Frozen product of three-dimensional cell culture, container containing frozen product of three-dimensional cell culture, cell freezing kit, cryopreservation liquid, method for producing frozen product of three-dimensional cell culture, method for using frozen product of three-dimensional cell culture, and method for using cryopreservation liquid

Abstract

A frozen product of a three-dimensional cell culture according to the present invention comprises: a three-dimensional cell culture having a cell-to-cell connection structure; and a cryopreservation liquid. The cryopreservation liquid contains a calcium salt and water, wherein the content of the calcium salt is 3.5 ppm to 500 ppm (both inclusive) in terms of calcium ion on the basis of the total weight of the cryopreservation liquid.

Description

Frozen three-dimensional cell culture, container containing frozen three-dimensional cell culture, cell freezing kit, cryopreservation solution, method for producing frozen three-dimensional cell culture, method for using frozen three-dimensional cell culture, and method for using cryopreservation solution

[0001] The present invention relates to a frozen three-dimensional cell culture, a container containing the frozen three-dimensional cell culture, a cell freezing kit, a cryopreservation solution, a method for producing a frozen three-dimensional cell culture, a method for using the frozen three-dimensional cell culture, and a method for using the cryopreservation solution.

[0002] Known techniques for cryopreserving cells are described in Patent Documents 1 to 3. Patent Document 1 describes a method for freezing single cells contained in a cell suspension in a cryopreservation solution containing purified albumin, dimethyl sulfoxide (DMSO), calcium, and magnesium. Patent Document 2 describes a buffer solution for diluting a cell suspension obtained by thawing cryopreserved cells. It describes that because a cell suspension may contain cytotoxic components (e.g., DMSO), the effects of the cytotoxic components can be reduced by diluting the cell suspension with a buffer solution containing human serum albumin. Patent Document 3 describes a method for freezing cell sheets. It describes the use of Stem Cell Banker (registered trademark), which contains DMSO, as the cryopreservation solution.

[0003] Patent No. 4385158 International Publication No. 2021 / 065971 Japanese Patent Application Laid-Open No. 2022-8269

[0004] When single cells are frozen, the extracellular osmotic pressure increases, causing dehydration and shrinkage. When cell sheets are frozen, the same dehydration and shrinkage phenomena as single cells occur. In addition, it has been reported that the intercellular junctions and extracellular matrix of cell sheets are destroyed by ice crystal formation. While the mechanism is unclear, it is speculated that when distortion occurs due to dehydration and shrinkage during freezing, single cells have a high degree of freedom of the cell membrane due to the lack of constraints from surrounding cells. On the other hand, in three-dimensional cell culture constructs with cell-to-cell interconnections, such as cell sheets, cells are constrained by other cells around them, resulting in a low degree of freedom of individual cell membranes, making them more susceptible to membrane destruction. The inventors' studies have revealed that there is room for improvement in three-dimensional cell culture constructs, firstly, in terms of cell viability after freezing and thawing, and secondly, in terms of metabolic activity.

[0005] Regarding repair of the cell membrane, when the cell membrane is locally damaged, Ca ２＋ flows into the cell, and high concentrations of Ca ２＋ The signal activates the fusion of intracellular vesicles with the cell membrane (exocytosis), and this Ca ２＋ It is known that new membrane components are supplied by ATP-dependent exocytosis, facilitating repair of the damaged part of the cell membrane. However, the above-mentioned mechanism of cell membrane repair has not been fully elucidated. For example, in the case of a single cell, the surrounding area is in contact with the cryopreservation solution, so calcium ions (Ca ２＋) can easily penetrate into the three-dimensional cell culture medium, but if the connection between cells (extracellular matrix, etc.) in the three-dimensional cell culture is damaged, it is expected that calcium ions and the cryopreservation solution will have difficulty penetrating into this area. As a result of extensive research, the present inventors discovered that the cell survival rate after the first freeze-thaw can be improved by using a calcium ion concentration higher than that contained in commercially available cryopreservation solutions, and thus completed the present invention. Although the detailed mechanism is unclear, when frozen cells are thawed, calcium ions outside the cell membrane flow into the interior from around the cell membrane portion that has been subjected to mechanical stress or damage. However, by using high-concentration calcium ions, it is possible to increase the calcium ion content even in damaged areas of the connection between cells (extracellular matrix, etc.) that are not in contact with the cryopreservation solution (where calcium ions have not flowed in). ２＋ It is speculated that cell membrane repair by calcium-dependent exocytosis occurs, and cell viability after freeze-thawing in three-dimensional cell cultures can be improved. Similarly, we discovered that using a magnesium ion concentration higher than that contained in commercially available cryopreservation solutions can improve metabolic activity after the second freeze-thawing, leading to the completion of the present invention. Furthermore, the combined use of high concentrations of calcium ions and magnesium ions improves metabolic activity, further increasing cell viability.

[0006] According to one aspect of the present invention, there are provided the following frozen three-dimensional cell culture bodies, a container containing the frozen three-dimensional cell culture bodies, a cell freezing kit, a cryopreservation solution, a method for producing a frozen three-dimensional cell culture body, a method for using the frozen three-dimensional cell culture body, and a method for using the cryopreservation solution.

[0007] 1. A frozen product of a three-dimensional cell culture comprising a three-dimensional cell culture having a cell-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm or more and 500 ppm or less in terms of calcium ions, based on the total weight of the cryopreservation solution. 3. A frozen product of a three-dimensional cell culture comprising a three-dimensional cell culture having a cell-to-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution comprises water and one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion based on the total weight of the cryopreservation solution. 3. A frozen product of the three-dimensional cell culture according to any one of 1. to 3., wherein the cryopreservation solution contains one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion, based on the total weight of the cryopreservation solution. 4. A frozen product of the three-dimensional cell culture according to any one of 1. to 3., wherein the cryopreservation solution contains a cryoprotectant.5. A frozen product of the three-dimensional cell culture according to 4., wherein the cryoprotectant comprises one or more selected from the group consisting of DMSO, glycerin, propylene glycol, and ethylene glycol. 6. A frozen product of the three-dimensional cell culture according to any one of 1. to 5., wherein the three-dimensional cell culture comprises a cell sheet or a spheroid. 7. A frozen product of the three-dimensional cell culture according to 6., wherein the three-dimensional cell culture comprises a substrate and the cell sheet adhered onto the substrate. 8. A frozen product of the three-dimensional cell culture according to any one of 1. to 7., wherein the survival rate of cells contained in the three-dimensional cell culture after thawing the frozen product is 35% or more. 9. A container comprising the frozen product of the three-dimensional cell culture according to any one of 1. to 8.. 10. A cell freezing kit comprising a three-dimensional cell culture construct having a cell-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution. (ii) A cell freezing kit comprising one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains the divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion, based on the total weight of the cryopreservation solution.11. A cryopreservation solution used for freezing a three-dimensional cell culture having a cell-cell connection structure, wherein the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) comprising a calcium salt and water, wherein the content of the calcium salt is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution; (ii) comprising one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein, when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ions based on the total weight of the cryopreservation solution; and / or, when the cryopreservation solution contains a divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ions based on the total weight of the cryopreservation solution. 12. A method for producing a frozen product of a three-dimensional cell culture, comprising a freezing step of freezing a three-dimensional cell culture having a cell-cell connection structure in a cryopreservation solution, wherein the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm or more and 500 ppm or less in terms of calcium ions, based on the total weight of the cryopreservation solution; (ii) A method for producing a frozen product, comprising one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains the divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion, based on the total weight of the cryopreservation solution.13. A method for producing a frozen product according to 12., wherein the freezing step freezes the three-dimensional cell culture at a temperature below the freezing temperature of the cryopreservation solution. 14. A method for producing a thawed product of a three-dimensional cell culture, comprising a thawing step of thawing the frozen product of the three-dimensional cell culture obtained by the method for producing a frozen product according to 12. or 13. 15. A method for producing a thawed product according to 14., wherein the thawing step maintains at least a portion of the three-dimensional cell culture in contact with the cryopreservation solution in a liquid state. 16. A method for using a frozen product of a three-dimensional cell culture according to any one of 1. to 8., wherein at least a portion of the three-dimensional cell culture is maintained in contact with the cryopreservation solution in a liquid state when thawing the frozen product. 17. A method for using a frozen product according to 11. 18. A method for using the cryopreservation solution described in 1., comprising the step of improving the viability of cells in the three-dimensional cell culture by maintaining the cryopreservation solution in contact with at least a portion of the three-dimensional cell culture during freezing and thawing. 19. A method for using the cryopreservation solution described in 1., comprising the step of protecting cells in the three-dimensional cell culture from cell damage associated with freezing by maintaining the cryopreservation solution in contact with at least a portion of the three-dimensional cell culture during freezing and thawing.

[0008] According to the present invention, there are provided frozen three-dimensional cell cultures that have excellent cell viability and / or metabolic activity after freezing and thawing, containers containing frozen three-dimensional cell cultures using the same, cell freezing kits, cryopreservation solutions, methods for producing frozen three-dimensional cell cultures, methods for using frozen three-dimensional cell cultures, and methods for using cryopreservation solutions.

[0009] An overview of the frozen three-dimensional cell culture construct of this embodiment will be described.

[0010] The frozen three-dimensional cell culture of the first embodiment is a frozen three-dimensional cell culture comprising a three-dimensional cell culture having a cell-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution contains a calcium salt and water, and the calcium salt content is 3.5 ppm or more and 500 ppm or less in terms of calcium ions, based on the total weight of the cryopreservation solution.

[0011] The frozen product of the three-dimensional cell culture construct of the second embodiment is a frozen product of the three-dimensional cell culture construct, comprising a three-dimensional cell culture construct having a cell-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution comprises one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, and when the cryopreservation solution contains a magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion based on the total weight of the cryopreservation solution.

[0012] The frozen material in the first and second embodiments includes a three-dimensional cell culture frozen in a cryopreservation solution. The calcium salts and divalent metal salts other than calcium salts contained in the cryopreservation solution are at least partially or almost completely ionized in water before freezing or after freeze-thawing, and exist in the ionic state of calcium ions or divalent metal ions.

[0013] According to the first embodiment, by adjusting the calcium ion concentration to be equal to or greater than the lower limit and equal to or less than the upper limit, the cell viability of the three-dimensional cell culture construct after freezing and thawing can be improved. The calcium concentration in the cryopreservation solution can be adjusted, for example, by adding a predetermined calcium salt, and preferably by mixing a commercially available cryopreservation solution with a predetermined calcium salt. Commercially available cryopreservation solutions do not substantially contain calcium ions, and even if they do, the calcium ion concentration is at most about 3.0 ppm or less. By adding a predetermined calcium salt to a commercially available cryopreservation solution, the calcium ion concentration can be adjusted to be equal to or greater than the lower limit.

[0014] The cryopreservation solution may also contain a cryoprotectant, and in one preferred embodiment, DMSO may be included. In the first embodiment, even when a cryopreservation solution containing DMSO is used, a decrease in cell viability can be suppressed. Although the detailed mechanism is unclear, it is speculated that, although there is a possibility of toxic damage due to DMSO during freezing and thawing, exocytosis due to a high concentration of calcium ions induces early cell membrane repair, suppressing toxic damage and thereby suppressing a decrease in cell viability.

[0015] According to the second embodiment, by adjusting the magnesium ion concentration to be equal to or greater than the lower limit and equal to or less than the upper limit, the metabolic activity rate of the three-dimensional cell culture after freezing and thawing can be improved. Furthermore, according to the second embodiment, by adjusting the concentration of divalent metal ions other than magnesium ions to be equal to or greater than the lower limit and equal to or less than the upper limit, the metabolic activity rate of the three-dimensional cell culture after freezing and thawing can be improved. Although the detailed mechanism is unclear, it is presumed that magnesium ions and other divalent metal ions are involved in metabolic enzymes and can accelerate the recovery of cell membranes, thereby improving metabolic activity.

[0016] The magnesium concentration in the cryopreservation solution can be adjusted, for example, by adding a predetermined magnesium salt, and preferably by mixing a commercially available cryopreservation solution with a predetermined magnesium salt. Commercially available cryopreservation solutions do not substantially contain magnesium ions, and even if they do, the concentration is at most about 1.0 ppm or less. By adding a predetermined magnesium salt to a commercially available cryopreservation solution, the calcium ion concentration can be adjusted to be above the above lower limit.

[0017] In a third embodiment, the cryopreservation solution contains a calcium salt and one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, wherein the content of the calcium salt in the cryopreservation solution of the third embodiment is 3.5 ppm to 500 ppm in terms of calcium ion based on the total weight of the cryopreservation solution, and when the cryopreservation solution of the third embodiment contains a magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution of the third embodiment contains a divalent metal salt other than a magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion based on the total weight of the cryopreservation solution.

[0018] According to the third embodiment, improvement in metabolic activity due to magnesium ions and other divalent metal ions can be expected, and the synergistic effect with calcium ions can further improve cell viability and metabolic activity.

[0019] In medical fields such as regenerative medicine, medical technology has been rapidly advancing in recent years to perform cell transplantation by preparing a three-dimensional cell culture such as a cell sheet formed from cells in a sheet form and transplanting this into the affected area in order to repair damaged tissue, etc. In such cell transplantation, after culturing the three-dimensional cell culture and proliferating the cells, it is necessary to maintain good cell viability and store the cells for a long period of time until transplantation into the patient.

[0020] In contrast, by using a frozen three-dimensional cell culture of this embodiment, a three-dimensional cell culture with excellent cell viability and / or metabolic activity can be provided even after freezing and thawing, and therefore this can be suitably used for transplantation.

[0021] Furthermore, the present inventors have conducted extensive research into the components of cryopreservation solutions and methods for freezing and thawing three-dimensional cell cultures in order to obtain three-dimensional cell cultures with improved cell viability after cryopreservation. As a result, they have surprisingly found that when a cryopreservation solution containing a specific concentration of calcium salt is placed in contact with the frozen-thawed three-dimensional cell culture during thawing, cell viability immediately after freezing and thawing is significantly improved compared to when the cryopreservation solution does not contain the specific concentration of calcium salt. Furthermore, they have found that cell viability after freezing and thawing is further improved or maintained at a high level. This finding led to the completion of the present invention. Furthermore, they have found that the addition of magnesium salt to the specific concentration of calcium salt can further improve cell viability.

[0022] Furthermore, according to the present invention, when a three-dimensional cell culture is cryopreserved using a cryopreservation solution containing a predetermined concentration of calcium salt and, optionally, a predetermined concentration of magnesium salt, the cell viability after freezing and thawing is significantly improved compared to the cell viability in a three-dimensional cell culture obtained under the same conditions except that the cryopreservation solution contains calcium salt and, optionally, a predetermined concentration of magnesium salt. Furthermore, according to the present invention, high cell viability is maintained for the cells in the three-dimensional cell culture after freezing and thawing, so there is no need to culture the cells again after freezing and thawing, and there is also no need to remove the cryopreservation solution containing cytotoxic substances such as DMSO after thawing the cells, which simplifies the process and reduces production costs.

[0023] Furthermore, this embodiment can provide a frozen three-dimensional cell culture body, a container containing the frozen three-dimensional cell culture body, a cell freezing kit, a cryopreservation solution, a method for producing a frozen three-dimensional cell culture body, a method for using the frozen three-dimensional cell culture body, and a method for using the cryopreservation solution.

[0024] The container of this embodiment may contain the above-mentioned frozen three-dimensional cell culture body, and may contain a frozen three-dimensional cell culture body packaged in a package.

[0025] The cell freezing kit of this embodiment includes a three-dimensional cell culture construct having a cell-cell connection structure and a cryopreservation solution. The cryopreservation solution in the cell freezing kit satisfies at least one of the following conditions (i) and (ii): (i) it contains a calcium salt and water, and the calcium salt content is 3.5 ppm to 500 ppm, in terms of calcium ions, based on the total weight of the cryopreservation solution; (ii) it contains one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, and when the cryopreservation solution contains a magnesium salt, the magnesium salt content is 2 ppm to 300 ppm, in terms of magnesium ions, based on the total weight of the cryopreservation solution; and / or when the cryopreservation solution contains a divalent metal salt other than a magnesium salt, the divalent metal salt content is 0.01 ppm to 100 ppm, in terms of metal ions, based on the total weight of the cryopreservation solution.

[0026] The cryopreservation solution of this embodiment is used for freezing a three-dimensional cell culture having a cell-cell connection structure, and the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) it contains a calcium salt and water, and the calcium salt content is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution; (ii) it contains one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, and when the cryopreservation solution contains a magnesium salt, the magnesium salt content is 2 ppm to 300 ppm in terms of magnesium ions based on the total weight of the cryopreservation solution; and / or when the cryopreservation solution contains a divalent metal salt other than a magnesium salt, the divalent metal salt content is 0.01 ppm to 100 ppm in terms of metal ions based on the total weight of the cryopreservation solution.

[0027] The method for producing a frozen three-dimensional cell culture of this embodiment includes a freezing step of freezing a three-dimensional cell culture having a cell-cell connection structure in a cryopreservation solution, wherein the cryopreservation solution used in the freezing step satisfies at least one of the following (i) and (ii): (i) A cryopreservation solution containing a calcium salt and water, wherein the calcium salt content is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution; (ii) A cryopreservation solution containing one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein, when the cryopreservation solution contains a magnesium salt, the magnesium salt content is 2 ppm to 300 ppm in terms of magnesium ions based on the total weight of the cryopreservation solution, and / or, when the cryopreservation solution contains a divalent metal salt other than a magnesium salt, the divalent metal salt content is 0.01 ppm to 100 ppm in terms of metal ions based on the total weight of the cryopreservation solution.

[0028] The method for using the frozen three-dimensional cell culture of this embodiment includes a step of holding at least a portion of the three-dimensional cell culture in contact with a liquid cryopreservation solution when thawing the frozen three-dimensional cell culture.

[0029] The method of using the cryopreservation solution of this embodiment includes a step of improving the viability of cells in the three-dimensional cell culture by maintaining the cryopreservation solution in contact with at least a portion of the three-dimensional cell culture during freezing and thawing.

[0030] The method of using the cryopreservation solution of this embodiment includes a step of protecting cells in the three-dimensional cell culture from cell damage associated with freezing by maintaining the cryopreservation solution in contact with at least a portion of the three-dimensional cell culture during freezing and thawing.

[0031] Hereinafter, each component of the frozen three-dimensional cell culture construct of this embodiment will be described in detail.

[0032] (Definition) As used herein, the term "frozen" refers to a solid state in which no intracellular ice crystals are formed or only a small amount of ice crystals are formed within the cells. To reduce damage to cells caused by freezing and thawing, it is preferable that no intracellular ice crystals are formed. The term "three-dimensional cell culture frozen together with a cryopreservation solution" refers to the freezing of a three-dimensional cell culture placed or immersed in a cryopreservation solution, or a three-dimensional cell culture in contact with a cryopreservation solution in any manner, resulting in the three-dimensional cell culture and the cryopreservation solution being frozen together. The term "frozen" includes cases in which the three-dimensional cell culture and the cryopreservation solution are completely frozen, as well as cases in which only a portion of them is frozen. The water molecules contained in the frozen product may be crystalline (ice crystals), or at least a portion of them may be amorphous.

[0033] As used herein, "freezing treatment" refers to a process for freezing cells, specifically, placing a three-dimensional cell culture 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. Furthermore, "freezing a three-dimensional cell culture in a cryopreservation solution below the freezing temperature of the cryopreservation solution" is synonymous with placing the three-dimensional cell culture in the cryopreservation solution in an environment below the freezing temperature of the cryopreservation solution to freeze the cells present in the three-dimensional cell culture. Furthermore, "cryopreservation" refers to preserving frozen cells for a certain period of time (including both long and short periods).

[0034] As used herein, "freezing and thawing" refers to thawing frozen cells. Specifically, freezing and thawing refers to placing a three-dimensional cell culture frozen together with a cryopreservation solution in an environment at or above the freezing temperature of the cryopreservation solution to thaw the cells in the three-dimensional cell culture. When the frozen cryopreservation solution thaws and becomes liquid (this can be confirmed visually) and the liquid cryopreservation solution is in contact with the three-dimensional cell culture, the cells in the three-dimensional cell culture can be considered to have thawed. As used herein, the term "during freezing and thawing" can include cases where the entire cryopreservation solution is in a liquid state after freezing and thawing, as well as cases where only a portion of the cryopreservation solution becomes liquid.

[0035] "Placing in contact" refers to maintaining a contact state between the three-dimensional cell culture and the cryopreservation solution for a predetermined period of time, typically referring to a state in which the three-dimensional cell culture (all or part thereof) is placed or immersed in the cryopreservation solution. This "contact state" may be distinguished from a situation in which, after thawing a frozen cryopreservation solution, (i) the cryopreservation solution containing the cytotoxic substance is immediately discarded, preventing the three-dimensional cell culture from coming into contact with the cytotoxic substance, for example, to avoid adverse effects of the cytotoxic substance (DMSO, etc.) in the cryopreservation solution. Furthermore, this "contact state" may be distinguished from a situation in which, after thawing a frozen cryopreservation solution, (ii) the three-dimensional cell culture is brought into contact with a diluted solution of the cryopreservation solution in which the concentration of the cytotoxic substance has been diluted. Details of the freezing method and the freeze-thawing method are described below.

[0036] (Cryopreservation Solution) As used herein, the term "cryopreservation solution" (also referred to as "cell cryopreservation solution") refers to a solution used for cryopreserving cells.

[0037] The cryopreservation solution of the first embodiment contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution. However, the cryopreservation solution of the first embodiment may or may not contain one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts. When a magnesium salt is contained, the content of the magnesium salt may be less than 2 ppm and may be 1.9 ppm or less in terms of magnesium ions based on the total weight of the cryopreservation solution. A cryopreservation solution according to a second embodiment comprises water and one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and when the cryopreservation solution contains a magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm, in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than a magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm, in terms of metal ion, based on the total weight of the cryopreservation solution. However, the cryopreservation solution according to the second embodiment may or may not contain a calcium salt, and when a calcium salt is contained, the content of the calcium salt may be less than 3.5 ppm, and may be 3.4 ppm or less, in terms of calcium ion, based on the total weight of the cryopreservation solution.A cryopreservation solution of a third embodiment comprises a calcium salt and one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, wherein the content of the calcium salt is 3.5 ppm to 500 ppm in terms of calcium ion based on the total weight of the cryopreservation solution, and when the cryopreservation solution contains a magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than a magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion based on the total weight of the cryopreservation solution. In the present embodiment, the frozen three-dimensional cell culture medium, the container containing the frozen three-dimensional cell culture medium, the cell freezing kit, the cryopreservation solution, the method for producing the frozen three-dimensional cell culture medium, the method for using the frozen three-dimensional cell culture medium, and the method for using the cryopreservation solution may be any of the cryopreservation solutions of the first embodiment, the second embodiment, and the third embodiment.

[0038] The calcium salt content calculated as calcium ions means the concentration of calcium ions. Similarly, for the magnesium salt content and the divalent metal salt content, calculated as ions, the term "calcium salt content" refers to the magnesium ion concentration and the divalent metal ion concentration, respectively. Hereinafter, the calcium salt content, magnesium salt content, and divalent metal salt content may be referred to as the calcium ion concentration, magnesium ion concentration, and divalent metal ion concentration, respectively. The "content based on the total weight of the cryopreservation solution" refers to the proportion of each element contained in the total amount (100% by mass) of the cryopreservation solution, calculated by mass conversion.

[0039] The calcium salt may be a calcium salt with an anion such as hydrochloric acid, nitric acid, or sulfuric acid, and examples thereof include calcium chloride and calcium nitrate. The magnesium salt may be a magnesium salt with an anion such as hydrochloric acid, nitric acid, or sulfuric acid, and examples thereof include magnesium sulfate and magnesium chloride. Divalent metal salts other than magnesium salts include divalent metal salts of anions such as hydrochloric acid, nitric acid, sulfuric acid, or ammonia with other anions such as copper ions, manganese ions, iron ions, molybdenum ions, nickel ions, or cobalt ions.

[0040] The calcium ion concentration, magnesium ion concentration, and other divalent metal ion concentrations in the cryopreservation solution may be calculated from the raw material ratios, and can be measured by ion chromatography before freezing or after freeze-thawing. When calculated from the raw material ratios, the calcium ion concentration, magnesium ion concentration, and other divalent metal ion concentrations may be calculated on the assumption that all salts present in the cryopreservation solution are dissociated into ions.

[0041] <Ion Chromatography Analysis> An example of a method for measuring calcium ions and magnesium ions in a cryopreservation solution by ion chromatography is as follows. Calcium ions and magnesium ions in a cryopreservation solution are measured by ion chromatography (ICS2100, manufactured by Thermo Fisher Scientific). A measurement sample is used from which proteins have been removed by ultrafiltration, and measurement is performed in matrix illumination (MI) mode. Ion chromatography is performed 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: Electric 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 flow time: -1 to 0 min Sample introduction amount: 25 μL Calibration curve: Prepared by arbitrarily diluting cation mixed standard solution II (07197-96, Kanto Chemical Co., Ltd.) with ultrapure water.

[0042] The calcium ion concentration of 3.5 ppm to 500 ppm includes any concentration range between any values ​​within this range in increments of 0.1 ppm or 1 ppm. For example, the calcium ion concentration may be, based on the total weight of the cryopreservation solution, 3.5 to 500 ppm, 3.5 to 450 ppm, 3.5 to 400 ppm, 3.5 to 350 ppm, 3.5 to 300 ppm, 3.5 to 250 ppm, 3.5 to 200 ppm, 3.5 to 150 ppm, 3.5 to 100 ppm, 5 to 500 ppm, 5 to 450 ppm, 5 to 400 ppm, 5 to 350 ppm, 5 to 300 ppm, 5 to 250 ppm, 5 to 200 ppm, 5 to 150 ppm, 5 to 100 ppm, 7 to 500 ppm, 7 to 450 ppm, 7 to 400 ppm, 5 to 350 ppm, 7 to 300 ppm, 7 to 250 ppm, 7 to 200 ppm, 7 to 150 ppm, 7 to 100 ppm, etc.Alternatively, the lower limit of the calcium ion concentration may be, for example, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, based on the total weight of the cryopreservation solution. ,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,15,20,25,30,35,40,45, The upper limit of the calcium ion concentration may be, 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, 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, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 107 It may be 0, 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 ppm (including any value in increments of 0.1 ppm or 1 ppm between these values).

[0043] The magnesium ion concentration may be 2 to 300 ppm, 2 to 200 ppm, 2 to 150 ppm, 2 to 100 ppm, or 2 to 20 ppm, based on the total weight of the cryopreservation solution, or any value in increments of 0.1 ppm or 1 ppm within these ranges. Alternatively, the lower limit of the magnesium ion concentration may be 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 ppm (including any value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution. The upper concentration limit may be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 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 ppm (including any value in increments of 0.1 ppm or 1 ppm between these values).

[0044] The concentration of divalent metal ions other than magnesium ions is 0.01 ppm to 100 ppm based on the total weight of the cryopreservation solution, and may be the total concentration of copper ions, manganese ions, zinc ions, iron ions, molybdenum ions, nickel ions, and cobalt ions.

[0045] The copper ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. Alternatively, the lower limit of the copper ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any value in increments of 0.01 ppm or 0.1 ppm therebetween) based on the total weight of the cryopreservation 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 value in increments of 0.1 ppm or 1 ppm therebetween).

[0046] The manganese ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. Alternatively, the lower limit of the manganese ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any value in increments of 0.01 ppm or 0.1 ppm therebetween), 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 value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution.

[0047] The zinc ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. Alternatively, the lower limit of the zinc ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any value in increments of 0.01 ppm or 0.1 ppm therebetween), 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 value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution.

[0048] The iron (II) ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. 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 value in increments of 0.01 ppm or 0.1 ppm therebetween), 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 value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution.

[0049] The molybdenum (II) ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. 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 value in increments of 0.01 ppm or 0.1 ppm therebetween), 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 value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution.

[0050] The nickel ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. Alternatively, the lower limit of the nickel ion concentration may be 0.01, 0.02, 0.03, 0.04, 0.05 ppm (including any value in increments of 0.01 ppm or 0.1 ppm therebetween), 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 value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution.

[0051] The cobalt (II) ion concentration is, based on the total weight of the cryopreservation solution, 0.01 ppm or more, 0.02 ppm or more, 0.02 to 100 ppm, 0.02 to 50 ppm, 0.02 to 10 ppm, 0.02 to 8 ppm, or 0.02 to 5 ppm, and also includes concentration ranges between any values ​​within these numerical ranges in increments of 0.01 ppm, 0.1 ppm, or 1 ppm. 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 value in increments of 0.01 ppm or 0.1 ppm therebetween), 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 value in increments of 0.1 ppm or 1 ppm therebetween), based on the total weight of the cryopreservation solution.

[0052] The cryopreservation solution may further contain inorganic salts other than calcium salts, magnesium salts, and the above-mentioned divalent metal ions. Examples of inorganic salts include metal salts, including alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (excluding calcium salts and magnesium salts). The metal salt may be a divalent or trivalent metal salt. Specific examples include metal salts of hydrochloric acid, nitric acid, sulfuric acid, etc., such as potassium chloride and sodium chloride. These may be added separately from the metal salt contained in the cell culture solution. The type and concentration of the metal salt may be appropriately determined based on the cell viability. The concentration is not limited, but may be in the range of 0.01 to 10,000 ppm, preferably 1 to 1,000 ppm.

[0053] The cryopreservation solution may contain a cryoprotectant, which is a substance used to reduce damage to cells caused by freezing and thawing during cryopreservation.

[0054] Examples of cryoprotectants include cell-impermeant cryoprotectants and cell-permeant cryoprotectants. Specific examples of cell-impermeant cryoprotectants include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeant cryoprotectants include DMSO, polyols, glycerin (glycerol), propylene glycol, ethylene glycol, and propanediol. These cryoprotectants may be formulated alone or in combination. Preferably, the cryoprotectant may contain one or more selected from the group consisting of DMSO, glycerin, propylene glycol, and ethylene glycol. These cryoprotectants may be appropriately selected from known cryoprotectants depending on the type of cell, the composition of the cryopreservation solution, and the like. The content of the cryoprotectant can be appropriately adjusted depending on the type of components used.

[0055] While prior art teaches that DMSO may affect cell differentiation and therefore is preferably not included from the perspective of cell viability, the present invention achieves good cell viability even when DMSO is included. When DMSO is included in the cryopreservation solution, the upper limit of the weight of DMSO, based on the volume of the cryopreservation solution, is 30 w / v%, preferably 25 w / v%, more preferably 20 w / v%, and even more preferably 15 w / v%, and the lower limit is 1 w / v%, more preferably 2 w / v%, and even more preferably 3 w / v%. Therefore, the weight of DMSO, based on the volume of the cryopreservation solution, is preferably in a numerical range defined by 1 to 30 w / v% or any numerical value within this range in 1 w / v% increments, such as 1 to 25 w / v%, 2 to 25 w / v%, 3 to 20 w / v%, 4 to 20 w / v%, 5 to 20 w / v%, 5 to 15 w / v%, or 5 to 10 w / v%.

[0056] The cryopreservation solution may include commercially available cryopreservation solutions, such as Stem Cell Banker (registered trademark) GMP Grade (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) hRM (CG Lymphotec), Bambanker (registered trademark) (CG Lymphotec), and iStock (registered trademark) (CG Lymphotec). Commercially available cryopreservation solutions that do not contain DMSO include, for example, StemCellBanker (registered trademark) DMSO-free GMP grade (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) DMSO-free (CG Lymphotec Co., Ltd.), Cryoscarless (registered trademark) DMSO-free (BioVerde Co., Ltd.), StemCellKeep (BioVerde Co., Ltd.), CryoNovo (registered trademark) X12 (Akron BioProducts LCC), CryoNovo (registered trademark) P24 (Akron BioProducts LCC), DMSO-free cryopreservation solution for cryopreservation of human ES / iPS cells (ReproCell Co., Ltd.), Cell Reservoir One (Nacalai Tesque Co., Ltd.), TheliKeep (registered trademark: BioVerde Co., Ltd.), and Cellvation (registered trademark: Protide Co., Ltd.). Examples include ReproCryo RM (ReproCell Pharmaceuticals), and SOFORO Cryo (Saraya).

[0057] The cryopreservation solution may contain natural animal-derived components, but is preferably free of natural animal-derived components, for example, from the perspective described below. Examples of natural animal-derived components include albumin, serum (calf serum, newborn calf serum, fetal bovine serum, horse serum, etc.), plasma, and basal medium. Freezing the solution without natural animal-derived components can avoid changes in cell properties due to components unnecessary for cell preservation, such as various cytokines and growth factors contained in serum, or the effects of components in the basal medium of unknown origin, and is therefore useful from the perspective of safe application to living organisms in clinical use. Furthermore, as shown in Examples 10 and 11 described below, the present invention has been found to achieve good cell survival rates after freezing and thawing, even when natural animal-derived components such as albumin are not contained.

[0058] The cryopreservation solution may contain a pH adjuster. The term "pH adjuster" refers to a substance that maintains the pH within a certain range in an aqueous solution and provides a buffering effect to the aqueous solution. Examples of pH adjusters include carbonate buffers (sodium carbonate, sodium bicarbonate), phosphate buffers (phosphoric acid, sodium phosphate, potassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, tripotassium phosphate), Tris, Good's buffer (HEPES, MES, PIPES, etc.), citrate buffers (trisodium citrate), acetate buffers (sodium acetate, potassium acetate), borate buffers (sodium borate, sodium tetraborate), tartaric acid (sodium tartrate), and amino acid buffers (histidine, taurine, aspartic acid). The pH adjuster may be a combination of two or more pH adjusters. The pH adjuster is used to adjust the pH of the cryopreservation solution to, for example, about 6.0 to 9.0, preferably 6.5 to 8. The pH adjuster may be one that does not contain at least one element selected from the group consisting of calcium, magnesium, copper, manganese, zinc, iron, molybdenum, nickel, and cobalt, and preferably does not contain calcium or magnesium.

[0059] In addition to the substances or components listed above, the cryopreservation solution may further contain any other substances or components known in the art to improve cell viability after cell cryopreservation.

[0060] The freezing 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, and is preferably -15°C to -5°C.

[0061] The cryopreservation solution before freezing may be provided in a container such as a cold-resistant container or a package (including a kit), and the container or package may be accompanied by or include instructions for use that describe the cryopreservation method, etc.

[0062] (Cells) The "cells" contained in the three-dimensional cell culture construct may be any cell type suitable for transplantation, as long as they are in the form of a three-dimensional cell aggregate with intercellular connectivity. Examples of such cells include, but are not limited to, clinically useful cells for treating or preventing symptoms associated with cell, tissue, or organ loss, dysfunction, or dysfunction, and culturable cells for use in non-clinical trials, which are isolated from living organisms. Examples include biological tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissue, pluripotent stem cells capable of differentiating into various biological tissues, and stem and progenitor cells that can be induced to differentiate. Cells used for cell transplantation may be autologous, allogeneic non-autologous, or xenogeneic. Furthermore, preferred cells are human autologous cells from the standpoint of clinical application and safety, and human non-autologous cells from the standpoint of clinical application and productivity.

[0063] Specific examples of biological tissue cells include fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiac muscle cells, myoblasts, bone cells, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, and endothelial cells. Specific examples of mesenchymal stem cells include 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 induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured alone 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.

[0064] The origin of the cells is not particularly limited, and examples thereof include mammals, birds, amphibians, fish, insects, plants, microorganisms, etc. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, chickens, etc.

[0065] (Three-dimensional cell culture construct) The "three-dimensional cell culture construct" is preferably a three-dimensional cell culture construct used for transplantation to treat a disease or disorder or to ameliorate its symptoms. The subject to be transplanted is preferably a mammal, such as a human or a household pet (dog, cat, etc.), and particularly preferably a human.

[0066] Specific examples of "three-dimensional cell cultures" include aggregates of cultured cells that have a three-dimensional structure, where cells are physically and / or functionally connected to each other via adhesion molecules or extracellular matrix. "Three-dimensional cell cultures" include, but are not limited to, three-dimensional cell sheets, spheroids, organoids, and the like. Shapes of three-dimensional cell cultures include, but are not limited to, three-dimensional sheets, organ-like, spherical, tissue-like, hollow, and block-like. Typically, adherent cells (cells that adhere to and grow on a culture substrate, e.g., fibroblasts) tend to form three-dimensional cell aggregates through physical and / or functional connections, as in cell sheets. However, even suspension cells (i.e., cells that grow suspended in a medium, e.g., hematopoietic cells, blood cells), such as spheroids, are considered three-dimensional cell cultures when they form three-dimensional cell aggregates through physical and / or functional connections.

[0067] The method for producing a three-dimensional cell culture construct can be performed by a method known to those skilled in the art and is not particularly limited. Examples of the method for producing a three-dimensional cell culture construct include methods taught in documents such as JP 2012-120696 A, JP 2017-176025 A, and JP 2015-149905 A. The density of cells cultured in the three-dimensional cell culture construct is not particularly limited as long as it is suitable for the cells to be cultured, the culture vessel (e.g., a cell culture multiwell plate), or the use of the cultured cells, but may be, for example, 5 x 10 ２ cells / cm ２ 1x10 or more ９ cells / cm ２ The lower limit is more preferably 1×10 ３ cells / cm ２ More preferably, 5×10 ３ cells / cm２ More preferably, 5×10 ４ cells / cm ２ On the other hand, the upper limit is more preferably 1×10 ８ cells / cm ２ More preferably, 5 × 10 ７ cells / cm ２ Below, particularly preferably 1 × 10 ７ cells / cm ２ The following is the result.

[0068] The following can be used as cell culture media for the three-dimensional cell culture construct. The cell culture medium is not particularly limited, and a medium that allows the cells to be cultured can be appropriately selected. Examples of media include basal media such as AIM V 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 basal culture media may be used alone or in combination of two or more. Furthermore, depending on the type and condition of the cells, culture media components may be added, removed, increased, or decreased. These basal culture media may be appropriately selected from known media depending on the cells to be cultured.

[0069] The substances or components contained in the cell culture medium are not particularly limited as long as they are suitable for the cells to be cultured, and include, for example, amino acids, sugars, vitamins, inorganic salts, antioxidants, etc. "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. "Sugars" include, for example, 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, vitamin B12, and the like. Inorganic salts include metal salts, including alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), copper, zinc, iron, and the like. Metal salts may be divalent or trivalent metal salts. Specific examples include metal salts such as hydrochlorides, carbonates, nitrates, and sulfates, such as potassium chloride, sodium chloride, calcium chloride, sodium sulfate, magnesium sulfate, magnesium carbonate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate. When calcium and magnesium salts are contained in the cell culture medium, the concentrations of calcium and magnesium salts in the cryopreservation solution specified above include the concentrations of the calcium and magnesium salts contained in the medium. Examples of "antioxidants" include ascorbic acid, glutathione, and the like.Other additives to the medium include antibiotics such as penicillin and streptomycin, lipids such as cholesterol, fatty acids such as linolenic acid, amines such as ethanolamine and putrescine, reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol, thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan, pH indicators such as phenol red, etc. These components may also be added exogenously, separately from the cell culture medium.

[0070] The three-dimensional cell culture may include a substrate and a three-dimensional cell aggregate formed on the substrate. The three-dimensional cell aggregate may include a cell sheet or a spheroid. The substrate may be a scaffold or a culture carrier substrate, as described below.

[0071] The three-dimensional cell culture may be three-dimensionally cultured using a scaffold; for example, adherent cells can adhere and proliferate on a scaffold having a mesh structure or the like. The scaffold material used for the scaffold is a material that can adhere or hold the cells to be cultured and enhance intercellular interactions for culture. The scaffold material may be appropriately selected from known materials depending on the cells to be cultured, and examples thereof 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), poly(ethylene glycol), poly(propylene glycol), polyisoprene, ...(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), poly(propylene glycol), Examples of suitable scaffold 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 polymer, acrylate resin, methacrylate resin, and derivatives and copolymers thereof. The scaffold material may be used alone or in combination of two or more materials. Its shape is not particularly limited, and examples include films, fibers, nonwoven fabrics, etc. Furthermore, to improve cell adhesion and spreadability, an extracellular matrix (ECM) with high cell affinity may be used. The extracellular matrix (ECM) is a non-cellular component present in all tissues and organs, and is composed primarily of two types of polymers: fibrous proteins and proteoglycans. The main fibrous proteins in the ECM are collagen, elastin, fibronectin, and laminin.On the other hand, the three-dimensional cell culture may be one that is three-dimensionally cultured without using a scaffold (scaffold-free). A scaffold-free three-dimensional cell culture, for example, a scaffold-free spheroid obtained by culturing suspended cells, has the advantage of reducing the risk of inflammatory reactions and infections due to foreign matter contamination that occurs when using a scaffold.

[0072] A "cell sheet" is a sheet structure in which cells are physically and / or functionally connected to one another via adhesion molecules, extracellular matrix, or the like. The method for producing a cell sheet can be performed using methods known to those skilled in the art and is not particularly limited. Examples of cell sheet production methods include those taught in literature such as International Publication No. 2016 / 068217, Japanese Patent Application Laid-Open No. 2019-000038, Japanese Patent Application Laid-Open No. 2011-006490, and International Publication No. 2015 / 068505. A cell sheet may have a single-layer structure consisting of one cell layer, or a laminated structure consisting of two or more cell layers (two, three, four, five, etc.). The thickness of the cell sheet can be appropriately set to ensure high cell viability and excellent shape retention, which is advantageous for cell transplantation, and may be, for example, 0.001 mm to 2.0 mm.

[0073] (Culture carrier substrate) When a cell sheet is used as a three-dimensional cell culture, it is preferable to form the cell sheet on a culture carrier substrate. By forming the cell sheet on a culture carrier substrate, the possibility of the cell sheet breaking when removed from a culture vessel or a cryopreservation container can be reduced, and it can be easily removed. The culture carrier substrate is used both as a cell sheet scaffold for culturing cells to form a cell sheet and as a cell sheet support for transporting the cultured cell sheet, and is a substrate that is detached from the cell sheet after attachment to the transplantation site. The area of ​​the culture carrier substrate is not particularly limited, but is preferably smaller than the culture vessel and the same as or larger than the cell sheet, for example, 0.3 cm ２ ~1000cm ２or less. 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 hydrophilization treatment, for example, UV ozone treatment, plasma treatment, etc., in order to improve cell adhesion. The materials constituting the culture carrier substrate can be the same as those usable for scaffolding materials. In particular, polyether ether ketone (PEEK), polyethylene terephthalate (PET), and polybutylene terephthalate (PBT) are preferred. Note that the culture carrier substrate can be packaged in a packaging material before being used for cell sheet cultivation.

[0074] An example of a method for producing a cell sheet-attached culture carrier substrate may include a culture step in which a plurality of cells, a culture medium (culture solution), and a culture carrier substrate are introduced into a culture vessel (e.g., each well of a cell culture multi-well plate) and a 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 detaching the cell sheet from the cell sheet-attached culture carrier substrate, the cell sheet can be used for treatment, prevention, etc., such as cell transplantation therapy.

[0075] The term "spheroid" generally refers to a mass (typically ball-shaped) of cells aggregated together, or a simple cluster (aggregate) of cells. Spheroids are typically formed by the aggregation of several thousand cells into a spherical structure. Three-dimensional cultures can maintain high levels of functional expression over longer periods than two-dimensional cell cultures. Spheroids may be composed of, for example, 1,000 or more cells, 10,000 or more cells, or even 10,000 or more cells. Spheroids can be produced and cultured using known methods, such as those taught in Scientific Reports 6(1):31063, "Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells," DOI:10.1038 / srep31063. Spheroids are preferably cultured in three-dimensional culture, where they grow three-dimensionally while maintaining their in vivo shape.

[0076] An "organoid" refers to a collection of cells that have been given the function of an organ. While actual organs cannot be cultured due to the difficulty of continuously providing nutrients, organoids can be cultured because they are smaller and simpler than actual organs. For example, organoids can be produced by passage of human organ stem cells or by differentiating organ stem cells to obtain hollow structures composed of multiple organ-specific cell types. The difference between organoids and spheroids is that spheroids are typically three-dimensionally cultured masses of simple cell types, packed to the core, whereas organoids are typically hollow structures composed of multiple cells that have specific organ functions. Organoids can be produced and cultured using known methods, such as those taught in Stem Cells International, Volume 2021, Article ID: 9929461, "Development of a Human Intestinal Organoid Model for In Vitro Studies on Gut Inflammation and Fibrosis", doi: 10.1155 / 2021 / 9929461.

[0077] The steps from freezing to thawing the three-dimensional cell culture construct are described below.

[0078] (Freezing Preparation Step) Three-dimensional cell cultures can be obtained by culturing transplantation cells formed into a desired three-dimensional shape, such as a three-dimensional sheet, sphere, or hollow shape, in a medium containing the above-mentioned culture solution (see, for example, the publicly known literature regarding the production of cell sheets, spheroids, and organoids exemplified above). The above-mentioned cryopreservation solution is also prepared. After cell culture, the three-dimensional cell culture is placed or immersed in the cryopreservation solution, for example, by adding the cryopreservation solution to the culture vessel containing the three-dimensional cell culture, or the three-dimensional cell culture is placed in contact with the cryopreservation solution in any manner. When cells are cultured in a state adhered 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 the culture vessel. Alternatively, to facilitate 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 left to stand in the culture vessel. Detachment may be achieved by enzymatic treatment using trypsin or physical techniques such as direct use of tweezers.

[0079] Methods for placing or immersing cultured cells in a cryopreservation solution include replacing the culture solution in a culture vessel with a cryopreservation solution and then placing or immersing the cultured cells in the cryopreservation solution. The method for replacing the culture solution in the culture vessel with a cryopreservation solution is not particularly limited, and known methods can be used. Specific examples of replacement methods include, once the cultured cells have reached the desired state, removing the culture solution from the culture vessel using a pipette and adding a cryopreservation solution to the culture vessel, removing a portion of the culture solution from the culture vessel and adding a cryopreservation solution, or adding a cryopreservation solution to the culture solution in the culture vessel. Alternatively, a culture vessel other than the one used for culturing the three-dimensional cell culture may be prepared, the cultured three-dimensional cell culture may be detached and transferred to the other culture vessel, and the three-dimensional cell culture may be immersed in a cryopreservation solution in the other culture vessel.

[0080] (Freezing Step) The freezing step is a step of freezing a three-dimensional cell culture together with a cryopreservation solution. The three-dimensional cell culture is placed or immersed in a cryopreservation solution above the freezing temperature, or the three-dimensional cell culture is placed in contact with the cryopreservation solution in any manner, and the three-dimensional cell culture and the cryopreservation solution are frozen together by placing the three-dimensional cell culture placed or immersed in a cryopreservation solution above the freezing temperature in an environment below the freezing temperature of the cryopreservation solution. This results in a frozen three-dimensional cell culture. The freezing method is not particularly limited, and any known freezing method can be used, but examples include a method of rapidly cooling a culture vessel containing the cryopreservation solution and the three-dimensional cell culture in an environment above the freezing temperature to the freezing temperature.

[0081] The freezing temperature is any temperature below the freezing temperature of the cryopreservation solution, and is not particularly limited as long as it can freeze the cultured cells and the cryopreservation solution. 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.

[0082] The freezing method is not particularly limited, and known freezing methods can be used. Examples include contact with the liquid or gas phase of a coolant and the use of a cooling device. Examples of coolants used in freezing include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice. The cooling device used in freezing is not particularly limited, and examples include a quick freezing device and a cryogenic refrigeration device. Note that, as a cooling device, a freezing device that does not come into contact with the heat transfer means and sprays cold air onto the culture vessel from multiple directions, preferably all directions, rather than one direction, rather than one direction, is preferable from the perspective of freezing cells at a uniform temperature and increasing the survival rate of the cells after thawing, rather than a cooling device that freezes the culture vessel and cells by bringing a heat transfer means into contact with the culture vessel. The storage time after freezing can be appropriately selected depending on the use, and may be, for example, 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. The three-dimensional cell culture frozen together with the cryopreservation solution can be stored and transported in the form of a container or package containing them.

[0083] (Freezing and thawing process) The freezing and thawing method is not particularly limited, and known thawing methods can be used. The freezing and thawing method can be performed by simply placing a container containing a frozen material containing a three-dimensional cell culture and a cryopreservation solution in an environment above the freezing point of the cryopreservation solution, or by using a water bath, incubator, hot plate, or the like. The thawing temperature or the environmental temperature in contact with the frozen material during thawing is not particularly limited, as long as it is above the freezing temperature of the cryopreservation solution and 45°C or lower. A thawing temperature higher than 45°C is not preferred because it may cause thermal damage to the cells. 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, provided that the thawing temperature is equal to or higher than the freezing temperature of the cell preservation solution; the lower limit of the thawing temperature may be selected from 0°C, 5°C, 10°C, 15°C, or 20°C; or, provided that the thawing temperature is equal to or higher than the freezing temperature of the cell preservation solution, the range of 0 to 45°C (including any value in 1°C increments therein), more preferably 10 to 35°C or 15 to 30°C, and even more preferably 15 to 25°C or 20 to 25°C.

[0084] The freeze-thawing process of the present invention involves placing the thawed three-dimensional cell culture in contact with a thawed cryopreservation solution. Typically, the process involves maintaining the thawed three-dimensional cell culture in a thawed, liquid state, or immersed in the cryopreservation solution for a predetermined period of time. The advantage of placing the three-dimensional cell culture in contact with the cryopreservation solution is that, as shown in the examples below, significant or substantial improvements in cell viability, as measured by trypan blue staining, are obtained compared to cryopreservation under the same conditions except for the inclusion of a specific concentration of calcium salt (and, if a magnesium salt is additionally included, a specific concentration of magnesium salt). The cell viability obtained after such cryopreservation is sufficient for clinical use and has not been achieved by conventional techniques. Furthermore, in conventional cryopreservation techniques, when the cryopreservation solution contains a component that is toxic to cells, such as DMSO, it is common to remove the cryopreservation solution early after thawing. However, the present inventors, contrary to this, deliberately maintained contact with the cells after thawing. Surprisingly, they found an excellent effect of significantly improving cell viability even after a certain period of time had passed. Therefore, the present invention can be used even when a component that is toxic to cells, such as DMSO, is contained, and there is also the advantage that there is no need to remove the cryopreservation solution after thawing. Another advantage is that conventional cryopreservation techniques have low cell viability after freezing and thawing, requiring cell proliferation by culturing after freezing and thawing. However, the present invention achieves high cell viability after freezing and thawing, eliminating the need for culturing after thawing (although this does not exclude the possibility of culturing after thawing). Another advantage is that the present invention allows for the use of known simple freezing and thawing processes, eliminating the need for complex freezing and thawing processes, thereby simplifying the processing steps and reducing costs.As will be shown in the Examples below, the cell viability after thawing improves over time, such as 60 minutes after the "starting point" (the point at which the frozen cryopreservation solution is thawed and becomes completely liquid). This is thought to be because cells that are not actually dead immediately after thawing but are counted as (apparently) dead by trypan blue staining are revived by trypan blue staining over time and newly counted as live cells through some mechanism. On the other hand, considering that cell viability decreases over time when a cytotoxic substance is contained in the preservation solution, as is commonly seen in conventional techniques, in the present invention, the addition of a specific concentration of calcium salt (and optionally a specific concentration of magnesium salt) improves cell protection through some mechanism, thereby improving cell viability. However, one possible reason for this is that, when the concentration is outside the specific range, this cytoprotective mechanism ceases to function or becomes weaker for some reason. However, the present invention is not bound by such a theory.

[0085] The time for which the thawed cell culture and the thawed cryopreservation solution are kept in contact with each other can be selected appropriately. For example, it may be at least 0.5 minutes, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, or more from the time when the frozen cryopreservation solution is thawed and becomes completely liquid (the "starting point"). Furthermore, the time for keeping the cells in contact with each other may be, for example, until the cell viability measured by trypan blue staining becomes higher. After the thawing and contact, the three-dimensional cell culture may be removed from the incubator or the like and washed with medium or the like.

[0086] In the present invention, the cell viability in the three-dimensional cell culture construct after freezing and thawing is preferably 35% or more or 40% or more at the time of calculation and / or after a certain time has elapsed since the time of calculation (e.g., 30, 60, 120, 180, or 240 minutes), more preferably 45% or more, 50% or more, or 55% or more, even more preferably 60% or more, 65% or more, or 70% or more, and particularly preferably 75% or more or 80% or more. Cell viability can be assessed by known methods, such as staining with trypan blue and counting live and dead cells using a hemocytometer. In the present invention, a cell evaluation method using trypan blue staining is used. In this case, cell viability is defined as the number of live cells / (number of live cells + number of dead cells) × 100%. More specifically, this is as described in the Examples below.

[0087] Immersion of a three-dimensional cell culture in the liquid cryopreservation solution of the present invention results in a three-dimensional cell culture for transplantation with improved post-thaw cell viability or metabolic activity. Here, the "metabolic activity" when immersed in the solution of the present invention is defined as the metabolic activity C1 of the three-dimensional cell culture obtained by immersing the three-dimensional cell culture in the solution of the present invention divided by the metabolic activity C0 obtained by immersing the three-dimensional cell culture for transplantation in a control solution (a solution that does not contain the calcium salt and magnesium salt at the predetermined concentrations specified for the solution of the present invention; a typical example of a control solution that does not contain the calcium salt and magnesium salt at the predetermined concentrations includes a commercially available cryopreservation solution that does not contain the specified metal salt). Specifically, metabolic activity is measured herein using the tetrazolium salt WST-8 (Cell Count Reagent SF, manufactured by Nacalai Tesque, Inc., product number 07553-44), a viable cell count measurement reagent. This is a cell counting method that uses intracellular reductase activity as an index, utilizing the fact that reduction in the presence of an electron carrier produces a highly water-soluble orange formazan dye. Those skilled in the art will understand that the "metabolic activity rate" measured when the solution of the present invention is used compared to a control solution is an index for determining whether or not the "cell viability rate" (defined as the ratio of the number of viable cells to the reference value (C0) in this specification) has been improved. The specific procedure for measuring "metabolic activity" involves first immersing the above-mentioned three-dimensional culture for transplantation in the solution of the present invention, then removing the solution, adding a 2% serum-containing medium, and incubating at 37°C and 5% CO. ２ After re-cultivation, the viable cell count measurement reagent WST-8 is diluted 20-fold with 2% serum-containing medium (reagent / medium = 1 / 19 volume ratio) to prepare a test solution. Next, a certain amount of the test solution is added to the container containing the three-dimensional cell culture construct, and the three-dimensional cell culture construct is incubated at 37°C and 5% CO ２The cells are incubated for 1 hour in an environment of 0.1%. After incubation, the supernatant of the test solution is added to a multi-well cell culture plate, and the supernatant is then measured using a plate reader to determine the metabolic activity using the following formula: metabolic activity C = {absorbance of measurement sample (450 nm) - absorbance of measurement sample (630 nm)} - {absorbance of blank (450 nm) - absorbance of blank (630 nm)]}. The test solution is used as the blank. The above measurement method is used in all of the examples described below.

[0088] Alternatively, "metabolic activity" may be evaluated by an ATP assay that measures ATP derived from living cells using the luminescence reaction of the firefly luciferase enzyme.

[0089] As described above, the present invention provides a three-dimensional cell culture with improved cell viability after freezing and thawing, using the freezing and freeze-thawing processes. Accordingly, one aspect of the present invention provides a three-dimensional cell culture frozen with a cryopreservation solution, the cryopreservation solution containing 3.5 to 500 ppm of calcium salt (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution. Furthermore, another aspect of the present invention provides a three-dimensional cell culture after freezing the three-dimensional cell culture with a cryopreservation solution and then placing the three-dimensional cell culture in contact with the cryopreservation solution, which is in a liquid state or in whole, during freezing and thawing, the three-dimensional cell culture containing 3.5 to 500 ppm of calcium salt (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution. As described above, this embodiment includes a three-dimensional cell culture in which a cryopreservation solution that is entirely in a liquid state when frozen and thawed is placed in contact with the three-dimensional cell culture, as well as a three-dimensional cell culture in which a cryopreservation solution that is partially in a liquid state when frozen and thawed is placed in contact with the three-dimensional cell culture (i.e., a three-dimensional cell culture in which the cryopreservation solution, in which some of the ice has melted and become liquid, is in contact with the thawed three-dimensional cell culture, even if the ice in the cryopreservation solution has not completely melted).

[0090] Furthermore, in one aspect of the present invention, the frozen three-dimensional cell culture can be produced by using the freezing step and the freeze-thaw step to freeze the three-dimensional cell culture in a cryopreservation solution containing 3.5 to 500 ppm of calcium ions (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution, and freezing the three-dimensional cell culture in the cryopreservation solution below the freezing temperature of the cryopreservation solution. Thus, in one aspect of the present invention, there is provided a method for producing a frozen three-dimensional cell culture, comprising the step of freezing the three-dimensional cell culture in the cryopreservation solution below the freezing temperature of the cryopreservation solution, wherein the cryopreservation solution contains 3.5 to 500 ppm of calcium ions (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution.

[0091] Furthermore, by utilizing the above-mentioned freezing step and freeze-thawing step, the three-dimensional cell culture of the present invention can be obtained by freezing the three-dimensional cell culture together with a cryopreservation solution containing 3.5 to 500 ppm of calcium ions (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution, and then placing the frozen-thawed three-dimensional cell culture in contact with the frozen-thawed cryopreservation solution. Thus, in one aspect of the present invention, a method is provided, comprising the steps of: freezing the three-dimensional cell culture in a cryopreservation solution at a temperature below the freezing temperature of the cryopreservation solution using a cryopreservation solution containing 3.5 to 500 ppm of calcium ions (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution; and freezing and thawing the frozen three-dimensional cell culture and the cryopreservation solution at a temperature above the freezing temperature of the cryopreservation solution, and placing the frozen-thawed three-dimensional cell culture in contact with the frozen-thawed cryopreservation solution.

[0092] Furthermore, the three-dimensional cell culture frozen with the cryopreservation solution, or the frozen-thawed three-dimensional cell culture and the frozen-thawed cryopreservation solution (or the thawed three-dimensional cell culture in the cryopreservation solution) may be provided in a container or package such as a cryo-resistant container or culture vessel (e.g., a multi-well cell culture plate). The term "package" includes any package capable of housing the three-dimensional cell culture and the cryopreservation solution, including kits. The container or package may also be accompanied by an instruction manual describing the cryopreservation method. Thus, in one aspect of the present invention, there is provided a container or package containing a frozen-thawed three-dimensional cell culture and a frozen-thawed cryopreservation solution, wherein the frozen-thawed three-dimensional cell culture is in contact with the frozen-thawed cryopreservation solution, and the cryopreservation solution contains 3.5 to 500 ppm of calcium ions (and 1 ppm or more of magnesium salt if a magnesium salt is further contained) based on the total weight of the cryopreservation solution.

[0093] Furthermore, in one aspect of the present invention, there is provided a method for improving the viability of cells in a frozen-thawed three-dimensional cell culture construct, the method comprising the freezing step and the freeze-thawing step, wherein the improved cell viability refers to an improvement compared to the viability of cells in a frozen-thawed three-dimensional cell culture construct obtained under the same conditions except that the cryopreservation solution contains 3.5 to 500 ppm of calcium ions based on the total weight of the cryopreservation solution (and, if a magnesium salt is further contained, contains a magnesium salt at a specific concentration).

[0094] Furthermore, in one aspect of the present invention, there is provided a method for protecting cells in a frozen three-dimensional cell culture construct from freezing-induced cell damage, which includes the above-mentioned freezing step and freeze-thaw step. Freezing-induced cell damage refers to any cell damage caused by freezing, such as cell damage due to ice crystal formation in a cryopreservation solution during freezing, and also includes cell damage caused by a toxic cryopreservation solution used for cell cryopreservation.

[0095] Although the embodiments of the present invention have been described above, these are merely illustrative examples, and various other configurations may be employed. Furthermore, the present invention is not limited to the above-described embodiments, and modifications, improvements, and the like that achieve the object of the present invention are encompassed within the scope of the present invention. Examples of reference embodiments are provided below. [Embodiment 1] A frozen three-dimensional cell culture for transplantation, wherein the frozen three-dimensional cell culture for transplantation is frozen together with a cryopreservation solution, and the cryopreservation solution contains 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 2] A frozen-thawed three-dimensional cell culture for transplantation, wherein the frozen three-dimensional cell culture for transplantation is a three-dimensional cell culture for transplantation that has been frozen together with a cryopreservation solution, and then the three-dimensional cell culture for transplantation is placed in contact with the cryopreservation solution, which is in a liquid state or in whole, during freezing and thawing, and the cryopreservation solution contains 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 3] The three-dimensional cell culture construct according to embodiment 1 or 2, wherein the cryopreservation solution comprises 5 to 300 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 4] The three-dimensional cell culture construct according to embodiment 1 or 2, wherein the cryopreservation solution comprises 7 to 100 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 5] The three-dimensional cell culture construct according to any one of embodiments 1 to 4, wherein the cryopreservation solution further comprises 1 ppm or more of magnesium salt based on the total weight of the cryopreservation solution. [Embodiment 6] The three-dimensional cell culture construct according to any one of embodiments 1 to 4, wherein the cryopreservation solution further comprises 2 to 100 ppm of magnesium salt based on the total weight of the cryopreservation solution. [Embodiment 7] The three-dimensional cell culture construct according to any one of embodiments 1 to 4, wherein the cryopreservation solution further comprises 2 to 20 ppm of magnesium salt based on the total weight of the cryopreservation solution. [Embodiment 8] The three-dimensional cell culture construct according to any one of embodiments 1 to 7, wherein the cryopreservation solution comprises a cryoprotectant.[Embodiment 9] The three-dimensional cell culture construct according to embodiment 8, wherein the cryoprotectant comprises DMSO, glycerin, propylene glycol, ethylene glycol, or a combination thereof. [Embodiment 10] The three-dimensional cell culture construct according to any one of embodiments 1 to 9, wherein the three-dimensional cell culture construct is a cell sheet, a spheroid, an organoid, or a combination thereof. [Embodiment 11] The three-dimensional cell culture construct according to embodiment 10, wherein the cell sheet is on a scaffold or a culture carrier substrate. [Embodiment 12] The three-dimensional cell culture construct according to any one of embodiments 1 to 11, wherein the cells in the three-dimensional cell culture construct are mammalian-derived cells. [Embodiment 13] The three-dimensional cell culture construct according to embodiment 12, wherein the mammalian-derived cells are at least one selected from the group consisting of biological tissue cells, mesenchymal stem cells, and pluripotent stem cells. [Embodiment 14] The three-dimensional cell culture construct according to embodiment 13, wherein the biological tissue cells are fibroblasts. [Embodiment 15] The three-dimensional cell culture construct according to any one of embodiments 1 to 14, wherein the viability of cells in the three-dimensional cell culture construct or the frozen-thawed three-dimensional cell culture construct after freezing and thawing is 35% or more. [Embodiment 16] The three-dimensional cell culture construct according to any one of embodiments 1 to 14, wherein the viability of cells in the three-dimensional cell culture construct or the frozen-thawed three-dimensional cell culture construct after freezing and thawing is 50% or more. [Embodiment 17] The three-dimensional cell culture construct according to any one of embodiments 1 to 14, wherein the viability of cells in the three-dimensional cell culture construct or the frozen-thawed three-dimensional cell culture construct after freezing and thawing is 60% or more. [Embodiment 18] The three-dimensional cell culture construct according to any one of embodiments 1 to 17, wherein the frozen three-dimensional cell culture construct and the frozen cryopreservation solution are thawed at 0 to 45°C. [Embodiment 19] The three-dimensional cell culture construct according to any one of embodiments 1 to 18, which is not cultured after freezing and thawing. [Embodiment 20] A container or package containing a frozen-thawed three-dimensional cell culture for transplantation and a frozen-thawed cryopreservation solution, wherein the three-dimensional cell culture for transplantation is placed in contact with the cryopreservation solution, and the cryopreservation solution contains 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution.[Embodiment 21] The container or package according to embodiment 20, comprising 5 to 300 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 22] The container or package according to embodiment 20, comprising 7 to 100 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 23] The container or package according to any one of embodiments 20 to 22, further comprising 1 ppm or more of magnesium salt based on the total weight of the cryopreservation solution. [Embodiment 24] A cryopreservation solution for use in cryopreserving a three-dimensional cell culture for transplantation, comprising 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 25] The cryopreservation solution according to embodiment 24, comprising 5 to 300 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 26] The cryopreservation solution according to embodiment 24, comprising 7 to 100 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 27] The cryopreservation solution of any one of embodiments 24 to 26, further comprising 1 ppm or more of a magnesium salt based on the total weight of the cryopreservation solution. [Embodiment 28] A method for producing a frozen three-dimensional cell culture for transplantation, comprising freezing a three-dimensional cell culture for transplantation in a cryopreservation solution below the freezing temperature of the cryopreservation solution, wherein the cryopreservation solution comprises 3.5 to 500 ppm of a calcium salt based on the total weight of the cryopreservation solution. [Embodiment 29] A method for producing a frozen-thawed three-dimensional cell culture for transplantation, comprising: freezing a three-dimensional cell culture for transplantation in a cryopreservation solution below the freezing temperature of the cryopreservation solution; and freezing and thawing the frozen three-dimensional cell culture for transplantation and the cryopreservation solution at a temperature equal to or higher than the freezing temperature of the cryopreservation solution, and placing the frozen-thawed three-dimensional cell culture for transplantation in contact with the frozen-thawed cryopreservation solution, wherein the cryopreservation solution comprises 3.5 to 500 ppm of a calcium salt based on the total weight of the cryopreservation solution.[Embodiment 30] A method for improving the viability of cells in a frozen-thawed three-dimensional cell culture for transplantation, comprising the steps of: freezing the three-dimensional cell culture for transplantation in a cryopreservation solution at a temperature below the freezing temperature of the cryopreservation solution; and freezing and thawing the frozen three-dimensional cell culture for transplantation and the frozen cryopreservation solution at a temperature equal to or higher than the freezing temperature of the cryopreservation solution, and placing the frozen-thawed three-dimensional cell culture for transplantation and the frozen-thawed cryopreservation solution in contact with each other, wherein the cryopreservation solution contains 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution, and the viability of the cells is improved compared to the viability of cells in a frozen-thawed three-dimensional cell culture for transplantation obtained under the same conditions except that the cryopreservation solution contains 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 31] A method for protecting cells in a frozen transplantable three-dimensional cell culture from cell damage associated with the freezing, comprising the steps of: freezing the transplantable three-dimensional cell culture in a cryopreservation solution at a temperature below the freezing temperature of the cryopreservation solution; and freezing and thawing the frozen transplantable three-dimensional cell culture and the cryopreservation solution at a temperature equal to or higher than the freezing temperature of the cryopreservation solution, and placing the frozen-thawed transplantable three-dimensional cell culture and the frozen-thawed cryopreservation solution in contact with each other, wherein the cryopreservation solution comprises 3.5 to 500 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 32] The method of any one of embodiments 28 to 31, wherein the cryopreservation solution comprises 5 to 300 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 33] The method of any one of embodiments 28 to 31, wherein the cryopreservation solution comprises 7 to 100 ppm of calcium salt based on the total weight of the cryopreservation solution. [Embodiment 34] The method according to any one of embodiments 28 to 31, wherein the cryopreservation solution further comprises 1 ppm or more of a magnesium salt based on the total weight of the cryopreservation solution. [Embodiment 35] The method according to any one of embodiments 28 to 34, wherein the three-dimensional cell culture for transplantation in the cryopreservation solution is frozen at -196°C to -25°C.

[0096] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0097] [Evaluation of cell viability of cell sheets after freezing and thawing] [Example 1] <Production of culture carrier substrate> A plasma treatment was performed on the culture surface (surface) of a PEEK film (Shin-Etsu Polymer Co., Ltd., polyether ether ketone film, Shin-Etsu Sepla Film (registered trademark), low crystallinity type, 12 μm thick). The surface-treated film was then cut into a Φ14.0 mm size to obtain a disk-shaped culture carrier substrate.

[0098] <Production of Cell Sheets> The culture carrier substrates produced as described in <Production of Culture Carrier Substrates> above were washed with 70% ethanol, phosphate buffer, and culture medium in that order, and then placed on the flat bottom of each well of a multi-well cell culture plate (24 wells). The culture surface of the culture carrier substrate was placed facing the opening of the well plate, i.e., the back surface of the culture carrier substrate was placed in contact with the flat bottom of each well of the multi-well cell culture plate. Cryopreserved human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with culture medium. 5 x 10 ５ The cells were suspended in a 5% serum-containing medium and placed in two dishes (60.1 cm ２ ) to 2.5 x 10 ５ The cultured cells were collected, suspended in a 5% serum-containing medium, and placed in 10 flasks (culture area 150 cm). ２ ) to 2.5 x 10 ５ The cultured cells were collected, suspended in a 2% serum-containing medium, and placed in a 27.9 × 10 cells / well in each well of a multi-well cell culture plate equipped with a culture carrier substrate. ４ pieces / cm ２ and incubated at 37°C and 5% CO ２ The culture medium was incubated for 1 day under the above conditions to prepare a cell sheet on the culture surface of the culture carrier substrate.

[0099] <Preparation of cryopreservation solution> Using an electronic balance (BalanceXS104, 11106012, manufactured by Mettler-Toledo), CaCl ２ ・2H ２ O (031-25035, Fujifilm Wako Pure Chemical Industries, Ltd.), and MgSO ４ Each sample (137-12335, Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed and added to 1 L of commercially available cryopreservation solution A (Bambanker (registered trademark) hRM, CG Lymphotec, containing 10 w / v% DMSO (cryoprotectant) in water and 1 wt% human albumin) to prepare a cryopreservation solution. At this time, the contents of calcium salt and magnesium salt contained in the cryopreservation solution after preparation were calculated as the concentrations of calcium ion and magnesium ion, respectively, and were calculated as the Ca ion and magnesium ion concentrations shown in Table 1 below. ２＋ Concentration, Mg ２＋ CaCl ２ ・2H ２ O and MgSO ４ The amount of added was changed.

[0100] <Freezing of Cell Sheet> After the above <Cell Sheet Production>, the cell culture multiwell plate was placed on ice, and the cell sheet with the culture carrier substrate was washed with phosphate buffer. The cryopreservation solution shown in Table 1 was then added at 0.22 mL / well, and the cell sheet was immersed in the cryopreservation solution in each well. The cell culture multiwell plate was then packaged in a zippered plastic bag, and the resulting package was placed in a non-throughflow cooling device (3D Freezer (registered trademark), KSS-40BLW-2400V, manufactured by Kogasan Co., Ltd.) precooled to -35°C and maintained for 30 minutes. The cooling rate from 0 to -5°C was 3.4°C / min. The cooled package was then promptly placed in a -150°C freezer (Freeze Ultra-Low Temperature Chamber CLN-17000WE, manufactured by Nippon Freezer Co., Ltd.) and maintained for 24 hours. In this manner, a frozen three-dimensional cell culture construct containing a frozen cell sheet was produced.

[0101] <Thawing of Cell Sheets> After the freezing period, the package was removed from the -150°C freezer, and the cell culture multiwell plate was left to thaw at room temperature (20-25°C). The point at which it was visually confirmed that the frozen cryopreservation solution had completely turned liquid (i.e., the ice in the cryopreservation solution had completely disappeared) was designated the "starting point" (0 minutes). The cell sheet was left to stand in the thawed cryopreservation solution for 60 minutes from the starting point, thereby bringing the cell sheet into contact with the cryopreservation solution. Temperature measurements were performed by placing only the cryopreservation solution in an unused well of the cell culture multiwell plate, placing a thermocouple in the cryopreservation solution, and measuring the temperature. This measured temperature was considered to be the same as the temperature of the cryopreservation solution in the well containing the cell sheet and the cryopreservation solution. The viability of cells present in the thawed cell sheet was then measured according to the procedure described below in <Measurement of Cell Viability>.

[0102] <Measurement of cell viability> The thawed cell sheet obtained in <Thawing of cell sheet> was washed with phosphate buffer, treated with r-TE (1210, Cytochemical Research Institute) for 3 minutes, and dispersed into single cells by pipetting. s-TI (1220, Cytochemical Research Institute) was added to the resulting single cell suspension, which was then stained with trypan blue. Live and dead cells were counted using a hemocytometer to evaluate cell viability (%).

[0103] [Examples 2 to 9 and Comparative Examples 1 and 2] In Examples 2 to 9 and Comparative Examples 1 and 2, the calcium salt and magnesium salt contained in the cryopreservation solution in the above <Preparation of cryopreservation solution> were changed to Ca ２＋ Concentration and Mg ２＋ Except for the change in concentration, the <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet> were carried out in the same manner as in Example 1.

[0104] For each of the Examples and Comparative Examples, the cell viability after thawing was evaluated as described above in <Measurement of cell viability>. The results are shown in Table 1.

[0105]

[0106] As shown in Table 1, in Examples 1 to 8, a very good improvement in cell viability was obtained both at the starting point and 60 minutes after the starting point compared to the results of Comparative Examples 1 and 2 (i.e., in these Examples, the cell viability was 44-70% at the starting point, and 55-74% at 60 minutes after the starting point). In addition, it was unexpectedly found that the cell viability at 60 minutes after the starting point was higher than that at the starting point. More specifically, as can be seen from a comparison of the results of Example 4 and Comparative Example 1 (these Examples have the same magnesium salt concentration), the Ca ２＋ It was surprising and unexpected that increasing the concentration from 3 ppm to 3.6 ppm significantly increased cell viability from 2% to 55%. Furthermore, in Example 9, similar to Examples 1 to 8, high cell viability (60%) was obtained at the initial time point, but at 60 minutes from the initial time point, the cell viability decreased, but was still higher than the cell viability measured in Comparative Examples 1 and 2.

[0107] Furthermore, in Example 2, in addition to measuring the cell viability at the starting point and 60 minutes after the starting point, the cell viability was also measured at 30 minutes, 180 minutes, and 240 minutes after the starting point. The results are shown in Table 2 below. For example, for "+240 minutes from the starting point" in Table 1, the cell sheet was left in the thawed cryopreservation solution for 240 minutes after the starting point in <Thawing the cell sheet>, thereby bringing the cell sheet into contact with the cryopreservation solution, and then the <Measurement of cell viability> was performed. Furthermore, for "1 day before freezing" in Table 2, the <Measurement of cell viability> was performed on the cell sheet obtained in the <Production of the cell sheet> one day before the <Freezing of the cell sheet> was performed.

[0108]

[0109] As shown in the results in Table 2 above, it was found that the cell viability increased up to 180 minutes from the starting point, and furthermore, that the cell viability remained high even after 240 minutes from the starting point.

[0110] Furthermore, in Comparative Example 1, the cell viability was further measured at 180 minutes and 240 minutes from the starting point in the same manner as in Example 2. The results are shown in Table 2. As shown in Table 2, in Comparative Example 1, the cell viability at 60 minutes from the starting point significantly decreased at 180 minutes and 240 minutes from the starting point. On the other hand, in Example 2, it was found that high cell viability was still maintained even at 180 to 240 minutes from the starting point, and it was found that high cell viability could be maintained for a longer period of time.

[0111] [Examples 10 and 11] In Examples 10 and 11, instead of cryopreservation solution A, a commercially available cryopreservation solution B (Stem Cell Banker (registered trademark) EX GMP grade (11936, manufactured by Nippon Zenyaku Kogyo Co., Ltd.), an aqueous solution containing 10 w / v% DMSO (cryoprotectant), not containing human-derived albumin) was used, and in the above <Preparation of cryopreservation solution>, the calcium salt and magnesium salt contained in the cryopreservation solution were each replaced with the Ca salt shown in Table 3. ２＋ Concentration and Mg ２＋ Except for the change in concentration, the same procedures as in Example 1 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0112] Comparative Examples 3 and 4 Comparative Examples 3 and 4 are the same as those in Comparative Examples 3 and 4 except that the amount of Ca in the cryopreservation solution in the above <Preparation of cryopreservation solution> was ２＋ Concentration and Mg ２＋ Except for changing the concentration to the value shown in Table 3, the same procedures as in Example 10 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0113] [Examples 12 to 13] In Examples 12 to 13, instead of cryopreservation solution A, a commercially available cryopreservation solution C (iStock (385-19451, manufactured by CG Lymphotec), an aqueous solution containing 11 w / v % DMSO (cryoprotectant)) was used, and in the above <Preparation of cryopreservation solution>, the calcium salt and magnesium salt contained in the cryopreservation solution were changed to the Ca salts shown in Table 4. ２＋ Concentration and Mg ２＋Except for the change in concentration, the same procedures as in Example 1 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0114] Comparative Examples 5 and 6 Comparative Examples 5 and 6 are the same as those in Comparative Examples 5 and 6, except that the amount of Ca in the freezing solution in the above <Preparation of cryopreservation solution> was ２＋ Concentration and Mg ２＋ Except for changing the concentration to the value shown in Table 4, the same procedures as in Example 12 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0115] The cell viability after thawing of Examples 10 to 13 and Comparative Examples 3 to 6 was evaluated as described in <Measurement of cell viability>. The respective results are shown in Tables 3 and 4.

[0116]

[0117]

[0118] From the results of Examples 10 to 13 shown in Tables 3 and 4 above, it was found that even when cryopreservation solution A was changed to B or C, the cell survival rate after thawing was improved by adjusting the calcium salt concentration in the cryopreservation solution to the concentrations shown in Tables 3 and 4. Furthermore, as shown in Examples 10 and 11, it was found that a high cell survival rate after thawing could be obtained even when the cryopreservation solution did not contain albumin.

[0119] [Examples 14 to 35] In Examples 14 to 35, in the above <Preparation of cryopreservation solution>, magnesium salt (MgSO ４ , 137-12335, Fujifilm Wako Pure Chemical Industries, Ltd.), copper salt (CuSO ４ ・5H ２ O, 031-044115, Fujifilm Wako Pure Chemical Industries, Ltd.), manganese salt (MnSO ４ ・5H ２ O, 1139-0082, Fujifilm Wako Pure Chemical Industries, Ltd.), zinc salt (ZnSO ４ ・7H ２ O, 268-00405, Fujifilm Wako Pure Chemical Industries, Ltd.), iron salt (FeSO ４ ・7H２ O, 098-01085, Fujifilm Wako Pure Chemical Industries, Ltd.), molybdenum salt ((NH ４ ) ６ Mo ７ O ２４ ・4H ２ O, 010-06905, Fujifilm Wako Pure Chemical Industries, Ltd.), nickel salt (NiCl ２ ・6H ２ O, 147-01042, Fujifilm Wako Pure Chemical Industries, Ltd.), and cobalt salt (CoCl ２ ・6H ２ 0, 036-03682, Fujifilm Wako Pure Chemical Industries, Ltd.) in the frozen solution. ２＋ The same procedures as in Example 7 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>, except that the concentration was set to the values ​​shown in Table 5. The content of the divalent metal salt contained in the cryopreservation solution after preparation was expressed as the concentration converted into the divalent metal ion of X, where X is the metal element contained in the divalent metal salt used. The cell viability after thawing in Examples 14 to 35 was evaluated as described in <Measurement of cell viability>. The respective results are shown in Table 5.

[0120]

[0121] [Evaluation of metabolic activity rate of cell sheets after freezing and thawing] [Examples 36 to 40] In Examples 36 to 40, in the above <Preparation of cryopreservation solution>, MgSO ４ By further adding Mg as shown in Table 6, the magnesium salt contained in the cryopreservation solution can be ２＋ Except for the change in concentration, the same procedures as in Comparative Example 1 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0122] <Measurement of metabolic activity (WST-8 assay)> In Comparative Example 1 and Examples 36 to 40, the cryopreservation solution was removed from the cell culture multiwell plate containing the cell sheet obtained in <Thawing of cell sheet>, and then a 2% serum-containing medium was added and the cells were thawed at 37°C under 5% CO２ After re-cultivation, the viable cell count measurement reagent WST-8 was diluted 20-fold with 2% serum-containing medium (reagent / medium = 1 / 19 volume ratio) to prepare a test solution. Next, a certain amount of the test solution was added to the cell culture multi-well plate containing the cell sheet, and the cells were incubated at 37°C and 5% CO ２ The cells were incubated for 1 hour in an environment of 0.1%. After incubation, 100 μL of the supernatant from the test solution was added to another cell culture multiwell plate (MS-8096R, manufactured by Sumitomo Bakelite Co., Ltd.), and the supernatant was then measured using a plate reader to determine the metabolic activity using the following formula: Metabolic activity C = {absorbance of measurement sample (450 nm) - absorbance of measurement sample (630 nm)} - {absorbance of blank (450 nm) - absorbance of blank (630 nm)]}. The test solution was used as the blank.

[0123] <Calculation of metabolic activity rate> In Examples 36 to 40, in <Thawing of cell sheet>, the cryopreservation solution shown in Table 6 was used, and the cell sheet was allowed to stand in the cryopreservation solution for 60 minutes from the starting point. The metabolic activity C1 of the cell sheet in contact with the cryopreservation solution was measured in accordance with the above <Measurement of metabolic activity>. The metabolic activity C0 of Comparative Example 1 was measured in the same manner as in the above Examples in accordance with the above <Measurement of metabolic activity>. The metabolic activity rate for each Example was calculated using the measured metabolic activities C1 and C0 based on the formula "C1 / C0". The results are shown in Table 6.

[0124]

[0125] [Examples 41 to 44] In Examples 41 to 44, in the above <Preparation of cryopreservation solution>, a copper salt (CuSO ４ ・5H ２ O, 031-044115, Fujifilm Wako Pure Chemical Industries, Ltd.), manganese salt (MnSO ４ ・5H ２ O, 1139-0082, Fujifilm Wako Pure Chemical Industries, Ltd.), iron salt (FeSO ４ ・7H ２ O, 098-01085, Fujifilm Wako Pure Chemical Industries, Ltd.), and nickel salt (NiCl ２ ・6H ２0, 147-01042, Fujifilm Wako Pure Chemical Industries, Ltd.) in Table 7, ２＋ Except for the addition of additional ingredients so that the concentrations were as shown in Table 7, the same procedures as in Comparative Example 1 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0126] In Examples 41 to 44, the metabolic activity rate (%) was calculated in the same manner as in <Calculation of metabolic activity rate> above. The results are shown in Table 7.

[0127]

[0128] [Examples 45 and 47] In Example 45, in the above <Preparation of cryopreservation solution>, CaCl was used as the calcium salt. ２ ・2H ２ Instead of O, Ca(NO ３ ) ２ ・4H ２ Example 47 was carried out in the same manner as Comparative Example 1, except that MgSO 4 was added as a magnesium salt in the above <Preparation of cryopreservation solution>. ４ Instead of MgCl ２ ・6H ２ The same procedures as in Comparative Example 1 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>, except that 135-00165 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added.

[0129] In Examples 45 and 47, the metabolic activity rate (%) was calculated in the same manner as in <Calculation of metabolic activity rate> above. The results are shown in Table 8.

[0130]

[0131] [Examples 48 to 54] In Examples 48 to 54, in the above <Preparation of cryopreservation solution>, manganese salt (MnSO ４ ・5H ２ O, 1139-0082, Fujifilm Wako Pure Chemical Industries, Ltd.), zinc salt (ZnSO ４ ・7H２ O, 268-00405, Fujifilm Wako Pure Chemical Industries, Ltd.), and nickel salt (NiCl ２ ・6H ２ O, 147-01042, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in the frozen solution. ２＋ Except for the addition of additional ingredients so that the concentrations were as shown in Table 9, the same procedures as in Comparative Example 1 were carried out as described in <Production of culture carrier substrate>, <Production of cell sheet>, <Preparation of cryopreservation solution>, <Freezing of cell sheet>, and <Thawing of cell sheet>.

[0132] In Examples 48 to 54, metabolic activity rates (%) were calculated in the same manner as in the above <Metabolic activity rate>. The results are shown in Table 9.

[0133]

[0134] [Examples 1 to 3, Comparative Examples 1 and 2] The metabolic activity rate (%) was calculated in the same manner as in <Calculation of metabolic activity rate> above for Examples 1 to 3 and Comparative Examples 1 and 2. The results are shown in Table 10.

[0135]

[0136] [Evaluation of metabolic activity rate of spheroids after freezing and thawing] [Example 55]

[0137] <Production of spheroids> Cryopreserved human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with medium. ５ The cells were suspended in a 5% serum-containing medium and placed in two dishes (60.1 cm ２ ) to 2.5 x 10 ５ The cultured cells were collected, suspended in a 5% serum-containing medium, and placed in 10 flasks (culture area 150 cm). ２ ) to 2.5 x 10 ５ The cultured cells were collected, suspended in a 2% serum-containing medium, and seeded at 1000 cells / well / 0.15 mL in each well of a cell culture multi-well plate (MS-9096U, manufactured by Sumitomo Bakelite Co., Ltd., U-bottom). ２The cells were incubated for 2 days under this environment to form spheroids (cell aggregates) on the U-shaped bottom of the wells.

[0138] <Preparation of cryopreservation solution> CaCl was measured using an electronic balance (BalanceXS104, 11106012, Mettler-Toledo). ２ ・2H ２ O (031-25035, Fujifilm Wako Pure Chemical Industries, Ltd.), and MgSO ４ Each sample (137-12335, Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed and added to 1 L of commercially available cryopreservation solution A (Bambanker (registered trademark) hRM, CG Lymphotec Inc.; containing an aqueous solution containing 10 w / v% DMSO (a cryoprotectant) and 1 wt% human albumin). The calcium salt (Ca ２＋ ) concentration and magnesium salt (Mg ２＋ ) were adjusted to the concentrations shown in Table 11 below.

[0139] <Freezing of Spheroids> After the above <Production of Spheroids>, the spheroids in the cell culture multiwell plate were added to a 1.5 mL tube (131-7155CS-N, manufactured by WATSON). After washing the spheroids with phosphate buffer, the cryopreservation solution prepared in <Preparation of Cryopreservation Solution> was added to each tube at 0.2 mL, and the spheroids were immersed in the cryopreservation solution in each tube. The tubes containing the spheroids were then placed in a package (AFB-25, manufactured by AS ONE Corporation) and placed in a non-throughflow cooling device (3D Freezer (registered trademark), KSS-40BLW-2400V, manufactured by Kogasan Co., Ltd.) cooled to -35 ° C and held for 30 minutes. The cooling rate at 0 to -5 ° C was 3.4 ° C / min. Thereafter, the cooled package was quickly placed in a freezer (ultra-low temperature freeze chamber CLN-17000WE, manufactured by Nippon Freezer Co., Ltd.) at −150° C. and kept there for 24 hours.

[0140] <Thawing of Spheroids> After the freezing period, the package was removed from the -150°C freezer, and the 1.5 mL tube contained therein was allowed to stand at room temperature (20-25°C) to thaw. The point at which it was visually confirmed that the frozen cryopreservation solution had completely turned liquid (i.e., the ice in the cryopreservation solution had completely disappeared) was designated the "starting point" (0 minutes). The cell sheet was allowed to stand in the thawed cryopreservation solution for 90 minutes from the starting point, thereby placing the cell sheet in contact with the cryopreservation solution. Temperature measurements were performed by placing only the cryopreservation solution in a 1.5 mL tube, placing a thermocouple in the cryopreservation solution, and measuring the temperature. This measured temperature was considered to be the same as the temperature of the cryopreservation solution in the well containing the spheroids and cryopreservation solution. After thawing, 1 mL of 2% serum-containing medium was added to the 1.5 mL tube. The supernatant was then aspirated, and 1 mL of 2% serum-containing medium was added again. A 0.15 mL aliquot of the supernatant and spheroids in the tube was added to a cell culture multi-well plate (MS-8048R, manufactured by Sumitomo Bakelite Co., Ltd.), with four spheroids added per well. The plate was incubated at 37°C and 5% CO ２ The cells were incubated for 2 days in a 0.5% CO2 environment. Cell viability could not be measured because the spheroids could not be detached into single cells.

[0141] <Measurement of metabolic activity (ATP assay)> The metabolic activity of spheroids was measured using an ATP reagent ("Lump" ATP measurement reagent Ver. 2.1, Toyo B-Net Co., Ltd., KA2.1-100) and a plate reader (iMark, Bio-Rad Laboratories, Inc.). The ATP reagent was diluted 10-fold with culture medium (reagent / culture medium = 1 / 9 volume ratio) and used as the test solution. 1 mL of 2% serum-containing medium was added to the 1.5 mL tube thawed in the above <Thawing of spheroids>. Then, the supernatant was aspirated and 1 mL of 2% serum-containing medium was added again. 0.15 mL of the supernatant and spheroids in the tube were taken and added to a cell culture multi-well plate (MS-8048R, Sumitomo Bakelite Co., Ltd.). Four spheroids were added per well and incubated at 37°C, 5% CO ２The plates were incubated for 2 days under the following conditions. After incubation, 0.09 mL of the supernatant and four spheroids were added to a luminescence measurement plate (MS-8096W, manufactured by Sumitomo Bakelite Co., Ltd.). 0.01 mL of test solution was added to each well. After shaking for 1 minute and leaving the plate to stand for 9 minutes, the luminescence measurement plate was placed in a plate reader and the amount of luminescence was measured. The metabolic activity was calculated as follows: The test solution was used as a blank. The accumulation time during measurement was 1000 ms. Metabolic activity [RLU] = (luminescence amount of measurement sample - luminescence amount of blank) / 4

[0142] Comparative Example 7 Comparative Example 7 is a comparative example in which the amount of Ca in the preservation solution in the above <Preparation of cryopreservation solution> was ２＋ Concentration and Mg ２＋ Except for changing the concentration to the value shown in Table 11, the same procedures as in Example 55 were carried out as described in <Production of spheroids>, <Preparation of cryopreservation solution>, <Freezing of spheroids>, and <Thawing of spheroids>.

[0143] For each Example and Comparative Example, metabolic activity C0 of Comparative Example 7 and metabolic activity C1 of Example 55 after thawing were evaluated as described above in <Measurement of metabolic activity (ATP assay)>. The metabolic activity rate was calculated based on the formula "C1 / C0" using the measured metabolic activities C1 and C0. The results are shown in Table 11.

[0144]

[0145] As shown in Table 11, Example 55 showed a very good improvement in metabolic activity rate at the starting point and 90 minutes after the starting point, compared to the results of Comparative Example 7. As described above, it was found that by including specific concentrations of calcium salt and magnesium salt in the cryopreservation solution, the metabolic activity rate after thawing was good, and high metabolic activity was maintained for a long period of time, not only for two-dimensionally expanding cell structures such as cell sheets, but also for three-dimensional cell structures such as spheroids, compared to when these salts were not included.

[0146] This application claims priority based on Japanese Patent Application No. 2024-031022, filed March 1, 2024, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A frozen three-dimensional cell culture comprising a three-dimensional cell culture having a cell-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution contains a calcium salt and water, and the calcium salt content is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution.

2. A frozen product of a three-dimensional cell culture comprising a three-dimensional cell culture having a cell-to-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution comprises water and one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion based on the total weight of the cryopreservation solution.

3. A frozen product of a three-dimensional cell culture according to claim 1, wherein the cryopreservation solution contains one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts and cobalt salts, and when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains a divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion based on the total weight of the cryopreservation solution.

4. A frozen three-dimensional cell culture according to claim 1 or 2, wherein the cryopreservation solution contains a cryoprotectant.

5. A frozen three-dimensional cell culture according to claim 4, wherein the cryoprotectant comprises one or more selected from the group consisting of DMSO, glycerin, propylene glycol, and ethylene glycol.

6. A frozen three-dimensional cell culture according to claim 1 or 2, wherein the three-dimensional cell culture comprises a cell sheet or a spheroid.

7. A frozen three-dimensional cell culture according to claim 6, wherein the three-dimensional cell culture comprises a substrate and the cell sheet adhered onto the substrate.

8. A frozen three-dimensional cell culture according to claim 1 or 2, wherein the survival rate of the cells contained in the three-dimensional cell culture after thawing the frozen three-dimensional cell culture is 35% or more.

9. A container containing a frozen three-dimensional cell culture construct according to claim 1 or 2.

10. A cell freezing kit comprising a three-dimensional cell culture construct having a cell-cell connection structure and a cryopreservation solution, wherein the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm to 500 ppm in terms of calcium ions based on the total weight of the cryopreservation solution. (ii) A cell freezing kit comprising one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains the divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion, based on the total weight of the cryopreservation solution.

11. A cryopreservation solution used to freeze a three-dimensional cell culture having a cell-cell connection structure, wherein the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) It contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm or more and 500 ppm or less in terms of calcium ions, based on the total weight of the cryopreservation solution. (ii) A cryopreservation solution comprising one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm or more and 300 ppm or less in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains the divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm or more and 100 ppm or less in terms of metal ion, based on the total weight of the cryopreservation solution.

12. A method for producing a frozen product of a three-dimensional cell culture, comprising a freezing step of freezing a three-dimensional cell culture having a cell-cell connection structure in a cryopreservation solution, wherein the cryopreservation solution satisfies at least one of the following (i) and (ii): (i) contains a calcium salt and water, and the content of the calcium salt is 3.5 ppm or more and 500 ppm or less in terms of calcium ions, based on the total weight of the cryopreservation solution. (ii) A method for producing a frozen product, comprising one or more divalent metal salts selected from the group consisting of magnesium salts, copper salts, manganese salts, zinc salts, iron salts, molybdenum salts, nickel salts, and cobalt salts, and water, wherein when the cryopreservation solution contains the magnesium salt, the content of the magnesium salt is 2 ppm to 300 ppm in terms of magnesium ion, based on the total weight of the cryopreservation solution, and / or when the cryopreservation solution contains the divalent metal salt other than the magnesium salt, the content of the divalent metal salt is 0.01 ppm to 100 ppm in terms of metal ion, based on the total weight of the cryopreservation solution.

13. A method for producing a frozen product according to claim 12, wherein the freezing step freezes the three-dimensional cell culture at a temperature lower than the freezing temperature of the cryopreservation solution.

14. A method for producing a thawed three-dimensional cell culture, comprising a thawing step of thawing the frozen three-dimensional cell culture obtained by the method for producing a frozen product according to claim 12.

15. A method for producing a thawed product according to claim 14, wherein the thawing step comprises maintaining at least a portion of the three-dimensional cell culture construct in contact with the cryopreservation solution in a liquid state.

16. A method for using a frozen three-dimensional cell culture according to claim 1 or 2, wherein when the frozen product is thawed, at least a portion of the three-dimensional cell culture is kept in contact with the liquid cryopreservation solution.

17. A method for using the cryopreservation solution according to claim 11, comprising the step of improving the viability of cells in the three-dimensional cell culture by maintaining the cryopreservation solution in contact with at least a portion of the three-dimensional cell culture during freezing and thawing.

18. A method for using the cryopreservation solution according to claim 11, comprising the step of protecting cells in the three-dimensional cell culture from cell damage associated with freezing by maintaining the cryopreservation solution in contact with at least a portion of the three-dimensional cell culture during freezing and thawing.

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