or higher, 2.05 or higher, or 2.1 or higher.

[0091] In another embodiment of the present invention, a method for treating or preventing a disease is provided by transplanting an effective amount of the three-dimensional cell culture obtained above to the affected area of ​​a patient with the disease. "Transplantation" refers to attaching the three-dimensional cell culture without excising the affected area, as well as excising all or part of the affected area and attaching or embedding the three-dimensional cell culture at the excised site. Examples of tissues that can be treated include, but are not limited to, skin, oral tissue, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, and bone. Examples of diseases that can be treated include spinal cord injury, knee joint cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell deficiency, aplastic anemia, severe lower limb ischemia, intractable skin ulcers, prevention of postoperative complications (e.g., anastomotic leakage of various organs, bronchial stump fistula, pancreatic fistula, bile leak), and burns.

[0092] Although embodiments of the present invention have been described above, these are merely examples, and various other configurations can be adopted. Furthermore, the present invention is not limited to the embodiments described above, and modifications, improvements, etc., within the scope that can achieve the objectives of the present invention are included in the present invention.

[0093] Examples of reference forms are provided below. [Embodiment 1] A solution for immersing a freeze-thawed three-dimensional cell culture for transplantation, wherein the solution contains 5 to 700 ppm of calcium salt and / or 5 to 1000 ppm of magnesium salt based on the total weight of the solution, and has an osmotic pressure of 150 to 1200 mOsm / L. [Embodiment 2] The solution according to Embodiment 1, wherein the solution contains 50 to 700 ppm of calcium and / or 10 to 700 ppm of magnesium salt based on the total weight of the solution. [Embodiment 3] The solution according to Embodiment 1 or 2, wherein the solution has an osmotic pressure of 200 to 1000 mOsm / L. [Embodiment 4] The solution according to any one of Embodiments 1 to 3, wherein the solution does not contain any animal-derived components. [Embodiment 5] The solution according to any one of Embodiments 1 to 4, wherein the solution does not contain any cryoprotective agent. [Embodiment 6] The solution according to any one of Embodiments 1 to 5, wherein the three-dimensional cell culture is a cell sheet, a spheroid, an organoid, or a combination thereof. [Embodiment 7] The solution according to Embodiment 6, wherein the cell sheet is on a scaffold or a culture carrier substrate. [Embodiment 8] The solution according to any one of Embodiments 1 to 7, wherein the cells in the three-dimensional cell culture are cells derived from mammals. [Embodiment 9] The solution according to Embodiment 8, wherein the cells derived from mammals are selected from the group consisting of living tissue cells, mesenchymal stem cells, and pluripotent stem cells. [Embodiment 10] A three-dimensional cell culture for transplantation that has been frozen and thawed, and is immersed in the solution according to any one of Embodiments 1 to 9. [Embodiment 11] The three-dimensional cell culture according to Embodiment 10, wherein a cryoprotective agent is mixed into the solution. [Embodiment 12] The three-dimensional cell culture according to Embodiment 11, wherein the concentration of the cryoprotective agent is less than 0.5 w / v% based on the volume of the solution. [Embodiment 13] A three-dimensional cell culture according to any one of Embodiments 10 to 12, wherein the metabolic activity rate (C1 / C0) obtained by dividing the cellular activity C1 of the three-dimensional cell culture after immersion in the solution by the metabolic activity C0 of the three-dimensional cell culture after immersion in physiological saline is 1.04 or more.[Embodiment 14] A method for obtaining a three-dimensional cell culture for transplantation immersed in a solution, comprising the steps of (a) freezing the three-dimensional cell culture for transplantation, and (b) thawing the three-dimensional cell culture and immersing the thawed three-dimensional cell culture in the solution described in any one of Embodiments 1 to 9. [Embodiment 15] The method according to Embodiment 14, comprising the steps of (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container, and (b) thawing the three-dimensional cell culture and the cryopreservation solution, removing the thawed cryopreservation solution from the container, then adding the solution described in any one of Embodiments 1 to 9 to the container and immersing the three-dimensional cell culture in the solution. [Embodiment 16] The method according to Embodiment 14, comprising the steps of (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container, and (b) thawing the three-dimensional cell culture and the cryopreservation solution, removing the thawed three-dimensional cell culture from the container, and then immersing it in the solution of the present invention in another container. [Embodiment 17] The method according to Embodiment 14, comprising the steps of (a) freezing the three-dimensional cell culture in a container together with a cryopreservation solution, and (b) thawing the three-dimensional cell culture and the cryopreservation solution, leaving some or all of the thawed cryopreservation solution, and adjusting the calcium salt concentration and / or magnesium salt concentration in the remaining cryopreservation solution and the osmotic pressure of the remaining cryopreservation solution so that the remaining cryopreservation solution contains 5 to 700 ppm of calcium salt and / or 5 to 1000 ppm of magnesium salt and has an osmotic pressure of 150 to 1200 mOsm / L based on the total weight of the remaining cryopreservation solution.[Embodiment 18] The method according to Embodiment 14, comprising the steps of (a) freezing the three-dimensional cell culture in a container together with the cryopreservation solution, and (b) thawing the three-dimensional cell culture and the cryopreservation solution, leaving some or all of the thawed cryopreservation solution, diluting the remaining cryopreservation solution with any liquid, and adjusting the calcium salt concentration and / or magnesium salt concentration in the diluted cryopreservation solution and the osmotic pressure of the diluted cryopreservation solution so that the diluted cryopreservation solution contains 5 to 700 ppm of calcium salt and / or 5 to 1000 ppm of magnesium salt based on the total weight of the diluted cryopreservation solution and has an osmotic pressure of 150 to 1200 mOsm / L. [Embodiment 19] The method according to any one of Embodiments 14 to 18, wherein in step (b), the three-dimensional cell culture is immersed in the solution for at least 30 seconds. [Embodiment 20] The method according to Embodiment 14, wherein in step (a), the three-dimensional cell culture is thawed at 0 to 45°C. [Embodiment 21] The method according to any one of Embodiments 14 to 20, wherein the metabolic activity rate (C1 / C0) obtained by dividing the cellular activity C1 of the three-dimensional cell culture after immersion in the solution by the metabolic activity C0 of the three-dimensional cell culture after immersion in physiological saline is 1.04 or more. [Embodiment 22] A kit comprising a frozen three-dimensional cell culture for transplantation and the solution according to any one of Embodiments 1 to 9.

[0094] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way to the descriptions of these examples.

[0095] Example 1 <Production of Culture Carrier Substrate> Plasma treatment was performed on the culture surface of a PEEK film (Shin-Etsu Polymer Co., Ltd., polyether ether ketone film, Shin-EtsuSeplaFilm®, low crystallinity type, 12 μm thickness). The surface-treated film was then cut to a size of Φ14.0 mm to obtain a disc-shaped culture carrier substrate.

[0096] <Cell Sheet Production> The culture carrier substrate produced in <Culture Carrier Substrate Production> above was washed in 70% ethanol, phosphate-buffered saline, and culture medium in that order, and then placed on the flat bottom of each well in a 24-well cell culture plate. At this time, the culture surface of the culture carrier substrate was positioned facing the opening of the well plate, that is, the back surface of the culture carrier substrate was in contact with the flat bottom of each well in the cell culture multiwell plate. Cryopreserved human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with culture medium. 5 × 10 ５ Suspend individual cells in a 5% serum-containing medium and place them on one dish (150 cm²). ２ After seeding, the cells were cultured for 4 days. The cultured cells were harvested, suspended in a 5% serum-containing medium, and placed in 10 dishes (culture area 150 cm²). ２ ) 5 x 10 ５ After seeding each cell individually and subculturing, the cells were cultured for 3 days. The cultured cells were harvested, suspended in a 2% serum-containing medium, and placed in each well of a multi-well cell culture plate with a culture carrier substrate, measuring 27.9 × 10⁴. ４ pieces / cm ２ Seeds were sown at the following density, at 37°C and 5% CO2. ２ The cells were incubated in the specified environment for two days, and a cell sheet was prepared on the culture surface of the culture carrier substrate.

[0097] <Cryopreservation of Cell Sheets> After the above <Cell Sheet Manufacturing> procedure, the cell sheets with culture carrier substrate were washed with phosphate-buffered saline. Then, 0.22 mL of cryopreservation solution (BanBanCar® hRM, manufactured by GC Lymphotek, product number CS-11-002; containing 10 w / v% DMSO and 1% by weight of human albumin) was added per well, and the cell sheets were immersed in the cryopreservation solution in each well. Note that the calcium salts and magnesium salts contained in BanBanCar® hRM are both less than 5 ppm. Subsequently, the cell culture multiwell plates were packaged in resealable poly bags and placed in a non-through-flow cooling device (3D Freezer®, KSS-40BLW-2400V, manufactured by Kogasan) cooled to -35°C and held for 30 minutes. The cooling rate at 0 to -5°C was 3.4°C / min. Subsequently, the cooled packages were promptly placed in a -150°C freezer (CLN-17000WE, manufactured by Nippon Freezer Co., Ltd.) and kept there for 24 hours.

[0098] <Thawing of Cell Sheets> After the freezing period described above, the packaging was removed from the -150°C freezer, and the cell culture multiwell plates inside were allowed to thaw at room temperature (20-25°C). After the cryopreservation solution had thawed completely and become a solution, the cryopreservation solution was aspirated and removed. The thawing time at this time was 15 minutes.

[0099] <Preparation of the solution of the present invention and measurement of metabolic activity> In this example, in order to investigate how the difference in calcium salt and magnesium salt concentrations in the solution of the present invention affects the metabolic activity rate or cell viability, calcium chloride (CaCl) was added to physiological saline (Otsuka Saline Injection, manufactured by Otsuka Pharmaceutical Co., Ltd., drug code 3311401A7028). ２ ・2H ２ By adding (O, Fujifilm Wako Pure Chemical Industries, Ltd., product code 031-25035), the solutions of the present invention having calcium salt concentrations of 1, 5, 10, 50, 100, 350, 700, 1000, and 2000 ppm are prepared. Also, magnesium sulfate (anhydrous) (MgSO4) is added to physiological saline. ４By adding (Fujifilm Wako Pure Chemical Industries, Ltd., product code 137-12335), solutions of the present invention having magnesium salt concentrations of 1, 5, 10, 50, 100, 350, 700, 1000, and 2000 ppm were prepared (the osmotic pressure of these solutions was set to 308-458 mOsm / L). After aspirating and removing the above cryopreservation solution, each solution of the present invention or physiological saline (control) was added to a total volume of 0.22 mL / well, and the cell sheets were immersed in each solution and allowed to stand at room temperature (20-25°C) for 60 minutes. Next, metabolic activity was measured using the viable cell count reagent WST-8 (details of the method for measuring metabolic activity are as described above), and the metabolic activity rate was obtained by dividing the metabolic activity when using the solution of the present invention by the metabolic activity when using physiological saline (i.e., the metabolic activity when using physiological saline (control) was used as the standard, and the metabolic activity rate C0 in this case was set to 1). The results are shown in Figure 1. As shown in Figure 1(A), compared to physiological saline (control), the solution of the present invention showed an improvement in metabolic activity when it contained 5 ppm of calcium salt, and it was found that the metabolic activity was significantly higher when it contained 50 to 700 ppm of calcium salt. Furthermore, as shown in Figure 1(B), compared to physiological saline (control), the solution of the present invention showed an improvement in metabolic activity when it contained 5 ppm of magnesium salt, and a significant improvement in metabolic activity was also observed at a magnesium salt concentration of 10 ppm, and it was found that the metabolic activity was significantly higher when the concentration was 50 to 700 ppm.

[0100] Example 2 The production of culture carrier substrates, cell sheets, cryopreservation of cell sheets, and thawing of cell sheets were carried out in the same manner as in Example 1. Preparation of the solutions of the present invention and measurement of metabolic activity In this implementation, we investigated how the differences in osmotic pressure of the solutions of the present invention affect the metabolic activity rate or cell viability. As shown in Table 2, by adding sodium chloride (NaCl, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code 191-01665) to distilled water, solutions having osmotic pressures of 10, 50, 100, 200, 300, 500, 1000, 2000, and 3000 mOsm / L were prepared. Furthermore, calcium chloride and magnesium sulfate (anhydrous) were added to each solution to achieve constant values ​​of calcium salt concentration of 35.0 ppm (1.7 mmol / L) and magnesium salt concentration of 18.9 ppm (0.8 mmol / L), respectively, to prepare the solutions of the present invention as shown in Table 2. Furthermore, as control solutions, as shown in Table 3, distilled water was mixed with only sodium chloride to prepare solutions (physiological saline) with osmotic pressures of 10, 50, 100, 200, 300, 500, 1000, 2000, and 3000 mOsm / L.

[0101]

[0102]

[0103] After thawing the cell sheets and removing the cryopreservation solution by aspirating, 0.22 mL / well of each solution of the present invention or each control solution was added, and the cell sheets were immersed in each solution and allowed to stand at room temperature (20-25°C) for 60 minutes. Next, metabolic activity was measured using the viable cell count reagent WST-8 (details of the method for measuring metabolic activity are as described above), and the metabolic activity rate was obtained by dividing the metabolic activity when using the solution of the present invention by the metabolic activity when using the control solution (i.e., the metabolic activity with the control solution (physiological saline) was used as the baseline, and the metabolic activity rate C0 in this case was set to 1). The results are shown in Figure 2. As shown in Figure 2, high metabolic activity rates were obtained when using each solution of the present invention with osmotic pressures of 200, 300, 500, and 1000 mOsm / L, while very low metabolic activity rates were obtained when the osmotic pressure was less than 100 mOsm / L and 2000 mOsm / L or more, indicating that osmotic pressure greatly affects metabolic activity rate or cell viability.

[0104] Example 3 The following steps were taken in the same manner as in Example 1: <Preparation of Culture Carrier Substrate>, <Preparation of Cell Sheet>, <Cryopreservation of Cell Sheet>, and <Thawing of Cell Sheet>. <Preparation of Solution of the Present Invention and Measurement of Metabolic Activity> In this example, we investigated how the immersion time of the cell sheet in the solution after freezing and thawing of a cell culture multiwell plate affected the metabolic activity rate or cell viability. After aspirating and removing the thawed cryopreservation solution, the solution of the present invention (phosphate-buffered saline (PBS(-), manufactured by Cell Science Institute, product code 1102P10B), calcium chloride and magnesium sulfate (anhydrous) were added to adjust the calcium salt concentration to 35.0 ppm and the magnesium salt concentration to 18.9 ppm, and the osmotic pressure to 315 mOsm / L) was added to 0.22 mL / well, the cell sheet was immersed in the solution, and allowed to stand at room temperature (20-25°C) for 30 seconds, 5 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes. As a control, after thawing and removing the cryopreservation solution, PBS(-) was added to a level of 0.22 mL / well, and the cell sheets were immersed in the solution and allowed to stand at room temperature (20-25°C) for 30 seconds, 5 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes. Furthermore, for comparison, without removing the cryopreservation solution after thawing, the cell sheets were immersed directly in the cryopreservation solution and allowed to stand at room temperature (20-25°C) for 30 seconds, 5 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes. Next, metabolic activity was measured using the WST-8 viable cell count reagent (details of the metabolic activity measurement method are as described above), and the metabolic activity rate was obtained by dividing the metabolic activity when using the solution of the present invention and when using the cryopreservation solution as is by the metabolic activity when using the control solution (physiological saline). The results are shown in Figure 3. In Figure 3, (A) shows the case when immersed in PBS(-) (the metabolic activity rate in this case is set to 1), (B) shows the case when immersed in the cryopreservation solution, and (C) shows the case when immersed in the solution of the present invention. As shown in Figure 3, after more than 5 minutes, it was found that when the solution of the present invention was used (case (C)), a significantly higher metabolic activity rate was obtained than in either case (A) or (B), and it was observed that a high metabolic activity rate was maintained even after a long period of time (e.g., 2 hours).

[0105] Example 4 The following steps were taken in the same manner as in Example 1: <Preparation of Culture Carrier Substrate>, <Preparation of Cell Sheet>, <Cryopreservation of Cell Sheet>, and <Thawing of Cell Sheet>. <Preparation of Solution of the Invention and Measurement of Metabolic Activity> In this example, after freezing and thawing a cell culture multiwell plate, the metabolic activity rate was compared between immersion in the solution of the invention and immersion in basal medium and basal medium containing animal-derived components. After aspirating and removing the thawed cryopreservation solution, PBS(-) and the solution of the invention (prepared by adding calcium chloride and magnesium sulfate (anhydrous) to PBS(-), having a calcium salt concentration of 70 ppm and a magnesium salt concentration of 19 ppm) were added to a total volume of 0.22 mL / well, and the cell sheets were immersed in each solution and left to stand at room temperature (20-25°C) for 60 minutes. For comparison, after aspirating and removing the above cryopreservation solution, 0.22 mL / well of a basal medium free of animal-derived components (HFDM-1(+) Medium, manufactured by the Institute of Cell Science, product code 2102P10) and a basal medium containing animal-derived components (NeoSERA®, a serum for cell culture containing adult bovine serum) (HFDM-1(+) Medium and AIMV Medium (manufactured by ThermoFisher, product number 12055083) mixed in a 1:1 ratio) were added, and the cell sheets were immersed in each solution and left to stand at room temperature (20-25°C) for 60 minutes. Next, metabolic activity was measured using the WST-8 viable cell counting reagent (details of the metabolic activity measurement method are as described above). The metabolic activity rate was obtained by dividing the metabolic activity when using the solution of the present invention, as well as when using basal media without animal-derived components and basal media containing animal-derived components, by the metabolic activity when using the control solution (PBS(-)). The results are shown in Figure 4. As shown in Figure 4, when immersed in the solution of the present invention, a metabolic activity rate similar to that when immersed in basal media without animal-derived components was obtained, while when immersed in basal media containing animal-derived components, a lower metabolic activity rate was obtained than when immersed in the solution of the present invention.

[0106] Example 5 The following steps were taken in the same manner as in Example 1: <Preparation of Culture Carrier Substrate>, <Preparation of Cell Sheet>, <Cryopreservation of Cell Sheet>, and <Thawing of Cell Sheet>. <Preparation of Solution of the Invention and Measurement of Metabolic Activity> In this example, the metabolic activity rate was investigated when the solution of the invention contained both calcium salt and magnesium salt. As the solution of the invention, calcium chloride and magnesium sulfate were added to PBS(-) (osmotic pressure 308 mOsm / L) to prepare a solution of the invention containing 70 ppm of calcium salt and 19 ppm of magnesium salt (the osmotic pressure of these solutions was 320 mOsm / L). After aspirating and removing the thawed cryopreservation solution, the solution of the invention was added to a container, and the cell culture multiwell plate was left to stand at room temperature (20-25°C) for 60 minutes. Next, the metabolic activity was measured using the viable cell count reagent WST-8 (details of the method for measuring metabolic activity are as described above), and the metabolic activity rate was obtained by dividing the metabolic activity when using the solution of the invention by the metabolic activity when using the control solution (PBS(-)). As an additional comparison, cell sheets were immersed in physiological saline alone (osmotic pressure 308 mOsm / L), distilled water alone, and distilled water containing the above concentrations of calcium and magnesium salts as the solution of the present invention, using the same procedure. The metabolic activity of each solution was measured and the metabolic activity rate was obtained by dividing by the metabolic activity when PBS(-) was used. Furthermore, the metabolic activity rate when cell sheets were immersed in the cryopreservation solution without removing it after freezing and thawing (PBS(-) was the control) was also obtained. The results are shown in Figure 5. As shown in Figure 5, the metabolic activity rate was greatly improved when using the solution of the present invention compared to when PBS(-) was used alone and when physiological saline alone. On the other hand, when physiological saline was used, a metabolic activity rate similar to that when PBS(-) was used was obtained. In addition, the metabolic activity rate was significantly lower when distilled water was used and when distilled water containing the above concentrations of calcium and magnesium salts was used, which is thought to be largely influenced by osmotic pressure.

[0107] Example 6 <Production of culture carrier substrate>, <Production of cell sheet>, <Cryopreservation of cell sheet>, and <Thawing of cell sheet> were carried out in the same manner as in Example 1. <Preparation of the solution of the present invention and measurement of metabolic activity> In this example, the effect of residual DMSO from the cryopreservation solution on the metabolic activity rate of the solution of the present invention was investigated. Calcium chloride and magnesium sulfate were added to physiological saline to prepare solution A. After the thawing process, the thawed cryopreservation solution (containing 10 w / v% DMSO) was not removed, and solution A was added to dilute the cryopreservation solution. At that time, solution A was added so that the volume of the cryopreservation solution was diluted to 1 / 2, 1 / 4, 1 / 8, and 1 / 10 (the DMSO content after dilution was 5 w / v%, 2.5 w / v%, 1.25 w / v%, and 1 w / v%, respectively). Multiple solutions of solution A were prepared, and each was adjusted to a concentration such that the solution after diluting the cryopreservation solution at the above ratios had a calcium salt concentration of 35.4 ppm and a magnesium salt concentration of 19.3 ppm. After diluting the cryopreservation solution with solution A at the above ratio, the osmotic pressure of each solution was in the range of 313 to 322 mOsm / L (each of these solutions is considered "the solution of the present invention"). Cell sheets immersed in each solution (the solution of the present invention) were left to stand at room temperature (20-25°C) for 60 minutes. For further comparison, after aspirating and removing the thawed cryopreservation solution, physiological saline (control) and an additional solution of the present invention (physiological saline to which calcium chloride and magnesium sulfate were added to adjust the calcium salt concentration to 35.4 ppm and the magnesium salt concentration to 19.3 ppm, with an osmotic pressure of 315 mOsm / L. This is shown as "buffer replacement" in Figure 6. The DMSO concentration was less than 0.5 w / v%) were added to a total volume of 0.22 mL / well, and cell sheets were immersed in physiological saline (control) and the additional solution of the present invention, respectively, and left to stand at room temperature (20-25°C) for 60 minutes. The concentrations of physiological saline (control) and the additional DMSO solution of the present invention after immersion of the cell sheets were both less than 0.5 w / v%. Furthermore, for an additional comparison, after thawing, the cell sheets were left standing for 60 minutes while immersed in the cryopreservation solution without removing it.Next, metabolic activity was measured using the WST-8 viable cell counting reagent (details of the method for measuring metabolic activity are as described above). The metabolic activity rate was obtained by dividing the metabolic activity when using each of the above solutions (solutions of the present invention), an additional solution of the present invention ("buffer replacement"), and the cryopreservation solution ("standing directly in cryopreservation solution") by the metabolic activity when using the control solution (physiological saline). The results are shown in Figure 6. As shown in Figure 6, even when DMSO was present in the solution in which the cell sheet was immersed (i.e., when using the above diluted solution and "buffer replacement"), a significantly higher metabolic activity rate was obtained than when using physiological saline (control).

[0108] The results of measuring the essential amino acid content in the culture medium using the method described below are shown below. The physiological saline, distilled water, and PBS(-) used in Examples 1 to 6 all contained 0 essential amino acids in the culture medium. In all of the basal media used in Example 4, the content of essential amino acids in the medium exceeded the upper limits specified above ((i) L-histidine greater than 20 mg / L, (ii) L-isoleucine greater than 40 mg / L, (iii) L-leucine greater than 40 mg / L, (iv) L-lysine greater than 50 mg / L, (v) L-methionine greater than 10 mg / L, (vi) L-phenylalanine greater than 20 mg / L, (vii) L-threonine greater than 7 mg / L, (viiii) L-valine greater than 40 mg / L, (ix) L-arginine greater than 40 mg / L, (x) L-cystine greater than 20 mg / L, (xi) L-tyrosine greater than 10 mg / L).

[0109] [Measurement of Essential Amino Acids in the Culture Medium] The concentrations of essential amino acids in the culture medium contained in the following cryopreservation solutions A to D were measured according to the procedure below. The results for cryopreservation solution A (BanBanKar® hRM) are shown in Table 3. In all of the cryopreservation solutions B (StemCell Banker® EX GMP Grade (11936, manufactured by Nippon Zenyaku Kogyo Co., Ltd.), C (iStock (385-19451, manufactured by GC Lymphotec), and D (SOFORO Cryo, manufactured by Saraya Co., Ltd.), which were measured using the same procedure as cryopreservation solution A, it was confirmed that the concentrations of the 11 essential amino acids in the culture medium shown in Table 3 were all lower than the concentrations of each essential amino acid shown in cryopreservation solution A. Note that a concentration of 0 means less than 0.5 mg / L.

[0110]

[0111] (Procedure for Measuring Amino Acid Concentration) To measure the concentration of essential amino acids in the culture media contained in cryopreservation solutions A to D, quantitative analysis was performed using liquid chromatography (LC)-fluorescence detection (FLD) according to the amino acid analysis method (AAA: AminoAcidAnalysis method) provided by Agilent. 1. Equipment and Reagents Used The equipment and reagents used are as follows: ・LC system: Agilent 1260 Infinity II LC system ・Detector: Agilent FLD (fluorescence detector), excitation wavelength λEx = 340 nm / fluorescence wavelength λEm = 450 nm (for OPA derivatives), and λEx = 266 nm / λEm = 305 nm (for FMOC derivatives) ・Column: AdvanceBio C18 (4.6 mm × 150 mm, particle size 2.7 μm, Agilent catalog number: p / n 655950-802) ・Derivatization reagents: Agilent OPA Reagent (p / n 5061-3335) Agilent FMOC Reagent (p / n 5061-3337) Borate buffer (p / n 5061-3339) • Amino acid standard solution: Agilent Amino Acid Standard Mix (p / n 5061-3330, 17 types x 250 μM) • Mobile phase: Solution A: 40 mM Na ２ HPO ４ / Na ２ B ４ O７ Buffer solution (pH 8.2) Solution B: Acetonitrile / methanol / water = 45 / 45 / 10 (v / v / v) mixture 2. Sample pretreatment The cryopreservation solution was vortexed and diluted 10-fold with mobile phase A. Dilution was performed similarly when the DMSO concentration was high (≥5%). Then, filtration was performed using a 0.22 μm PES filter (Millex-GP, Merck) to eliminate the effects of precipitation and matrix interference. 3. Preparation of calibration curves Standard solutions (Agilent Amino Acid Standard Mix) were prepared at concentrations of 10, 25, 100, and 250 μM, and each solution was derivatized before LC measurement. Linear regression analysis was performed based on the peak area of ​​each amino acid, and the coefficient of determination (r ２ ) ≥ 0.99 was confirmed. 4. Derivatization Procedure Sample pre-derivatization was performed according to the following procedure: 2.5 μL of borate buffer was added to 1 μL of sample and treated at room temperature for 0.2 minutes. Then, 0.5 μL of OPA reagent was added and reacted at room temperature. Furthermore, 0.4 μL of FMOC reagent and 32 μL of solution A were added and sample pre-derivatization was performed at room temperature. 1 μL of the prepared solution was injected into the LC instrument. 5. Analytical Conditions Flow rate: 0.62 mL / min Column temperature: 40°C Gradient program: Time (min): %B 0:2 0.35:2 13.4:57 13.5:100 15.7:100 15.8:2 18.0:2

[0112] [Evaluation of cell / tissue processing] Frozen human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with culture medium. 10 × 10 ５ Suspend individual cells in a 10% serum-containing medium and place them on a single dish (150 cm²). ２ After seeding, the cells were cultured for 3 days. The cultured cells were harvested, suspended in a 10% serum-containing medium, and cultured in 10 dishes (culture area 150 cm²). ２ ) 2.5 x 10 ５ After sowing individual seeds and subculturing, the cells were cultured for 4 days. The cultured cells were harvested and divided into 10 x 10⁻¹⁰⁻⁴ ５Each cell was dispersed in 1 mL of the cell treatment solution described in Tables 4 and 5 and added to a cryotube. The cryotube was then frozen overnight in a freezer set to -80°C. The frozen cryotube was removed from the freezer and thawed at 23°C. This solution was then seeded directly into a multi-well culture plate and cultured at 37°C in 5% CO2. ２ The cells were incubated in the specified environment for 3 or 24 hours. After incubation, the cells were observed using an inverted microscope to evaluate cell adhesion. Cell adhesion was evaluated by checking for cells that adhered to the culture multiwell plate; those that adhered were rated as "adherent," and those that did not adhere were rated as "not adhered." The results of the cell adhesion evaluation are shown in Tables 4 and 5.

[0113]

[0114]

[0115] The composition of culture medium A used in Tables 4 and 5 is that of a medium having the amino acid concentrations shown in Table 6. Note that the amino acid concentrations at 90 Vol% and 10 Vol% shown in Table 6 are calculated from the 100 Vol% concentration based on the volume ratios used in Tables 4 and 5.

[0116]

[0117] PBS(+) is a solution containing 485 volumes of PBS(-) (phosphate-buffered saline without added calcium and magnesium ions, 1102P05, manufactured by the Institute of Cell Science) with a calcium ion concentration of 3040 ppm. ２＋ 10 parts by volume of aqueous solution, and Mg with a magnesium ion concentration of 1740 ppm ２＋ It was prepared by adding 5 parts by volume of an aqueous solution and mixing. ２＋ Aqueous solution and Mg ２＋ For preparing the aqueous solution, use Otsuka distilled water and CaCl ２ ・2H ２ O (031-25035, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and MgSO ４ 7H ２O (137-12335, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The calcium ion concentration in the prepared PBS(+) was 60.8 ppm, and the magnesium ion concentration was 17.4 ppm.

[0118] The results in Tables 4 and 5 show that the solutions used to treat the cells in Reference Examples A1 to A3 exhibit cell adhesion because they contain essential amino acids in the culture medium at predetermined concentrations, whereas the solutions used to treat the cells in Reference Examples B1 to B3 either do not contain essential amino acids in the culture medium or contain them only at concentrations below the specified value, thus preventing cell adhesion. Since cell adhesion is a phenomenon that occurs prior to cell proliferation, the solutions used to treat the cells in Reference Examples B1 to B3 do not constitute cell or tissue processing, even when used for cell freezing, cell thawing, or immersion in frozen cells, suggesting they are suitable for non-cell processing applications.

[0119] This application claims priority based on Japanese Patent Application No. 2024-203403, filed on 21 November 2024, and incorporates all of its disclosures herein.

Claims

1. A solution for immersing a frozen-thawed three-dimensional cell culture for transplantation, wherein the solution comprises one or more divalent metal salts selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and an aqueous solvent, satisfying at least one of the following (a) to (c), and having an osmotic pressure of 150 mOsm / L or more and 1200 mOsm / L or less. (a) If the calcium salt is included, the calcium salt content is 5 ppm or more and 700 ppm or less in terms of calcium ions, based on the total weight of the solution. (b) If the magnesium salt is included, the magnesium salt content is 5 ppm or more and 1000 ppm or less in terms of magnesium ions, based on the total weight of the solution. (c) If other divalent metal salts other than the calcium salt and magnesium salt are included, the content of the other divalent metal salts is 0.01 ppm or more and 100 ppm or less in terms of metal ions, based on the total weight of the solution.

2. The solution according to claim 1, wherein the aqueous solvent comprises physiological saline.

3. The solution according to claim 1, wherein the aqueous solvent comprises a buffer solution.

4. A solution according to any one of claims 1 to 3, wherein at least one of the culture medium essential amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine is not contained, or its concentration (mg / L) in the solution is less than or equal to the specified values ​​shown in (i) to (xi) below. (i) L-histidine less than 20 mg / L (ii) L-isoleucine less than 40 mg / L (iii) L-leucine less than 40 mg / L (iv) L-lysine less than 50 mg / L (v) L-methionine less than 10 mg / L (vi) L-phenylalanine less than 20 mg / L (vii) L-threonine less than 7 mg / L (viiii) L-valine less than 40 mg / L (ix) L-arginine less than 40 mg / L (x) L-cystine less than 20 mg / L (xi) L-tyrosine less than 10 mg / L 5. The solution according to claim 4, wherein the solution contains two or more amino acids selected from the group consisting of L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-valine, L-arginine, L-cystine, and L-tyrosine, and the total concentration of two or more culture medium essential amino acids belonging to the group contained in the solution is 300 mg / L or less.

6. The solution according to claim 4, wherein the essential amino acid of the culture medium comprises at least one of (ii) L-isoleucine at a concentration of 40 mg / L or less, (iii) L-leucine at a concentration of 40 mg / L or less, (iv) L-lysine at a concentration of 50 mg / L or less, and (ix) L-arginine at a concentration of 40 mg / L or less.

7. A solution according to any one of claims 1 to 3, wherein the solution is for non-processing of cells.

8. A solution according to any one of claims 1 to 3, wherein the solution is substantially free of animal-derived components.

9. A solution according to any one of claims 1 to 3, wherein the solution is substantially free of cryoprotectants.

10. A solution according to any one of claims 1 to 3, wherein the three-dimensional cell culture is a cell sheet, a spheroid, an organoid, or a combination thereof.

11. The solution according to claim 10, wherein the cell sheet is located on a scaffold or culture carrier substrate.

12. A solution according to any one of claims 1 to 3, wherein the cells in the three-dimensional cell culture are cells derived from mammals.

13. The solution according to claim 12, wherein the mammalian-derived cells are selected from the group consisting of living tissue cells, mesenchymal stem cells, and pluripotent stem cells, to the extent that at least one is selected from this group.

14. A method for producing a solution according to any one of claims 1 to 3, comprising a compounding step of adding one or more inorganic salt powders selected from the group consisting of calcium salts, magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts to an aqueous solvent, or adding a solution obtained by dissolving the inorganic salt powder in a liquid to an aqueous solvent to obtain the solution.

15. A method for producing a solution according to claim 14, wherein in the blending step, 50% by volume or more of a basal culture medium is not added relative to 100% by volume of the solution.

16. A frozen-thawed product of a three-dimensional cell culture for transplantation, comprising: a solution according to any one of claims 1 to 3; and a three-dimensional cell culture immersed in the solution.

17. A frozen-thawed three-dimensional cell culture for transplantation according to claim 16, wherein a cryoprotective agent is mixed in the solution.

18. A frozen-thawed product of a three-dimensional cell culture for transplantation according to claim 17, wherein the concentration of the cryoprotective agent is 7 w / v% or more based on the volume of the solution.

19. A frozen-thawed product of a three-dimensional cell culture for transplantation according to claim 17, wherein the concentration of the cryoprotective agent is less than 0.5 w / v% based on the volume of the solution.

20. A freeze-thawed product of a three-dimensional cell culture for transplantation according to claim 16, wherein the three-dimensional cell culture is a cell sheet, and the cell sheet is located on a scaffold or culture carrier substrate.

21. A method for producing a frozen-thawed three-dimensional cell culture for transplantation, comprising: (a) a step of preparing a frozen three-dimensional cell culture for transplantation; and (b) a step of thawing the three-dimensional cell culture and immersing the thawed three-dimensional cell culture in the solution described in any one of claims 1 to 3.

22. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation as described in claim 21, comprising: (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container; and (b) thawing the three-dimensional cell culture and the cryopreservation solution, removing the thawed cryopreservation solution from the container, then adding the solution described in any one of claims 1 to 3 to the container, and immersing the three-dimensional cell culture in the solution.

23. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation as described in claim 21, comprising: (a) freezing the three-dimensional cell culture together with a cryopreservation solution in a container; and (b) thawing the three-dimensional cell culture and the cryopreservation solution, removing the thawed three-dimensional cell culture from the container, and then immersing it in a solution according to any one of claims 1 to 3 in another container.

24. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation according to claim 21, wherein in step (b), the three-dimensional cell culture is immersed in the solution for at least 30 seconds.

25. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation according to claim 21, wherein in step (b), the three-dimensional cell culture is thawed at 0 to 45°C.

26. A method for producing a freeze-thawed product of a three-dimensional cell culture for transplantation according to claim 21, wherein the three-dimensional cell culture is a cell sheet, and the cell sheet is located on a scaffold or culture carrier substrate.

27. A method for using the solution according to any one of claims 1 to 3, comprising the step of immersing a frozen and thawed three-dimensional cell culture for transplantation in the solution.

28. A transplantation kit comprising a frozen three-dimensional cell culture for transplantation and the solution according to any one of claims 1 to 3.

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