Cryopreservation preparation for corneal endothelial cells and method for producing said cryopreservation preparation

Abstract

The present disclosure provides a method for cryopreserving corneal endothelial cells and / or corneal-endothelial-like cells, and a freezing preparation for corneal endothelial cells and / or corneal-endothelial-like cells. The present disclosure provides a method for preserving corneal endothelial cells and / or corneal-endothelial-like cells, wherein the method includes: a freezing step for freezing unfrozen corneal endothelial cells and / or corneal-endothelial-like cells, the freezing step including at least one stage in which the temperature is reduced at a rate of less than 1°C / min when the temperature is changed from the unfrozen temperature to the freezing target temperature; and, if necessary, a step for maintaining the corneal endothelial cells and / or corneal-endothelial-like cells in a frozen state.

Description

Cryopreserved preparation of corneal endothelial cells and method for producing same

[0001] The present disclosure relates to a cryopreserved preparation of corneal endothelial cells, a manufacturing method thereof, and application techniques such as treatment using the same.

[0002] Damage to corneal endothelial cells leads to corneal opacity and severe visual impairment due to bullous keratopathy. The only treatment for bullous keratopathy is corneal transplantation, but issues such as a shortage of donors and transplant rejection pose a need for the development of new regenerative medicine treatments. At Doshisha University, we established a treatment method for regenerating corneal endothelium by growing corneal endothelial cells isolated from donor corneas in the presence of a Rho-associated coiled-coil forming kinase (ROCK) inhibitor and injecting these cells into a large number of patients. However, the limited time that cells can be stored while maintaining their quality poses a major challenge in supplying them throughout Japan and around the world. While cells can generally be cryopreserved in a preservation solution containing 10% dimethyl sulfoxide (DMSO), concerns about the toxicity of DMSO to cells and irritation upon administration to the eye have led to the need for cryopreservation using a reduced concentration of DMSO.

[0003] Furthermore, frozen cell preparations used in regenerative medicine contain at least 7% or more DMSO, and when administered to patients, due to concerns about the toxicity of DMSO to the patient, they are diluted with saline immediately before administration, or the administration rate when administered by intravenous infusion or the like is extremely slowed down to avoid administering high concentrations of DMSO to the patient.

[0004] In corneal endothelial cell injection therapy, a highly concentrated cell suspension needs to be administered in a small dose of 400 μL or less into the anterior chamber of the patient's eye. Therefore, dilution before administration is not possible, and a cell cryopreservation preparation with a reduced concentration of DMSO or no DMSO content is required.

[0005] The present inventors have conducted detailed studies on temperature conditions during cryopreservation and have found that a high viability of corneal endothelial cells can be maintained in a cryopreservation solution with a reduced DMSO concentration (for example, less than 7%) or no DMSO, by decreasing the temperature at a rate slower than -1°C / min. The present inventors have further found that cell viability increases when the temperature is first decreased at a slow rate and then the cooling rate is increased for freezing. Therefore, the present disclosure provides a method for freezing corneal endothelial cells in a cryopreservation solution with a reduced DMSO concentration or no DMSO, and a method for producing a cell cryopreservation preparation that can be administered directly to patients.

[0006] The present invention provides, for example, the following items. (Item 1) A method for preserving corneal endothelial cells and / or corneal endothelial-like cells, comprising: a freezing step of freezing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state, the freezing step comprising at least one step of lowering the temperature at a rate of less than 1°C per minute when changing the temperature from the unfreezing temperature to a target freezing temperature; and, if necessary, a step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. (Item 2) A method according to any one of the above items, comprising a step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. (Item 3) A method according to any one of the above items, wherein the step of maintaining the corneal endothelial cells in a frozen state comprises maintaining them at a freezing maintenance temperature. (Item 4) A method according to any one of the above items, wherein the freezing maintenance temperature is a temperature within the range of about -80°C to about -10°C. (Item 5) The method according to any one of the above items, wherein the freezing maintenance temperature is a temperature within the range of about -196°C to about -10°C. (Item 6) The method according to any one of the above items, wherein the freezing maintenance temperature is a temperature of about -30°C or less. (Item 7) The method according to any one of the above items, wherein the temperature of the corneal endothelial cells and / or corneal endothelial-like cells is reduced from a non-freezing temperature at a rate of about 0.1°C to about 0.9°C per minute. (Item 8) The method according to any one of the above items, wherein the temperature of the corneal endothelial cells and / or corneal endothelial-like cells is reduced from a non-freezing temperature at a rate of about 0.2°C to about 0.8°C per minute. (Item 9) The method according to any one of the above items, wherein the temperature of the corneal endothelial cells and / or corneal endothelial-like cells is reduced from a non-freezing temperature at a rate of about 0.7°C or less per minute. (Item 10) The method according to any one of the above items, wherein the temperature of the corneal endothelial cells and / or corneal endothelial-like cells is lowered from the non-freezing temperature at a rate of about 0.2°C to about 0.7°C per minute. (Item 11) The method according to any one of the above items, wherein the non-freezing temperature is a temperature within the range of about 0°C to about 42°C. (Item 12) The method according to any one of the above items, wherein the non-freezing temperature is a temperature within the range of about 0°C to about 37°C.(Item 13) The method according to any one of the above items, wherein the non-freezing temperature is a temperature within the range of about 4°C to about 23°C. (Item 14) The method according to any one of the above items, wherein the freezing step comprises at least one step of lowering the temperature at a rate of less than 1°C per minute in at least a part of a temperature range of about -20°C±10°C. (Item 15) The method according to any one of the above items, wherein the freezing step comprises at least one step of maintaining the temperature in a temperature range of about -20°C±10°C for a certain period of time or longer. (Item 16) The method according to any one of the above items, wherein the corneal endothelial cells and / or corneal endothelial-like cells are stored in a storage solution containing less than about 7% DMSO. (Item 17) The method according to any one of the above items, wherein the corneal endothelial cells and / or corneal endothelial-like cells are stored in a storage solution containing about 5% or less DMSO. (Item 18) The method according to any one of the above items, wherein the corneal endothelial cells and / or corneal endothelial-like cells are stored in a storage solution containing about 2% or less DMSO. (Item 19) The method according to any one of the above items, wherein the corneal endothelial cells and / or corneal endothelial-like cells are stored in a storage solution containing no DMSO. (Item 20) The method according to any one of the above items, wherein the freezing step comprises freezing the corneal endothelial cells and / or corneal endothelial-like cells in the presence of a ROCK inhibitor. (Item 21) The method according to any one of the above items, wherein the freezing step comprises lowering the temperature to a first target temperature at a first rate, and lowering the temperature from the first target temperature to a second target temperature at a second rate, wherein the first rate is a rate of less than 1°C per minute and is slower than the second rate. (Item 22) The method of any one of the above items, wherein the freezing step further comprises lowering the temperature to the first target temperature and then maintaining the temperature at the first target temperature. (Item 23) The method of any one of the above items, wherein the first target temperature is a temperature within a range of about -20°C to about -5°C. (Item 24) The method of any one of the above items, wherein the first target temperature is a temperature within a range of about -15°C to about -10°C. (Item 25) The method of any one of the above items, wherein the second target temperature is a temperature of about -20°C or lower.(Item 26) The method of any one of the above items, wherein the second target temperature is a temperature within the range of about -196°C to about -80°C. (Item 27) The method of any one of the above items, wherein the first rate is a rate of about 0.5°C to about 0.05°C per minute. (Item 28) The method of any one of the above items, wherein the first rate is a rate of about 0.3°C to about 0.1°C per minute. (Item 29) The method of any one of the above items, wherein the second rate is a rate of about 0.5 to about 5°C per minute. (Item 30) The method of any one of the above items, wherein the second rate is a rate of about 1 to about 3°C ​​per minute. (Item 31) A method for producing a frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells, comprising the steps of mixing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state with optionally pharmaceutically acceptable ingredients and freezing to produce a frozen preparation, the freezing step comprising at least one step of lowering the temperature at a rate of less than 1°C per minute when changing the temperature from the unfreezing temperature to a target freezing temperature, and optionally a step of maintaining the frozen preparation of the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. (Item 32) The method of any one of the above items, further comprising one or more features described in the methods of any one or more of items 2 to 30. (Item 33) A frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells produced by the method of any one of the above items. (Item 34) The corneal endothelial cells and / or corneal endothelial-like cells are about 1 x 10 ５ ~Approx. 3×10 ６(Item 35) The frozen formulation according to any one of the preceding items, wherein the volume of the frozen formulation is about 50 μL to about 600 μL. (Item 36) The frozen formulation according to any one of the preceding items, wherein the frozen formulation is administered in a volume of about 50 μL to about 350 μL per dose. (Item 37) An apparatus for preserving corneal endothelial cells and / or corneal endothelial-like cells, the apparatus comprising: a storage / storage section that houses a container that houses the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control section that commands control of the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container housed in the storage / storage section; and a temperature adjustment section that can adjust the temperature in the storage / storage section based on commands from the temperature control section, wherein the temperature control section can command temperature control to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and can command to maintain the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state as necessary. (Item 38) A program encoding a method for causing a computer to implement the method for preserving corneal endothelial cells and / or corneal endothelial-like cells in an apparatus, the apparatus comprising: a storage / storage unit that houses a container for storing the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control unit that issues commands to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container housed in the storage / storage unit; and a temperature adjustment unit that can adjust the temperature in the storage / storage unit based on commands from the temperature control unit, the program causing the temperature control unit to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state as necessary.(Item 39) A recording medium storing a program encoding a method for causing a computer to implement such a method for preserving corneal endothelial cells and / or corneal endothelial-like cells in an apparatus, the apparatus comprising: a storage / storage section that houses a container for storing the corneal endothelial cells and / or corneal endothelial-like cells, a temperature control section that gives instructions to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container housed in the storage / storage section, and a temperature adjustment section that can adjust the temperature in the storage / storage section based on instructions from the temperature control section, the program instructing the temperature control section to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and causing the corneal endothelial cells and / or corneal endothelial-like cells to be maintained in a frozen state as necessary. (Item 40) A frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells. (Item 41) A frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells, which can be administered directly to the eye after thawing. (Item 42) A frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells in a state frozen in a slow-freezing state. (Item 43) A frozen preparation comprising less than 7% DMSO, corneal endothelial cells and / or corneal endothelial-like cells, and physiological saline components in a frozen state. (Item 44) A frozen preparation comprising less than 7% DMSO, corneal endothelial cells and / or corneal endothelial-like cells, and medium components in a frozen state. (Item 45) A frozen cell preparation with long-term stability after thawing, comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells. (Item 46) The frozen preparation according to any one of the above items, which comprises about 5% or less DMSO. (Item 47) The frozen preparation according to any one of the above items, which comprises about 2% or less DMSO. (Item 48) The frozen preparation according to any one of the preceding items, which does not contain DMSO. (Item 49) The frozen preparation according to any one of the preceding items, further comprising a ROCK inhibitor. (Item 50) The frozen preparation according to any one of the preceding items, wherein the ROCK inhibitor is Y-27632. (Item 51) A frozen preparation comprising a ROCK inhibitor and corneal endothelial cells and / or corneal endothelial-like cells in a frozen state.(Item 52) A frozen preparation comprising a ROCK inhibitor, corneal endothelial cells and / or corneal endothelial-like cells, and a component of physiological saline in a frozen state. (Item 53) A frozen preparation comprising a ROCK inhibitor, corneal endothelial cells and / or corneal endothelial-like cells, and a medium component in a frozen state. (Item 54) A frozen cell preparation having long-term stability after thawing, said preparation comprising corneal endothelial cells and / or corneal endothelial-like cells and a ROCK inhibitor. (Item 55) The preparation according to any one of the preceding items, wherein the viability of the corneal endothelial cells and / or corneal endothelial-like cells is at least 80% for at least 6 hours at room temperature after thawing. (Item 56) A frozen preparation that does not inhibit the engraftment and in vivo survival of corneal endothelial cells and / or corneal endothelial-like cells when administered after thawing, the preparation comprising corneal endothelial cells and / or corneal endothelial-like cells and a ROCK inhibitor in a state frozen in a slow-freezing state. (Item 57) A frozen preparation comprising less than 7% DMSO, a ROCK inhibitor, and corneal endothelial cells and / or corneal endothelial-like cells in a state frozen in a slow-freezing state. (Item 58) The formulation according to any one of the above items, wherein the ROCK inhibitor is Y-27632. (Item 59) The formulation according to any one of the above items, comprising less than about 7% DMSO. (Item 60) The formulation according to any one of the above items, comprising about 5% or less DMSO. (Item 61) The formulation according to any one of the above items, comprising about 2% or less DMSO. (Item 62) The formulation according to any one of the above items, which does not contain DMSO. (Item 63) The preparation according to any one of the preceding items, wherein the preparation contains the cells in a state frozen in a slow-freezing state. (Item 64) The preparation according to any one of the preceding items, wherein the preparation is frozen by lowering the temperature from a non-freezing temperature at a rate of less than 1°C per minute. (Item 65) The preparation according to any one of the preceding items, wherein the corneal endothelial cells and / or corneal endothelial-like cells are used for cell injection therapy. (Item 66) The preparation according to any one of the preceding items, wherein the frozen preparation is administered without further processing or culturing after thawing. (Item 67) The corneal endothelial cells and / or corneal endothelial-like cells are about 1 x 10 ５~about 3×10 ６The formulation according to any one of the preceding items, comprising: (Item 68) The formulation according to any one of items 40 to 67, wherein the volume of the frozen formulation is about 50 μL to about 600 μL. (Item 69) The formulation according to any one of the preceding items, wherein the formulation is administered at a volume of about 50 μL to about 350 μL per administration. (Item 70) A frozen formulation kit comprising a container for housing a frozen formulation containing corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, and a container for housing the container while maintaining the container in a frozen state. (Item 71) The frozen formulation kit according to item 70, wherein the frozen formulation is the formulation according to any one of the preceding items. (Item 72) A frozen formulation kit comprising the formulation according to any one of the preceding items, a container for housing the formulation, and a container for housing the container while maintaining the formulation in a frozen state. (Item 73) A frozen formulation kit comprising a container and a container for housing the container, wherein the container is used to house the formulation according to any one of the preceding items, and the container is used to maintain the formulation in a frozen state. (Item 74) A frozen formulation kit comprising a container for housing a frozen formulation comprising a ROCK inhibitor and corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, and a container for housing the container while maintaining the container in a frozen state. (Item 75) Use of a kit comprising a container and a container for housing the container, wherein the container is used to house the formulation according to any one of the preceding items, and the container is used to maintain the formulation in a frozen state. (Item 76) A method for transporting and / or storing the formulation according to any one of the preceding items, comprising the steps of placing the formulation in a container of a kit comprising a container and a container for housing the container, and maintaining the formulation in the kit in a frozen state.(Item 77) A method for performing corneal endothelial cell injection therapy, comprising: a step of providing corneal endothelial cells and / or corneal endothelial-like cells suitable for the cell injection therapy; a freezing step comprising at least one step of lowering the temperature of the corneal endothelial cells and / or corneal endothelial-like cells from a non-freezing temperature at a rate of less than 1°C per minute; a step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state and transporting them to the injection therapy as needed; a step of thawing the corneal endothelial cells and / or corneal endothelial-like cells; and a step of administering the corneal endothelial cells and / or corneal endothelial-like cells to a subject. (Item 1A) A method for preserving corneal endothelial cells and / or corneal endothelial-like cells, the method comprising: a step of freezing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state, the freezing step comprising lowering the temperature to a first target temperature at a first rate and lowering the temperature from the first target temperature to a second target temperature at a second rate; and optionally a step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, wherein the first rate is a rate of less than 1°C per minute and is slower than the second rate. (Item 2A) The method according to any one of the above items, wherein the freezing step further comprises maintaining the temperature at the first target temperature after lowering it to the first target temperature. (Item 3A) The method according to any one of the above items, wherein the first target temperature is a temperature within the range of about -20°C to about -5°C. (Item 4A) The method of any one of the above items, wherein the first target temperature is a temperature within the range of about -15°C to about -10°C. (Item 5A) The method of any one of the above items, wherein the second target temperature is a temperature of about -20°C or lower. (Item 6A) The method of any one of the above items, wherein the second target temperature is a temperature within the range of about -196°C to about -80°C. (Item 7A) The method of any one of the above items, wherein the first rate is a rate of about 0.5°C to about 0.05°C per minute. (Item 8A) The method of any one of the above items, wherein the first rate is a rate of about 0.3°C to about 0.1°C per minute.(Item 9A) The method according to any one of the above items, wherein the second rate is a rate of about 0.5 to about 5°C per minute. (Item 10A) The method according to any one of the above items, wherein the second rate is a rate of about 1 to about 3°C ​​per minute.

[0007] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0008] The present disclosure provides a frozen corneal cell preparation that can be administered directly to the eye after thawing. The present invention makes it possible to provide corneal endothelial cells throughout Japan and overseas.

[0009] Figure 1 shows an overview of Example 1. Figure 2 shows a photograph of the culture morphology of the cells used in Example 1. Figure 3 shows a graph comparing the viability of cells after thawing in cryopreservation solutions containing 4% human serum albumin and 10% glycerin as the base components and varying DMSO concentrations. Figure 4 shows a graph comparing the viability of cells after thawing in cryopreservation solutions containing 4% human serum albumin and 10% polyethylene glycol as the base components and varying DMSO concentrations. Figure 5 shows data comparing the viability of cells after thawing frozen from 23°C at cooling rates of -1°C / min, -0.7°C / min, or -0.5°C / min. Error bars represent the mean ± SD. Statistical significance was based on Dunnett's t-test (vs. unfrozen) (n = 3, **p < 0.05). Figure 6 shows data comparing the cell density 7 days after seeding after storage in cells frozen from 4°C at cooling rates of -1°C / min or -0.5°C / min. Figure 7 shows an overview of Example 2. Figure 8 shows phase-contrast microscope images of cells cultured in medium containing 10% or 5% DMSO. Figure 9 shows phase-contrast microscope images of cells cultured in medium containing 2% DMSO or no DMSO. Figure 10 is a graph showing the results of recovering cells after replated and examining their viability. From left to right, each group shows 10%, 5%, 2%, and 0% DMSO. Figure 11 shows an overview of Example 3. Figure 12 shows phase-contrast microscope images of cells frozen in a Cryostor CS10 containing 10% DMSO, left at room temperature for 0 hours, 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours, and then replated into a T25 culture flask. The culture state of the cells was photographed 24 hours later. Figure 13 shows phase-contrast micrographs of cells frozen in a Cryostor CS5 containing 5% DMSO at room temperature for 0 hours, 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours, then reseeded into a T25 culture flask. 24 hours later, the cells were reseeded into a T25 culture flask. Figure 14 shows phase-contrast micrographs of cells frozen in a Cryostor CS2 containing 2% DMSO at room temperature for 0 hours, 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours, then reseeded into a T25 culture flask. Figure 15 shows a graph showing the results of recovering cells after reseeding and examining their viability. From the left, CS10, CS5, and CS2 are shown in each group.Figure 16 shows the viability and cell recovery rate when cells stored in a cold box for 5 days were thawed. Error bars indicate the mean ± SD. Statistical significance was based on Student's t-test (n=3). Figure 17 shows micrographs of cells cultured for 2 days after storage in a cold box. Figure 18 shows photographs of rabbit eyes injected with cells after storage in a cold box. Figure 19 shows photographs of immunohistochemical staining of CD166 in corneal endothelium 1 day after cell injection. Figure 20 shows photographs of immunohistochemical staining of ZO-1 and Na / K ATPase 1 day after cell injection. Figure 21 shows photographs of immunohistochemical staining of CD166, ZO-1, and Na / K ATPase in corneal endothelium 5 days after cell injection. Figure 22 shows an overview of cryopreservation using a cryopreservation solution of known ingredients in Example 6. FIG. 23 shows a graph comparing the viability of cells after thawing frozen at cooling rates of -1°C / min, -0.7°C / min, -0.5°C / min, or -0.2°C in cryopreservation solutions containing 4% human serum albumin and 10% glycerin as base components and varying DMSO concentrations. FIG. 24 shows an overview of cryopreservation using a commercially available cryopreservation solution from Example 6. FIG. 25 shows the viability and recovery rate of cells frozen at a cooling rate of -0.7°C / min from 4°C in a commercially available cryopreservation solution after thawing. FIG. 26 shows data comparing the viability of cells frozen at a cooling rate of -1°C / min or -0.7°C / min from 4°C in a commercially available cryopreservation solution. FIG. 27 shows data comparing the viability of cells frozen at a cooling rate of -0.5°C / min or -0.2°C / min from 4°C in a commercially available cryopreservation solution after thawing. FIG. 28 shows the cell viability of cells preserved in Example 7. Figure 29 shows the temperature change when cooled to -80°C at -0.5°C / min. Figure 30 shows the temperature change when cooled to -10°C at -0.5°C / min, held at -10°C for 110 minutes, and then cooled to -80°C at -1.0°C / min. Figure 31 shows the temperature change when cooled to -10°C at -0.1°C / min, then cooled to -80°C at -1.0°C / min.

[0010] The present disclosure will be described below. Throughout this specification, singular expressions should be understood to include the concept of the plural unless otherwise specified. Therefore, singular articles (for example, in English, "a", "an", "the", etc.) should be understood to include the concept of the plural unless otherwise specified. Furthermore, it should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of any conflict, the present specification (including definitions) shall prevail. In this specification, "about" means ±10% of the following value. Unless otherwise specified, "%" representing a composition means w / w% when referring to DMSO and human serum albumin (HSA), and v / v% when referring to glycerin and polyethylene glycol.

[0011] (Definition) In this specification, the term "corneal endothelial cells" is used in the usual sense used in the relevant field. The cornea is one of the layered tissues that make up the eye, is transparent, and is located closest to the outside world. In humans, the cornea is said to be made up of five layers, starting from the outside (body surface), and is composed of, from the outside, the corneal epithelium, Bowman's membrane, lamina propria, Descemet's membrane (corneal endothelial basement membrane), and corneal endothelium. Unless otherwise specified, parts other than the epithelium and endothelium are sometimes collectively referred to as the "corneal stroma," and will be referred to as such in this specification. In this specification, "HCECs" (human corneal endothelial cells) is an abbreviation for human corneal endothelial cells.

[0012] As used herein, the term "corneal endothelial-like cells" refers to cells differentiated from stem cells, for example, cells differentiated from iPS cells, and which have substantially the same functions as corneal endothelial cells. Methods for differentiating stem cells, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc., into corneal endothelial-like cells are well known in the art (McCabe et al., PLoS One. 2015 Dec 21; 10(12): e0145266; Ali et al., Invest Ophthalmol Vis Sci. 2018 May 1; 59(6): 2437-2444). Briefly, in a typical example, iPS cells are seeded onto 35 mm Matrigel-coated plates (Corning) at a 1:12 dilution using cell dissociation buffer (Life Technologies) on day 0 (80% confluent plates are split into 12 plates). iPS cells are grown in culture medium (mTeSR1; STEMCELL Technologies Inc.) for 4 days. On day 4, mTeSR1 medium was supplemented with 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (STEMCELL Technologies, Inc.), 0.1 mM β-mercaptoethanol (MilliporeSigma), and 8 ng / mL βFGF (MilliporeSigma) in a basal medium containing 500 ng / mL human recombinant Noggin (R&D The medium is replaced with Smad inhibitor medium containing 10 μM SB431542 (MilliporeSigma) and 10 μM SB431542 (Scientific Systems, Minneapolis, MN, USA).On day 6, Smad inhibitor medium was prepared by adding 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (STEMCELL Technologies, Inc.), 0.1 mM β-mercaptoethanol (MilliporeSigma), and 8 ng / mL βFGF (MilliporeSigma) to the basal medium, supplemented with 0.1× B27 supplement (Life Technologies), 10 ng / mL recombinant human platelet-derived growth factor-BB (PDGF-BB; PeproTech, Rocky Mountain). The corneal medium is replaced with corneal medium containing 10 ng / mL recombinant human Dickkopf-related protein-2 (DKK-2; R&D Systems) and 10 ng / mL recombinant human Dickkopf-related protein-2 (DKK-2; R&D Systems). On day 7, the differentiating CECs are transferred to a new Matrigel-coated plate (35 mm) and grown in corneal medium for an additional 13 days. The differentiated CECs are harvested on day 20. The above example is a typical example, and those skilled in the art may also use other methods well known in the art (Fukuta et al., PLoS One. 2014 Dec 2;9(12):e112291; Hayashi et al., Nature. 2016 Mar 17;531(7594):376-80). Furthermore, those skilled in the art can prepare corneal endothelial-like cells by appropriately adjusting the conditions of methods well known in the art.

[0013] "Corneal endothelial cells" and "corneal endothelial-like cells" may contain a magnetic material (e.g., iron). For example, when corneal endothelial cells containing a magnetic substance are injected into the anterior chamber, they can be attracted to the inside of the cornea (e.g., Descemet's membrane) by magnetic force, promoting adhesion (Patel et al., Invest Ophthalmol Vis Sci. 2009 May; 50(5): 2123-31; Mimura et al., Exp Eye Res. 2003 Jun; 76(6): 745-51; and Mimura et al., Exp Eye Res. 2005 Feb; 80(2): 149-57). A "magnetic material" refers to a substance that can be magnetized by a magnetic field, and examples include iron, cobalt, nickel, and ferrite.

[0014] As used herein, "storage" of cells means storing the cells in a container for a certain period of time for any purpose (e.g., cell infusion therapy or transportation therefor), and refers to maintaining the cells in the container while maintaining their function without proliferating the cells. Storage differs from "culturing," which aims to proliferate the cells. Furthermore, storage does not mean transferring the cells into a container such as a syringe immediately before administration, nor does it mean temporarily holding the cells in a container for preparation before administration. "Cryopreservation" means storing the cells in a frozen state.

[0015] As used herein, "non-freezing temperature" refers to a temperature at which freezing does not occur even when maintained, and "target freezing temperature" refers to a target temperature when freezing corneal endothelial cells and / or corneal endothelial-like cells in the freezing step of the method disclosed herein. "Freezing maintenance temperature" refers to a temperature at which frozen corneal endothelial cells and / or corneal endothelial-like cells are maintained in a frozen state for a certain period of time. The freezing maintenance temperature may be varied as long as the target cells or other subjects can be maintained in a frozen state.

[0016] As used herein, the term "slowly frozen state" refers to a state in which the material has been frozen by a freezing process that includes at least one step of lowering the temperature at a rate of less than 1° C. per minute.

[0017] As used herein, "long-term stability after thawing" refers to the maintenance of at least 80% cell viability for at least 6 hours when frozen cells are kept at room temperature after thawing.

[0018] As used herein, the term "frozen formulation" refers to a formulation that is stored in a frozen state and that is in a form suitable for use after thawing, or in a form that can be prepared just before use. "Preparation just before use" refers to preparing a formulation suitable for use by adding a drug or by diluting with a solvent just before administration.

[0019] As used herein, the term "processing," when applied to a cell or a cell population, refers to a specific operation that changes some state or property of the cell or cell population, and preferably refers to an operation that changes the property of the cell population, such as adding a drug, treating with a drug, or isolating specific cells, or an operation that changes the cell density, such as diluting or concentrating with a solvent.

[0020] In this specification, "constant temperature" refers to a temperature within a range of ±1°C of the set temperature.

[0021] As used herein, the term "corneal endothelial condition, disorder, or disease" refers to any condition, disorder, or disease occurring in the corneal endothelium. Examples of corneal endothelial conditions, disorders, or diseases include, but are not limited to, Fuchs' corneal endothelial dystrophy, post-corneal transplant disorders, corneal endotheliitis, trauma, disorders after ophthalmic surgery, disorders after ophthalmic laser surgery, aging, posterior polymorphous corneal dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), and idiopathic corneal endothelial disorders.

[0022] As used herein, the term "subject" refers to a subject to which the formulation of the present disclosure is administered. Examples of subjects include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.), with primates being preferred, and humans being particularly preferred.

[0023] As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually separated into two or more compartments. This kit format is preferred when providing a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Alternatively, when providing a compound that is unstable in solution, a kit format is preferred when it is necessary to prepare it immediately by dissolving a lyophilized powder in an appropriate solvent immediately before use. Such a kit advantageously includes instructions or manuals describing how to use the components to be provided (e.g., test agents, diagnostic agents, therapeutic agents) or how to handle the reagents.

[0024] In this specification, the term "program" is used in the ordinary sense of the term in this field, and refers to a sequence of processes to be performed by a computer. In Japan, this term is treated as a "product" under the Patent Act. All computers operate according to a program. In modern computers, programs are expressed as data in a broad sense and are stored on recording media or storage devices.

[0025] In this specification, the term "recording medium" refers to a recording medium that stores a program for executing the method of the present disclosure, and the recording medium may be any type of recording medium as long as it is capable of recording the program. For example, the recording medium may be an internally stored ROM, an HDD, a magnetic disk, or an external storage device such as a flash memory such as a USB memory, but is not limited to these.

[0026] In this specification, the term "system" refers to a configuration that executes the method or program disclosed herein, and originally means a system or organization for accomplishing a purpose, in which multiple elements are systematically configured and influence each other; in the computer field, it refers to the entire configuration, including hardware, software, an OS, and a network.

[0027] As used herein, "machine learning" refers to a technology that gives computers the ability to learn without explicit programming. It is the process by which functional units improve their performance by acquiring new knowledge and skills or by reconstructing existing knowledge and skills. Programming computers to learn from experience can greatly reduce the effort required for detailed programming. The field of machine learning discusses methods for building computer programs that can automatically improve through experience. Along with algorithms, data analysis and machine learning are fundamental technologies for intelligent processing. They are typically used in conjunction with other technologies and require knowledge of the relevant field (domain-specific knowledge; for example, medicine). Applications include prediction (collecting data and predicting future events), exploration (finding distinctive features from the collected data), and testing and description (examining the relationships between various elements in the data). Machine learning is based on metrics that indicate the degree of achievement of real-world goals, and machine learning users must understand those goals. Furthermore, it is necessary to formulate metrics that improve when the goal is achieved. Machine learning is an inverse problem, an ill-posed problem where it is unclear whether a solution has been found. The behavior of learned rules is not deterministic but probabilistic. Operational ingenuity is required, assuming that some uncontrollable aspects will remain, and the tailor-made method of the present invention can be said to be a solution to this. It is also useful for machine learning users to sequentially select and discard data and information according to real-world goals while observing performance indicators during training and operation.

[0028] As machine learning, linear regression, logistic regression, support vector machine, etc. can be used, and cross-validation (CV) can be performed to calculate the discrimination accuracy of each model. After ranking, feature amounts are added one by one, and machine learning (linear regression, logistic regression, support vector machine, etc.) and cross-validation can be performed to calculate the discrimination accuracy of each model. This makes it possible to select the model with the highest accuracy. In the present invention, any machine learning method can be used, and linear, logistic, support vector machine (SVM), etc. can be used as supervised machine learning.

[0029] (Preferred Embodiments) A description of preferred embodiments will be given below, but it should be understood that these embodiments are merely examples of the present disclosure and that the scope of the present disclosure is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present disclosure by referring to the following preferred examples. Those skilled in the art can combine any of these embodiments as appropriate.

[0030] (Storage Method) When corneal endothelial cells are cryopreserved, they can be preserved while maintaining a high viability by freezing them in a preservation solution containing 10% DMSO, which suppresses damage during freezing. However, there were concerns about the toxicity of DMSO to cells and irritation when administered to the eye, so the present inventors investigated storage conditions under which a high viability could be maintained in a preservation solution with reduced DMSO, and found that a high viability of corneal endothelial cells can be maintained in a cryopreservation solution with a reduced DMSO concentration (for example, less than 7%) or that does not contain DMSO, by decreasing the temperature at a rate slower than -1°C / min.

[0031] If the cooling rate is too slow, extracellular water freezes first, removing the extracellular water and causing water to flow out from within the cells. This increase in intracellular solute concentration has a detrimental effect on cell viability. If the cooling rate is too fast, the outflow of water from within the cells is suppressed, but damage due to surface crystals occurs within the cells, which has a detrimental effect on cell viability. The cooling rate during cell cryopreservation has a significant effect on cell damage. This effect can be minimized by an optimal cooling rate, and a cooling rate of -1°C / min is recommended. It was unexpected that the viability of corneal endothelial cells was maintained at a high level in a cryopreservation solution with a reduced DMSO concentration (e.g., less than 7%) or no DMSO, when the temperature was lowered at a rate slower than -1°C / min.

[0032] In one aspect, the present disclosure may provide a method for preserving corneal endothelial cells and / or corneal endothelial-like cells, the method comprising a freezing step of freezing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state, the freezing step comprising at least one step of lowering the temperature at a rate of less than 1°C per minute (at a cooling rate slower than -1°C / min) when changing the temperature from a non-freezing temperature to a target freezing temperature.

[0033] In one aspect, the present disclosure may provide a method for producing a frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells, the method comprising a freezing step of mixing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state with a pharmaceutically acceptable component as needed, and freezing the cells to produce a frozen preparation, the freezing step comprising at least one step of decreasing the temperature at a rate of less than 1° C. per minute when changing the temperature from a non-freezing temperature to a target freezing temperature.

[0034] In some embodiments, a cooling rate slower than -1°C / min can be at a temperature in the range of about 0.1°C to about 0.9°C per minute, preferably about 0.2°C to about 0.8°C per minute, and more preferably about 0.2°C to about 0.7°C per minute. In certain embodiments, a cooling rate slower than -1°C / min can be -0.9°C / min, -0.8°C / min, -0.7°C / min, -0.6°C / min, -0.5°C / min, -0.4°C / min, -0.3°C / min, -0.2°C / min, or -0.1°C / min.

[0035] In one embodiment, the cooling rate to the target freezing temperature (e.g., −80° C.) may be constant or may not be constant. In some embodiments, the method of the present disclosure includes at least a step of decreasing the temperature at a cooling rate slower than −1° C. / min in a specific temperature range, and the process of decreasing the temperature to the target freezing temperature may include a step of increasing the temperature, a step of decreasing the temperature at a cooling rate faster than −1° C. / min, or a step of maintaining the temperature at a constant temperature.

[0036] The target freezing temperature is set appropriately and may be, for example, a temperature within a range of about -20°C to -196°C, such as about -20°C, about -30°C, about -40°C, about -50°C, about -60°C, about -70°C, about -80°C, about -90°C, about -100°C, about -150°C, about -190°C, or about -196°C.

[0037] In one embodiment, the method of the present disclosure may include increasing the temperature, provided that the temperature is decreasing at an average cooling rate of less than −1° C. / min, decreasing the temperature at a cooling rate greater than −1° C. / min, or maintaining the temperature constant within a particular temperature range.

[0038] In one embodiment, the method of the present disclosure may achieve an average cooling rate of less than -1°C / min by decreasing the temperature at a rate faster than -1°C / min within a particular temperature range, then maintaining the temperature constant and then decreasing the temperature again at a rate faster than -1°C / min (this may be repeated).

[0039] In one embodiment, the method of the present disclosure may involve decreasing the temperature at a rate faster than -1°C / min within a particular temperature range, followed by increasing the temperature, then maintaining the temperature constant, and again decreasing the temperature at a rate faster than -1°C / min (this may be repeated) to achieve an average cooling rate of less than -1°C / min.

[0040] In one embodiment, the method of the present disclosure may involve decreasing the temperature at a rate faster than -1°C / min within a particular temperature range, then increasing the temperature, and then decreasing the temperature again at a rate faster than -1°C / min (this may be repeated) to achieve an average cooling rate of less than -1°C / min.

[0041] In one embodiment, the specific temperature range in which the temperature is lowered at a cooling rate slower than -1°C / min may be a temperature range that includes at least the temperature at which the temperature transitions from a non-frozen state to a frozen state. In a specific embodiment, the temperature range may be about -80°C to about 0°C, about -70°C to about 0°C, about -60°C to about 0°C, about -50°C to about 0°C, about -40°C to about 0°C, about -30°C to about 0°C, about -20°C to about 0°C, about -10°C to about 0°C, about -80°C to about -10°C, about -70°C to about -10°C, about -60°C to about -10°C, about -50°C to about -10°C, about -40°C to about -10°C, about -30°C to about -10°C, or about -20°C to about -10°C.

[0042] In one embodiment, the method of the present disclosure may further comprise the step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. In some embodiments, the frozen maintenance temperature may be within the range of about −196° C. to about −4° C., about −196° C. to about −10° C., about −196° C. to about −20° C., about −196° C. to about −30° C., about −196° C. to about −40° C., about −196° C. to about −50° C., about −196° C. to about −60° C., about −196° C. to about −70° C., about −196° C. to about −80° C., about −80° C. to about −4° C., about −80° C. to about −10° C., about −80° C. to about −20° C., about −80° C. to about −30° C., about −80° C. to about −40° C., about −80° C. to about −50° C., about −80° C. to about −60° C., or about −80° C. to about −70° C. In a preferred embodiment, the frozen maintenance temperature may comprise maintaining at about −80° C.

[0043] In some embodiments, the freezing step of the method of the present disclosure may be initiated from a non-freezing temperature within the ranges of about 0° C. to about 42° C., about 0° C. to about 37° C., about 4° C. to about 23° C., or about 4° C. to about 10° C. In a preferred embodiment, particularly when freezing is performed in the presence of DMSO, the freezing step may be initiated from a non-freezing temperature of 4° C. In certain embodiments, the method of the present disclosure may further comprise the step of incubating the corneal endothelial cells and / or corneal endothelial-like cells at the above-mentioned non-freezing temperature prior to the freezing step.

[0044] In one embodiment, the freezing step of the method of the present disclosure may include at least one step of decreasing the temperature at a rate of less than 1°C per minute or maintaining a constant temperature for a certain period of time within at least a portion or the entire temperature range of about -20°C±10°C. In one embodiment, the freezing step of the method of the present disclosure may include at least one step of continuously maintaining a temperature within the temperature range of about -20°C±10°C for at least a certain period of time, for example, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 1 hour and a half, or at least 2 hours. The temperature range may be -20°C±5°C. If ice crystals are formed unevenly and unaligned during the freezing step, this may cause significant damage to cells and reduce viability. Without wishing to be bound by theory, in the freezing process, slowly lowering the temperature near the eutectic point (e.g., −20° C.±10° C.) of the ice crystals and the solute (e.g., NaCl) in the preservation solution, or maintaining the temperature near the eutectic point for a certain period of time, may allow the crystals to align uniformly, thereby reducing damage to the cells.

[0045] The temperature may be changed at any rate outside the temperature range of -20°C±10°C, provided that the temperature is decreased at a rate of less than 1°C per minute within the temperature range of -20°C±10°C, or maintained at a constant temperature for a certain period of time.

[0046] In a further aspect, the present disclosure provides a method for preserving corneal endothelial cells and / or corneal endothelial-like cells, the method comprising a step of freezing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state, the freezing step comprising at least one stage of maintaining the cells at a temperature within a range of about −20° C.±10° C. for a certain period of time or longer, and, as necessary, a step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. Outside the temperature range of −20° C.±10° C., the temperature may be changed at any rate.

[0047] In some embodiments, the temperature may be lowered below −30° C. and then increased to slowly lower the temperature or maintain the temperature for a certain period of time in a temperature range near the eutectic point (−20° C.±10° C.). In light of the disclosures herein, those skilled in the art can appropriately adjust the time for maintaining the temperature below −30° C. as long as the effects of the present disclosure are achieved, and the time may be, for example, 2 hours or less, 1 hour or less, 30 minutes or less, or 20 minutes or less.

[0048] In one embodiment, corneal endothelial cells and / or corneal endothelial-like cells can be stored in a preservation solution containing less than about 7%, about 5% or less, or about 2% or less DMSO. Because DMSO contained in the preservation solution can have a deleterious effect on cells, it is preferably about 5% or less, more preferably about 2% or less, and most preferably no DMSO is contained in the preservation solution. In a specific embodiment, the DMSO contained in the preservation solution can be about 5%. In a specific embodiment, the DMSO contained in the preservation solution can be about 2%.

[0049] In one embodiment, corneal endothelial cells and / or corneal endothelial-like cells may be frozen in the presence of a ROCK inhibitor.

[0050] Examples of ROCK inhibitors include those described in the following documents: U.S. Patent No. 4,678,783, U.S. Patent No. 3,421,217, WO 95 / 28387, WO 99 / 20620, WO 99 / 61403, WO 02 / 076976, WO 02 / 076977, WO 2002 / 083175, WO 02 / 100833, WO 03 / 059913, WO 03 / 062227, WO 2004 / 009555, WO 2004 / 022541, WO 2004 / 108724, and WO 2 005 / 003101, International Publication No. 2005 / 039564, International Publication No. 2005 / 034866, International Publication No. 2005 / 037197, International Publication No. 2005 / 037198, International Publication No. 2005 / 035501, International Publication No. 2005 / 035503, International Publication No. 2005 / 035506, International Publication No. 2005 / 080394, International Publication No. 2005 / 103050, International Publication No. 2006 / 057270, International Publication No. 2007 / 026664, International Publication No. 2014 / 113620, International Publication No. 2019 / 089868, International Publication No. 2014 / 055996, International Publication No. 2019 / 014300, International Publication No. 2019 / 014304, International Publication No. 2018 / 138293, International Publication No. 2018 / 115383, International Publication No. 2018 / 118109, International Publication No. 2018 / 102325, International Publication No. 2018 / 009622, International Publication No. 2018 / 009625, International Publication No. 2018 / 009627, International Publication No. 2017 / 205709, International Publication No. 2017 / 123860, International Publication No. 2016 / 112236, International Publication No. 2016 / 028971, International Publication No. Examples include compounds disclosed in International Publication No. 2015 / 165341, International Publication No. 2015 / 054317, International Publication No. 2015 / 002926, International Publication No. 2015 / 002915, International Publication No. 2014 / 068035, International Publication No. 2014 / 055996, International Publication No. 2013 / 030366, International Publication No. 2012 / 146724, International Publication No. 2011 / 107608, International Publication No. 2010 / 104851, International Publication No. 2008 / 077550, International Publication No. 2008 / 036540, and International Publication No. 2005 / 097790. Such compounds can be produced by the methods described in the respective documents.Specific examples include 1-(5-isoquinolinesulfonyl)homopiperazine or a salt thereof (for example, fasudil (1-(5-isoquinolinesulfonyl)homopiperazine)), (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) or a salt thereof (for example, Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate)), and commercially available products of these compounds (FUJIFILM Wako Pure Chemical Industries, Ltd., Asahi Kasei Pharma, etc.) can also be suitably used.

[0051] In some embodiments, ROCK inhibitors that may be used include Y-27632 ((+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane), ripasudil (4-fluoro-5-{[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl}isoquinoline), fasudil (1-(5-isoquinolinesulfonyl)homopiperazine), berosudil (N-(1,2-dihydro-1-oxo-6-isoquinolinyl)-α-(dimethylamino)-3-thiopheneacetamide), belmosudil (2-[3-[4-[(1H-indazol-5-yl)amino]quinazolin-2-yl]phenoxy]-N-isopropylacetamide) and pharmaceutically acceptable salts thereof. The structure of belmosudil is as follows:

[0052]

[0053] In some embodiments, the ROCK inhibitor may be ripasudil, Y-27632, fasudil, netarsudil, belosudil, belmosudil, or a pharmaceutically acceptable salt thereof, and more preferably ripasudil, Y-27632, or a pharmaceutically acceptable salt thereof.

[0054] (Preparation) In another aspect, the present disclosure may provide a frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells produced by the above-described method for preserving corneal endothelial cells and / or corneal endothelial-like cells or the above-described method for producing a frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells.

[0055] In one aspect, the present disclosure may provide a frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells.

[0056] In one aspect, the present disclosure may provide a frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells in a state frozen under slow-freezing conditions.

[0057] In one aspect, the present disclosure may provide a frozen preparation containing less than 7% DMSO, corneal endothelial cells and / or corneal endothelial-like cells, and a physiological saline component in a frozen state.

[0058] In one aspect, the present disclosure may provide a frozen preparation comprising less than 7% DMSO, corneal endothelial cells and / or corneal endothelial-like cells, and medium components in a frozen state.

[0059] Frozen cell preparations used in regenerative medicine contain at least 7% DMSO, and when administered to patients, due to concerns about the toxicity of DMSO to the patient, it was necessary to dilute the preparation with saline immediately before administration or to extremely slow the administration rate during administration, such as intravenous infusion, to avoid administering high concentrations of DMSO to the patient. The method disclosed herein maintained a high viability even when stored in a preservation solution with a reduced DMSO concentration of less than 7%. The present disclosure achieved a frozen preparation containing a previously unattainable low concentration of DMSO, less than 7%.

[0060] In one aspect, the present disclosure provides a frozen cell preparation that is stable for a long period after thawing, the preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells.

[0061] In one aspect, the present disclosure may provide a frozen preparation comprising a ROCK inhibitor and corneal endothelial cells and / or corneal endothelial-like cells in a frozen state.

[0062] In one aspect, the present disclosure may provide a frozen preparation comprising a ROCK inhibitor, corneal endothelial cells and / or corneal endothelial-like cells, and a component of physiological saline (e.g., NaCl) in a frozen state.

[0063] In one aspect, the present disclosure may provide a frozen preparation containing a ROCK inhibitor, corneal endothelial cells and / or corneal endothelial-like cells, and medium components in a frozen state. Those skilled in the art can appropriately select medium components. Examples of medium components include, but are not limited to, carbon sources such as glucose, amino acids, vitamins, electrolytes, phosphate, buffers, growth factors, serum, and serum albumin. Amino acids contained in the basal medium components are not particularly limited, and examples include L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Vitamins contained in the basal medium components are not particularly limited, and examples thereof include calcium D-pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, and vitamin B 12 , adenine, thymidine, etc. The electrolytes contained in the medium components are not particularly limited, and examples thereof include CaCl ２ , KCl, MgSO ４ , NaCl, NaH ２ P.O. ４ , NaHCO ３ , Fe(NO ３ ) ３ , FeSO ４ , CuSO ４ , MnSO ４ , Na ２ SiO ３ , (NH ４ ) 6Mo ７ O ２４ , NaVO ３ , NiCl ２ , ZnSO ４and the like. When used in cell infusion therapy, the medium is preferably substantially free of xenogenic serum components. Here, "xenogenic serum components" refers to serum components derived from an organism of a species different from that of the recipient. For example, when the recipient is a human, sera derived from bovine or equine species, such as fetal bovine serum (FBS, FCS), calf serum (CS), and horse serum (HS), are examples of xenogenic serum components.

[0064] In one aspect, the present disclosure may provide a frozen cell preparation that is stable for a long period after thawing, the preparation comprising corneal endothelial cells and / or corneal endothelial-like cells and a ROCK inhibitor in a state frozen under slow freezing conditions.

[0065] In one aspect, the present disclosure can provide a frozen preparation that does not inhibit the engraftment and in vivo survival of corneal endothelial cells and / or corneal endothelial-like cells when administered after thawing, the preparation comprising corneal endothelial cells and / or corneal endothelial-like cells and a ROCK inhibitor in a state frozen under slow freezing conditions.

[0066] The formulations of the present disclosure may be administrable directly to the eye after thawing.

[0067] In one embodiment, the number of corneal endothelial cells and / or corneal endothelial-like cells contained in the preparation is about 1 x 10 ５ ~Approx. 3×10 ６ cells, preferably about 5 x 10 ５ ~Approx. 1×10 ６ In certain embodiments, the number of corneal endothelial cells and / or corneal endothelial-like cells contained in the preparation may be, for example, about 1 × 10 ５ cells, approximately 2 x 10 ５ cells, approximately 3 x 10 ５ cells, approximately 4 x 10 ５ cells, approximately 5 x 10 ５ cells, approximately 6 x 10 ５ cells, approximately 7 x 10 ５ cells, approximately 8 x 10 ５ cells, approximately 9 x 10 ５ cells, approximately 1 x 10 ６ cells, approximately 2 x 10 ６ cells, approximately 3 x 10 ６ cells, approximately 4 x 10６ cells, or approximately 5 x 10 ６ It may be a cell.

[0068] In one embodiment, the liquid volume of the formulation may be about 50 μl to about 2000 μl, about 50 μl to about 1000 μl, about 50 μl to about 800 μl, about 50 μl to about 600 μl, about 50 μl to about 300 μl, about 100 μl to about 2000 μl, preferably about 100 μl to about 1000 μl, more preferably about 200 μl to about 800 μl, and most preferably about 300 μl to about 600 μl, but can be changed appropriately depending on the purpose. Product specifications may be, for example, ± about 5%, ± about 10%, ± about 15%, ± about 20%, ± about 25%, ± about 50% of the reference volume (e.g., 300 μl). For example, the liquid volume of the formulation may be at least about 50 μl, e.g., about 100 μl, about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, or about 10 ml. In some embodiments, when injection into both eyes is intended for cell injection therapy, the product specifications may be two, three, or four times the dosage. Even when injection into both eyes is not intended, the product specifications may be two, three, or four times the dosage in case of administration failure. The liquid volume range may be any combination of the above values ​​as appropriate.

[0069] In one embodiment, the formulation of the present disclosure may be administered in a volume of about 50 μL to about 350 μL, about 250 μL to about 350 μL, about 300 μL to about 350 μL, or about 300 μL per dose. The formulation of the present disclosure may be administered into the anterior chamber.

[0070] The cell density of the formulation of the present disclosure is approximately 2 x 10 ４ Although not wishing to be bound by theory, when used for cell injection, if the cell density is too low, no therapeutic effect can be expected, and if the cell density is too high, the cell overlap increases, which may promote cell death during storage. Therefore, the cell density is typically about 2 × 10 ４ pieces / ml ~ approx. 8×10 ７The concentration can be appropriately determined within the range of about 2 × 10 ４ pieces / ml ~ approx. 8×10 ７ cells / ml, more preferably about 2 x 10 ５ pieces / ml ~ approx. 8×10 ６ / ml, more preferably about 1 x 10 ６ pieces / ml ~ approx. 8×10 ６ cells / ml, most preferably about 2 x 10 ６ pieces / ml ~ approx. 4×10 ６ Cells / ml can be obtained. Those skilled in the art can appropriately determine the appropriate cell density depending on the application. For example, when the preserved corneal endothelial cells and / or corneal endothelial-like cells are used in cell injection therapy, the cell density to be preserved may be determined so as to reduce the operation of adjusting the density after preservation, taking into consideration the volume of the suspension to be preserved, the optimal dose, the ROCK inhibitor to be optionally added, the volume of the suspension to be administered, and the like. Typically, the optimal dose is about 1 x 10 ５ ~Approx. 3×10 ６ cells, preferably about 5 x 10 ５ ~Approx. 1×10 ６ Those skilled in the art can appropriately determine the number of cells, liquid volume, and cell density contained in the formulation to achieve the optimal dosage.

[0071] In one embodiment, the corneal endothelial cells and / or corneal endothelial-like cells can be contained in a container. In some embodiments, any container may be used, including, but not limited to, a plate (12-, 24-, 48-, or 96-well plate), a tube, a vial (glass vial), a syringe, and a dish. The method of the present disclosure allows for preservation of cells with high cell viability, regardless of the type of container.

[0072] In one embodiment, the formulation may contain less than about 7%, about 5% or less, or about 2% or less DMSO. Preferably, the formulation contains about 5% or less DMSO, more preferably about 2% or less, and most preferably no DMSO. In certain embodiments, the DMSO contained in the formulation may be about 5%. In certain embodiments, the DMSO contained in the formulation may be about 2%.

[0073] In one embodiment, the formulation may contain a ROCK inhibitor. The ROCK inhibitor is as described above. Since the ROCK inhibitor promotes cell adhesion, if the ROCK inhibitor is included in the formulation from the time of storage, adhesion will be promoted, which is thought to have an adverse effect on storage. Therefore, the ROCK inhibitor is typically added to the formulation immediately before administration. However, unexpectedly, when the ROCK inhibitor is included in the formulation from the time of storage, the cell survival rate after storage was high, and the corneal endothelial cells and / or corneal endothelial-like cells injected into the anterior chamber were engrafted onto the corneal endothelium and functioned normally (Example 5).

[0074] In some embodiments, the viability of the corneal endothelial cells and / or corneal endothelial-like cells may be at least 80% or at least 90% for at least 6 hours at room temperature after thawing, hi some embodiments, the viability of the corneal endothelial cells and / or corneal endothelial-like cells may be at least 90% for at least 3 hours at room temperature after thawing.

[0075] In another aspect, the present disclosure provides a method for preserving corneal endothelial cells and / or corneal endothelial-like cells, the method comprising a step of freezing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state, the freezing step comprising lowering the temperature to a first target temperature at a first rate and lowering the temperature from the first target temperature to a second target temperature at a second rate, and, optionally, a step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, wherein the first rate is a rate of less than 1° C. per minute and is slower than the second rate. The "first target temperature" refers to the temperature at which a supercooled state is maintained. The first target temperature may preferably be the temperature at which freezing begins when cooled at a rate faster than the cooling rate to the first target temperature. The second target temperature refers to a final target temperature reached by further lowering the temperature from the first target temperature. The inventors have found that slowly lowering the temperature at a first rate under supercooling to a first target temperature, changing to a second rate to initiate freezing, and lowering the temperature to a second target temperature further improves cell viability. The method may have one or more embodiments described in the present disclosure.

[0076] In another aspect, the freezing step can include decreasing the temperature at a first rate to a first target temperature and decreasing the temperature from the first target temperature to a second target temperature at a second rate. The first rate can be less than 1° C. per minute and slower than the second rate. The method can have one or more embodiments described herein.

[0077] In some embodiments, the freezing step may further include lowering the temperature to a first target temperature and then maintaining the temperature at the first target temperature. The time for maintaining the temperature at the first target temperature may be set as appropriate as long as the supercooled state is maintained, and may be, for example, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 150 minutes, at least about 180 minutes, or up to about 240 minutes.

[0078] The first target temperature may be any temperature at which the supercooled state is maintained, and may be, for example, a temperature of about −20° C. to about −5° C., preferably a temperature of about −15° C. to about −10° C., and more preferably a temperature of −13° C. to −10° C.

[0079] The second target temperature is a temperature lower than the first target temperature and can be set as appropriate, for example, a temperature of about −20° C. or lower, preferably a temperature of about −196° C. to about −80° C., and more preferably about −196° C. or about −80° C.

[0080] The first rate may be set as appropriate so long as it is a slow rate, for example, a rate of about 0.9° C. per minute or less, preferably about 0.5° C. to about 0.05° C. per minute, and more preferably about 0.3° C. to about 0.1° C. per minute. The second rate may be set as appropriate so long as it is faster than the first rate and the temperature at which freezing begins when the rate is changed from the first rate to the second rate, for example, a rate of about 0.5 to about 5° C. per minute, and preferably about 1 to about 3° C. per minute.

[0081] In one embodiment, the corneal endothelial cells and / or corneal endothelial-like cells may be used in cell injection therapy. In one embodiment, the preparation may be administered after thawing without further processing or culturing.

[0082] (Storage Device) In a further aspect, the present disclosure can provide an apparatus for storing corneal endothelial cells and / or corneal endothelial-like cells, the apparatus comprising: a storage / storage unit that houses a container that houses the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control unit that gives instructions to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container housed in the storage / storage unit; and a temperature adjustment unit that can adjust the temperature in the storage / storage unit based on instructions from the temperature control unit, wherein the temperature control unit can give instructions to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and can give instructions to maintain the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, as necessary.

[0083] In another aspect, the temperature controller of the device of the present disclosure may command, when lowering the temperature from a non-freezing temperature to a freezing target temperature, to lower the temperature at a first rate to a first target temperature, and then to lower the temperature from the first target temperature to a second target temperature at a second rate, and the device may have one or more embodiments described herein.

[0084] In a further aspect, the present disclosure can provide a program encoding a method for causing a computer to implement the method so that corneal endothelial cells and / or corneal endothelial-like cells can be preserved in an apparatus, the apparatus comprising: a storage / storage unit that houses a container that houses the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control unit that issues commands to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container housed in the storage / storage unit; and a temperature adjustment unit that can adjust the temperature in the storage / storage unit based on commands from the temperature control unit, the program causing the temperature control unit to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state as necessary.

[0085] In another aspect, the program of the present disclosure may instruct, when lowering the temperature from a non-freezing temperature to a freezing target temperature, to lower the temperature at a first rate to a first target temperature, and then to lower the temperature from the first target temperature to a second target temperature at a second rate, and the program may have one or more embodiments described herein.

[0086] In a further aspect, the present disclosure can provide a recording medium storing a program encoding a method for causing a computer to implement the method so as to be able to preserve corneal endothelial cells and / or corneal endothelial-like cells in an apparatus, wherein the apparatus comprises a storage / storage unit that houses a container that houses the corneal endothelial cells and / or corneal endothelial-like cells, a temperature control unit that issues commands to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container housed in the storage / storage unit, and a temperature adjustment unit that can adjust the temperature in the storage / storage unit based on commands from the temperature control unit, and the program causes the temperature control unit to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and, as necessary, maintains the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state.

[0087] In another aspect, a program stored on a recording medium of the present disclosure may instruct, when lowering the temperature from a non-freezing temperature to a freezing target temperature, to lower the temperature at a first rate to a first target temperature, and then lower the temperature from the first target temperature to a second target temperature at a second rate. The program may have one or more embodiments described herein.

[0088] The various functions realized by the device or program of the present disclosure may be realized partially or entirely manually.

[0089] The various functions realized by the device or program of the present disclosure may be realized or optimized in part or in whole by artificial intelligence (AI) or machine learning.

[0090] The program according to the present disclosure may be stored on a computer-readable recording medium or configured as a program product. Here, the term "recording medium" includes any "portable physical medium" such as a memory card, USB memory, SD card, flexible disk, magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, DVD, and Blu-ray (registered trademark) Disc.

[0091] Furthermore, a "program" is a data processing method written in any language or description method, and does not matter whether it is in the form of source code or binary code. Note that a "program" is not necessarily limited to a single structure, but also includes a structure that is distributed as multiple modules or libraries, or a structure that achieves its function by cooperating with a separate program, such as an OS (Operating System). Note that well-known structures and procedures can be used for the specific structure, reading procedure, and installation procedure after reading for each device shown in the embodiments for reading a recording medium.

[0092] Various databases, etc. are storage means such as memory devices such as RAM, ROM, fixed disk devices such as hard disks, flexible disks, optical disks, etc., and store various programs, tables, databases, web page files, etc. used for various processes and providing websites.

[0093] Furthermore, the specific form of distribution and integration of the devices is not limited to that shown in the drawings, and all or part of them can be functionally or physically distributed and integrated in any unit depending on various additions, etc., or functional load. In other words, the above-mentioned embodiments can be implemented in any combination, or embodiments can be implemented selectively.

[0094] (Kit) In a further aspect, the present disclosure may provide a frozen preparation kit comprising a container that holds a frozen preparation containing a ROCK inhibitor and corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, and a container that holds the container while maintaining the container in a frozen state.

[0095] In one aspect, the present disclosure may provide a frozen preparation kit comprising a container for holding a frozen preparation containing corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, and a container for holding the container while maintaining the container in a frozen state.

[0096] In one aspect, the present disclosure may provide a frozen formulation kit comprising a formulation of the present disclosure, a container for housing the formulation, and a receptacle for housing the container while maintaining the formulation in a frozen state.

[0097] In one aspect, the present disclosure may provide a frozen formulation kit comprising a container and a receptacle for housing the container, wherein the container is adapted to house a formulation of the present disclosure and the receptacle is adapted to maintain the formulation in a frozen state.

[0098] In one aspect, the present disclosure may provide the use of a kit comprising a container and a receptacle for housing the container, wherein the container is adapted to house a formulation of the present disclosure and the receptacle is adapted to maintain the formulation in a frozen state.

[0099] In one embodiment, the container can maintain the container it contains at a temperature in the range of about -80°C to about -20°C. In some embodiments, the container can maintain the container it contains at about -80°C.

[0100] Storage, Transportation, and Treatment In a further aspect, the present disclosure may provide a method of transporting and / or storing a formulation of the present disclosure, comprising the steps of placing the formulation in a container of a kit comprising a container and a receptacle for housing the container, and maintaining the formulation in the kit in a frozen state.

[0101] In a further aspect, there may be provided a method for performing corneal endothelial cell injection therapy, the method comprising the steps of providing corneal endothelial cells and / or corneal endothelial-like cells suitable for the cell injection therapy, a freezing step including at least one step of lowering the temperature of the corneal endothelial cells and / or corneal endothelial-like cells from a non-freezing temperature at a rate of less than 1° C. per minute, maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state and transporting them to the injection therapy as needed, thawing the corneal endothelial cells and / or corneal endothelial-like cells, and administering the corneal endothelial cells and / or corneal endothelial-like cells to a subject. In another aspect, the method of the present disclosure may comprise, when lowering the temperature from a non-freezing temperature to a freezing target temperature, lowering the temperature at a first rate to a first target temperature, and lowering the temperature from the first target temperature to a second target temperature at a second rate. The method may have one or more embodiments described herein.

[0102] In one embodiment, the transport may be carried out while maintaining the temperature within the range of about -80°C to about -20°C, preferably at -80°C.

[0103] Administration to a subject is preferably within 6 hours after thawing, and may be, for example, within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. Administration to a subject may be into the anterior chamber of the eye.

[0104] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0105] The present disclosure will be described in more detail below with reference to examples. It will be understood that the various reagents used in these examples may include those specifically shown, as well as those available from Sigma-Aldrich, BASF Japan Ltd., and the like.

[0106] (Example 1: Freezing in a cryopreservation solution with known components) Glycerin and polyethylene glycol are well-known cell cryopreservation agents, but because these components alone have low preservation effects, the addition of a protein component such as albumin or 10% DMSO is commonly performed. This example aims to confirm whether HCECs can be preserved under these general conditions, and further to confirm whether cryopreservation is possible even when the DMSO concentration is low when the cooling rate is slower than the commonly known -1°C / min.

[0107] (Materials) Human corneal endothelial cells after 4 passages OptiMEM ＴＭ -I (Invitrogen 21585-070) ・Tryple ＴＭSelect Enzyme (10x) (Thermo Fisher Scientific A12177-01) ・Red Cross Albumin 25% Intravenous Injection 12.5g / 50mL (Japan Blood Products Organization) ・2.0ml Cryogenic Vials (Corning 430488) ・Programmable Freezer (NEPAGENE PF-NP-200) ・Bicell (Japan Freezer Co., Ltd.) ・iMatrix-511 (nippi 892012) ・Bambanker hRM (Japan Genetics CS-11-001) ・Cryostor CS10 preformulated with 10% DMSO (Hemacare 210102) ・Cryostor CS5 preformulated with 5% DMSO (Hemacare 205102) ・Cryostor CS2 preformulated with 2% DMSO (Hemacare 202102) ・CP-1 high grade (Kyokuto Pharmaceutical 27207) ・Bambanker DMSO Free (Nippon Genetics CS-09-001) ・CryoScarless DMSO Free (Bioverde CPL-A1) ・12-well plate (Costar 3513) ・Proteosave SS centrifuge tube 50 mL (Sumitomo Bakelite MS-52550) Stemful centrifuge tube 15 mL (Sumitomo Bakelite MS-90150) OptiMEM containing 4% HSA, 10% glycerin or 10% polyethylene glycol, and 10%, 5%, 2% or 0% DMSO was used as the storage solution.

[0108] (Method) 1. Human corneal endothelial cells after passage 4 were used. The medium was removed from the culture dish during culturing, and OptiMEM was added and washed. This procedure was repeated twice. 2. After removing OptiMEM, Tryple ＴＭ Select Enzyme (10x) was added, and the mixture was incubated at 37°C (5% CO ２) for 20 minutes. 3. After 20 minutes, the cells were suspended in medium and collected in a 50 ml stem-full. 4. Centrifuged at 300 G for 5 minutes. 5. The supernatant was removed and the cells were suspended in OptiMEM (2% HSA). 6. Centrifuged at 300 G for 5 minutes. 7. Steps 5-6 were repeated again. 8. The supernatant was removed and the cells were suspended in OptiMEM (2% HSA), and the cell number was counted by trypan blue staining. 9. 1.2 x 10 cells were added to the cryopreservation reagent to be examined. ６ Each well was dispensed into a 15 ml stem-fill. As an unfrozen control, 1000 cells / mm ２ The cells were seeded at a cell density of 1000 x ...

[0109] An outline of Example 1 is shown in FIG.

[0110] (Results) Figure 2 shows a photograph of the culture morphology of the cells used in this example. It was confirmed that the lot had no morphological abnormalities.

[0111] Figure 3 shows a graph comparing the viability of cells after thawing after storage in cryopreservation solutions containing 4% human serum albumin and 10% glycerin at varying DMSO concentrations. When DMSO was present at 5% or higher, high viability was maintained regardless of the cooling rate. However, at 2% or 0%, the cooling rate had a significant effect on cell viability, with over 90% viability maintained at -0.5°C / min. BiCell is a container for storing tubes of cells to be frozen. When placed in a deep freezer at -80°C, the internal temperature drops at a rate of approximately -1°C / min. Cells stored in BiCell had lower viability than those stored in a programmable freezer at -1°C / min.

[0112] Figure 4 shows a graph comparing the viability of cells after thawing after preservation in cryopreservation solutions containing 4% human serum albumin and 10% polyethylene glycol at varying DMSO concentrations. Unlike the results for cryopreservation solutions containing glycerin, the cooling rate also affected cell viability in cryopreservation solutions containing 5% DMSO. For compositions with 2% DMSO or less, freezing at a cooling rate of -0.5°C / min yielded a viability of approximately 80%, far higher than freezing at a cooling rate of -1°C / min. These results indicate that slowing the cooling rate improves viability.

[0113] (Example 2: Study of cryopreservatives and freezing rates) (Materials) Human corneal endothelial cells after 4 passages OptiMEM ＴＭ -I (Invitrogen 21585-070) ・Tryple ＴＭSelect Enzyme (10x) (Thermo Fisher Scientific A12177-01) ・Red Cross Albumin 25% Intravenous Injection 12.5g / 50mL (Japan Blood Products Organization) ・2.0ml Cryogenic Vials (Corning 430488) ・Programmable Freezer (NEPAGENE PF-NP-200) ・Bicell (Japan Freezer Co., Ltd.) ・iMatrix-511 (nippi 892012) ・Bambanker hRM (Japan Genetics CS-11-001) ・Cryostor CS10 preformulated with 10% DMSO (Hemacare 210102) ・Cryostor CS5 preformulated with 5% DMSO (Hemacare 205102) ・Cryostor CS2 preformulated with 2% DMSO (Hemacare 202102) ・CP-1 high grade (Kyokuto Pharmaceutical 27207) (contains 10% DMSO) ・Bambanker DMSO Free (Nippon Genetics CS-09-001) ・Cryoscarless DMSO Free (Bioverde CPL-A1) ・12-well plate (costar 3513) ・ProteoSave SS Centrifuge tube 50 mL (Sumitomo Bakelite MS-52550) Stemful centrifuge tube 15 mL (Sumitomo Bakelite MS-90150) (Method) 1. Human corneal endothelial cells after passage 4 were used. The medium was removed from the culture dish during cultivation, and OptiMEM was added and washed. This procedure was repeated twice. 2. After removing OptiMEM, Tryple ＴＭ Select Enzyme (10x) was added, and the mixture was incubated at 37°C (5% CO ２) for 20 minutes. 3. After 20 minutes, the cells were suspended in medium and collected in a 50 ml stem-full. 4. Centrifuged at 300 G for 5 minutes. 5. The supernatant was removed and the cells were suspended in OptiMEM (2% HSA). 6. Centrifuged at 300 G for 5 minutes. 7. Steps 5-6 were repeated again. 8. The supernatant was removed and the cells were suspended in OptiMEM (2% HSA), and the cell number was counted by trypan blue staining. 9. 1.2 x 10 cells were added to the cryopreservation reagent to be examined. ６ Each well was dispensed into a 15 ml stem-fill. As an unfrozen control, 1000 cells / mm ２ The cells were seeded at a cell density of 100 μM and cultured for 2 weeks, changing the medium every 2 days. 10. The 15 ml stem full was centrifuged at 300 G for 5 minutes. 11. The cells were suspended in 350 μl of various cryopreservation reagents. 12. Y-27632 (100 μl), which had been adjusted to a final concentration of 100 μM in various cryopreservation reagents, was added to the 15 ml stem full from 11, bringing the total volume to 450 μl. 13. 450 μl aliquots (1.2 x 10) were dispensed into cryotubes. ６ (cells / 450 μl). 14. The cells were frozen in a programmable freezer from 4°C to -80°C at a rate of -1°C / min, -0.5°C / min, or -0.2°C / min. 15. Once the temperature had dropped to -80°C, the cells were transferred to a bicelle-treatment container pre-cooled at -80°C and frozen for 3 days in a -80°C freezer. 16. After 3 days of frozen storage, the cryotube containing the cells was thawed for 1-2 minutes in a 37°C water bath. 17. The cells were recovered in medium pre-warmed to 37°C, and the number of recovered cells and cell viability were measured using trypan blue staining. 18. The recovered cells were plated at 1000 cells / mm in a 12-well plate. ２ 19. The cells were cultured for 2 weeks with the medium changed every 2 days. 20. On the 7th day of culture, cells were photographed in 5 fields using a phase contrast microscope (200x magnification) and the cell density was calculated.

[0114] (Results) Figure 5 shows data comparing the post-thaw viability of cells frozen from 4°C at cooling rates of -1°C / min, -0.5°C / min, or -0.2°C / min. With the DMSO-containing cryopreservative, high viability was achieved at all conditions: -1°C / min, -0.5°C / min, and -0.2°C / min. With the DMSO-free cryopreservative, cell viability improved inversely proportional to the cooling rate, demonstrating that slower cooling rates are effective in increasing cell viability. Furthermore, when cells frozen at a cooling rate of -1°C / min were thawed and seeded in an incubator, many cells failed to adhere to the bottom, suggesting a decrease in corneal endothelial cell function. These findings suggest that freezing at a cooling rate slower than -1°C / min is important for improving the viability and maintaining the function of corneal endothelial cells.

[0115] Figure 6 shows data comparing the cell density on day 7 after seeding when cells were frozen from 4°C at a cooling rate of -1°C / min or -0.5°C / min. When cells were frozen at -1°C / min in CS2 containing 2% DMSO, Bambanker DMSO-Free containing no DMSO, or Cryoscarless DMSO-Free, the cell density was low, but when cells were frozen at a cooling rate of -0.5°C / min, the cell density was high. This indicates that the cooling rate also affects the cell density in culture after storage.

[0116] (Example 3: Study of culture with DMSO added) (Method) 1. Cells are collected using the same method as in Example 2 (1-10) and stored in cryotubes using a Cryostor CS2. 2. Freeze from 4°C to -80°C at -0.5°C / min using a programmed freezer. 3. Once the temperature has dropped to -80°C, transfer to a bicelle processing container that has been pre-cooled to -80°C and store frozen in the -80°C freezer for 3 days. 4. After 3 days of frozen storage, thaw the cryotube containing the cells in a 37°C water bath for 1-2 minutes. 5. Cells are collected in medium that has been pre-warmed to 37°C, and the number of collected cells and cell viability are measured using trypan blue staining. 6. The collected cells are dispensed into four 15ml stem-fill tubes and centrifuged at 300G for 5 minutes. 7. After centrifugation, the supernatant was removed, and the cells were cultured at 1000 cells / mm using media containing 10%, 5%, 2%, or 0% DMSO. ２ 7. The cells are then adjusted to a cell density of 1000 μg / well and replated onto a 12-well plate. 8. Photographs of the cells are taken using a phase-contrast microscope 1 hour, 3 hours, 6 hours, and 24 hours after seeding. 9. After photographing the cells at each time point, all cells in the container, including floating cells, are collected and cell viability is measured by trypan blue staining.

[0117] An outline of Example 3 is shown in FIG.

[0118] (Results) Figure 8 shows phase-contrast micrographs of cells cultured in medium containing 10% or 5% DMSO. Cell numbers were significantly lower in cells cultured in medium containing 10% or 5% DMSO than in cells cultured in medium without DMSO, indicating a large number of non-adherent cells on the laminin-coated bottom of the culture vessel. For cells cultured in medium without DMSO, the majority of cells adhered even 1 hour after inoculation, and all cells adhered by 3 hours. In contrast, no cell adhesion was observed up to 24 hours after inoculation with 10% DMSO. Even with 5% DMSO, almost no adherent cells were observed after 1 hour, and although less than half of the cells adhered by 3 and 6 hours, almost no adherent cells remained after 24 hours. In the micrographs in Figure 8, white cells indicate non-adherent cells, and black, non-circular cells indicate adherent cells.

[0119] Figure 9 shows phase-contrast microscopic images of cells cultured in medium containing 2% DMSO or without DMSO. The adhesion rate of cells cultured in medium containing 2% DMSO was not different from that of cells cultured in medium without DMSO.

[0120] In the environment within the anterior chamber of the eye, although the anterior chamber fluid is gradually replaced, it is thought that when cells are injected with a composition containing DMSO, they will be exposed to high concentrations of DMSO for a considerable period of time. Therefore, if cells are injected into the anterior chamber in a cryopreservative containing high concentrations of DMSO, such as 10%, it is expected that the survival rate and adhesion of corneal endothelial cells will be significantly impaired.

[0121] Figure 10 is a graph showing the results of recovering cells after replated and examining their viability. Cells cultured in a medium containing 10% DMSO, which showed little adhesion in the micrographs (Figure 8), also showed no decrease in viability at 1 hour. However, since viability subsequently decreased, it is presumed that the cells cultured in a medium containing 10% DMSO had already suffered significant damage, which prevented them from adhering, even though no decrease in viability was observed at 1 hour.

[0122] Based on these results, the amount of DMSO contained in the cell preparation injected into the anterior chamber is preferably 5% or less, and most preferably 2% or less or no DMSO is contained.

[0123] (Example 4: Examination of stability after freezing and thawing) (Method) 1. Cells are collected using the same method as in Example 2 (1-10) and stored in cryotubes, six for each preservation solution, using a Cryostor CS10, CS5, or CS2. 2. Freeze from 4°C to -80°C at -0.5°C / min using a programmable freezer. 3. Once the temperature has dropped to -80°C, transfer to a bicell-processing container pre-cooled at -80°C and store frozen in the -80°C freezer for three days. 4. After three days of frozen storage, thaw the cryotubes containing the cells in a 37°C water bath for 1-2 minutes. 5. Immediately collect the cells from one tube per preservation solution in medium pre-warmed to 37°C, and measure cell viability by trypan blue staining (0 h). The remaining five tubes were left at room temperature for 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours, after which the cells were collected in the same manner and the cell viability was measured by trypan blue staining. 6. The cells collected at each time point were plated in a 12-well plate at 1000 cells / mm ２ 7. After culturing for 24 hours from the time of reseeding, photographs of the cells are taken using a phase contrast microscope.

[0124] An outline of Example 4 is shown in FIG.

[0125] 12 shows phase-contrast micrographs of cells frozen in Cryostor CS10 containing 10% DMSO, left at room temperature for 0 hours, 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours, then reseeded into a T25 culture flask, and the culture state of the cells was photographed 24 hours later. When left at room temperature for 6 hours or longer, a decrease in cell proliferation and adhesion ability was observed.

[0126] 13 shows phase-contrast microscopic images of cells frozen in Cryostor CS5 containing 5% DMSO, left at room temperature for 0 hours, 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours, then reseeded into a T25 culture flask. While some reduction in cell proliferation and adhesion was observed in the cells left at room temperature for 6 hours, the degree of reduction was less than in CS5.

[0127] 14 shows phase-contrast micrographs of cells frozen in a Cryostor CS2 containing 2% DMSO, left at room temperature for 0 hours, 30 minutes, 1 hour, 3 hours, 6 hours, or 24 hours, then reseeded into a T25 culture flask. No changes were observed even after 6 hours at room temperature.

[0128] The graph in Figure 15 shows the results of examining the viability of cells recovered after reseeding. These results suggest that cell preparations frozen in a cryopreservative containing DMSO at 5% or less, preferably 2% or less, are stable even after thawing and are therefore highly convenient for clinical use.

[0129] Example 5: VIXELL ＴＭ Cryopreservation and post-preservation corneal endothelial cell injection in VIXELL ＴＭ By filling it with dry ice, VIXELL can maintain a temperature of -75°C ± 15°C for 18 days. ＴＭ The corneal endothelial cells are then stored and transported using the device, and then injected.

[0130] (Method) (Cryopreservation) 1. The cultured corneal endothelial cells were collected and cryopreserved at 1.2 × 10 in a CryoStor® CS2 containing 100 μM Y27632. ６ 2. Using a programmable freezer, the vials were frozen from 4°C to -80°C at a rate of -0.5°C / min. 3. The stored vials were frozen in BICELL. ＴＭ 4. The stored vials were taken out and placed in a VIXELL container containing dry ice. ＴＭ It was stored in a box for five days.

[0131] (Cell injection) Cultured human corneal endothelial cells, cryopreserved in Cryostor CS2, were placed in a medical transport refrigerated box (VIXELL) for the model in which 8 mm diameter corneal endothelial cells were exfoliated. ＴＭ After preserving the tissue in the corneal endothelium for 5 days, aqueous humor was injected into the tissue without perfusion to observe the dynamics of corneal endothelial regeneration in vivo.

[0132]

[0133] After the three-hour prone position, the anterior segment was observed with a slit lamp microscope on days 1, 2, 3, and 5 to check for the presence or absence of inflammation or infection. The day before and the day of surgery, and on days 2 and 4 after surgery, Prograf injection 5 mg / mL was diluted with 100 mL of saline, and a total of 6 mL was injected into the posterior auricular vein. The animals were euthanized on days 1 and 5 after surgery, and immunostaining was performed.

[0134] (Results) Figure 16 shows the viability and cell recovery rate when cells stored in a cool box for 5 days were thawed. The viability was calculated as the number of live cells at the time point of 5 days divided by the total number of cells. The cell recovery rate was calculated by taking the theoretical number of cells (number of cells loaded) as 100%.

[0135] To confirm whether the cells stored in the ice box maintained their normal phenotype, the cells were thawed and centrifuged to remove the cryopreservative. They were then resuspended in standard OPTI-MEM medium (containing Y27632) containing 8% FBS and seeded into a T25 culture flask for two days of culture. Figure 17 shows a micrograph of cells cultured for two days after storage in the ice box. The cells adhered to the bottom of the incubator in the same shape as unfrozen cells, confirming that their normal phenotype was maintained.

[0136] Figure 18 shows a photograph of the eye of a rabbit into which cells were injected after storage in a cool box. Corneal transparency was maintained due to the engraftment of corneal endothelial cells.

[0137] Figure 19 shows photographs of immunohistochemical staining of CD166 in corneal endothelium one day after cell injection. The corneal endothelial tissue was fixed, and an antibody against CD166, one of the expression markers of corneal endothelial cells, was bound as the primary antibody, followed by binding of a fluorescently labeled secondary antibody, for immunohistochemical staining. It was confirmed that the injected cells had successfully engrafted into the monolayer, and that CD166 was strongly expressed. The top row shows the central cornea, and the bottom row shows the peripheral area. Because staining was performed with an antibody that binds only to human CD166, the rabbit corneal endothelium was not stained, and a clear boundary was confirmed.

[0138] Figure 20 shows photographs of immunohistochemical staining for ZO-1 and Na / K ATPase one day after cell injection. ZO-1 and Na / K ATPase are expressed as functional molecules in corneal endothelial cells. Figure 21 shows photographs of immunohistochemical staining for CD166, ZO-1, and Na / K ATPase in corneal endothelium five days after cell injection.

[0139] These results indicate that the injected corneal endothelial cells took root in the corneal endothelium and functioned normally.

[0140] (Example 6: Freezing at a cooling rate of -0.7°C / min) The purpose of this example is to confirm the viability of corneal endothelial cells when frozen from 4°C at a cooling rate of -0.7°C / min in a cryopreservation solution of known ingredients similar to that in Example 1 and a commercially available preservation solution similar to that in Example 2.

[0141] 22 and 24 show an outline of this embodiment.

[0142] (Results) Figure 23 shows a graph comparing the viability of cells after thawing after freezing at cooling rates of -1 ° C. / min, -0.7 ° C. / min, -0.5 ° C. / min, or -0.2 ° C. in cryopreservation solutions containing 4% human serum albumin and 10% glycerin and varying DMSO concentrations. A trend toward improved viability was observed when frozen at cooling rates of -0.5 ° C. / min and -0.2 ° C. in cryopreservation solutions containing 10% DMSO. Furthermore, a trend toward improved viability was observed when frozen at cooling rates of -0.7 ° C. / min, -0.5 ° C. / min, and -0.2 ° C. in cryopreservation solutions containing 5% DMSO. A significant improvement in viability was observed when frozen at cooling rates of -0.7 ° C. / min, -0.5 ° C. / min, and -0.2 ° C. in cryopreservation solutions containing 2% DMSO and cryopreservation solutions without DMSO.

[0143] Figure 25 shows the post-thaw viability and recovery rate of cells frozen from 4°C at a cooling rate of -0.7°C / min in a commercially available cryopreservation solution. Viability was calculated as the number of viable cells divided by the total number of cells. Cell recovery was calculated as the recovery rate when the theoretical cell number (number of cells loaded) was set at 100%. Figure 26 shows data comparing the post-thaw viability of cells frozen from 4°C at a cooling rate of -1°C / min or -0.7°C / min in a commercially available cryopreservation solution. Figure 27 shows data comparing the post-thaw viability of cells frozen from 4°C at a cooling rate of -0.5°C / min or -0.2°C / min in a commercially available cryopreservation solution. When using a cryopreservation solution without DMSO, significant improvements in viability were observed when cells were frozen at cooling rates of -0.7°C / min, -0.5°C / min, and -0.2°C / min compared to cells frozen at a cooling rate of -1°C / min.

[0144] Based on the results of Examples 1 and 2 and the results of this Example, cell viability is improved by freezing and preserving cells at a cooling rate of -0.7°C / min or slower. In particular, a significant improvement in viability was observed in cryopreservation solutions containing as little as 2% DMSO and in cryopreservation solutions containing no DMSO. Therefore, the method of the present disclosure makes it possible to reduce the amount of DMSO used during cryopreservation.

[0145] (Example 7: Cryopreservation in glass vial formulation) In this example, cryopreservation was carried out in glass vials. As will be shown below, regardless of the container, preservation can be achieved with a high viability by freezing at a slow cooling rate.

[0146] (Materials and Methods) Cells were collected in the same manner as in Example 2 (1-10) and stored in glass vials using the following storage solutions and cooling rates. Control group: Storage solution: CS2 + Y-27632 (100 μM), freezing rate: -0.5°C / min (up to -80°C) HSA-added group: Storage solution: CS2 + Y-27632 (100 μM) + 4% HSA, freezing rate: -0.5°C / min (up to -80°C) Rate (1) group: Storage solution: CS2 + Y-27632 (100 μM), freezing rate: -0.5°C / min (up to -10°C), 110 min hold (-10°C), -1.0°C / min (up to -80°C) Rate (2) group: Storage solution: CS2 + Y-27632 (100 μM), freezing rate: -0.1°C / min (up to -10°C), -1.0°C / min (up to -80°C)

[0147] (Results) Figures 29-31 show the temperature transition. The rapid temperature rise of the samples is due to the latent heat observed during cooling. When frozen by cooling to -80°C at -0.5°C / min, the samples were preserved with a high viability of over 85%. Furthermore, when HSA was added and the samples were preserved under the same cooling conditions, the viability further increased, reaching over 90%. Similarly, when the samples were slowly cooled to -10°C (and then maintained at -10°C for a certain period of time), and then cooled at a rate of -1.0°C / min, the viability also increased compared to the control group, reaching over 90% (Figure 28). No abnormalities in cell shape were observed in any of the preservation groups.

[0148] Thus, regardless of the container, we demonstrated that cells can be preserved with a high viability by freezing and storing them at a slow cooling rate. We also demonstrated that cell viability can be further improved by cooling to a specific temperature at a slow rate (which may then be maintained at that temperature for a certain period of time) and then cooling at a faster rate for storage.

[0149] As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the claims. It is understood that the patents, patent applications, and literature cited in this specification should be incorporated by reference in their entirety as if the contents themselves were specifically set forth herein. This application claims the benefit of priority from Japanese Patent Application No. 2021-184246, filed on November 11, 2021, the contents of which are incorporated herein by reference.

[0150] The present invention provides a method for freezing corneal endothelial cells in a cryopreservation solution containing reduced DMSO or no DMSO, and a method for producing a frozen cell preparation that can be administered directly to patients. Since the preparation can be used for cell transplantation, etc., it can be used in fields such as pharmaceuticals.

Claims

1. A method for preserving corneal endothelial cells and / or corneal endothelial-like cells, comprising: a step of freezing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state, the step including at least one step of lowering the temperature at a rate of less than 1° C. per minute when changing the temperature from a non-freezing temperature to a target freezing temperature; and If necessary, maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state A method comprising:

2. The method according to claim 1, further comprising the step of maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state.

3. The method of claim 1 , wherein the step of maintaining in a frozen state comprises maintaining at a frozen maintenance temperature.

4. 4. The method of claim 3, wherein the frozen maintenance temperature is within the range of about -80°C to about -10°C.

5. 4. The method of claim 3, wherein the frozen maintenance temperature is a temperature within the range of about -196°C to about -10°C.

6. The method of claim 3, wherein the frozen maintenance temperature is a temperature of about -30°C or lower.

7. 2. The method of claim 1, wherein the corneal endothelial cells and / or corneal endothelial-like cells are cooled from a non-freezing temperature at a rate of about 0.1°C to about 0.9°C per minute.

8. 2. The method of claim 1, wherein the corneal endothelial cells and / or corneal endothelial-like cells are cooled from a non-freezing temperature at a rate of about 0.2°C to about 0.8°C per minute.

9. The method of claim 1, wherein the corneal endothelial cells and / or corneal endothelial-like cells are cooled from a non-freezing temperature at a rate of about 0.7°C per minute or less.

10. 2. The method of claim 1, wherein the corneal endothelial cells and / or corneal endothelial-like cells are cooled from a non-freezing temperature at a rate of about 0.2°C to about 0.7°C per minute.

11. 10. The method of claim 1, wherein the non-freezing temperature is a temperature within the range of about 0°C to about 42°C.

12. 10. The method of claim 1, wherein the non-freezing temperature is a temperature within the range of about 0°C to about 37°C.

13. 10. The method of claim 1, wherein the non-freezing temperature is a temperature within the range of about 4°C to about 23°C.

14. 2. The method of claim 1, wherein the freezing step comprises at least one step of decreasing the temperature at a rate of less than 1° C. per minute in at least a portion of a temperature range of about −20° C.±10° C.

15. The method according to claim 1, wherein the freezing step includes at least one step of maintaining the temperature in the range of about -20°C ± 10°C for a certain period of time or longer.

16. The method of claim 1 , wherein the corneal endothelial cells and / or corneal endothelial-like cells are preserved in a preservation solution containing less than about 7% DMSO.

17. The method according to claim 1, wherein the corneal endothelial cells and / or corneal endothelial-like cells are preserved in a preservation solution containing about 5% or less DMSO.

18. The method according to claim 1, wherein the corneal endothelial cells and / or corneal endothelial-like cells are preserved in a preservation solution containing about 2% or less DMSO.

19. The method according to claim 1 , wherein the corneal endothelial cells and / or corneal endothelial-like cells are preserved in a preservation solution that does not contain DMSO.

20. The method according to claim 1 , wherein the freezing step comprises freezing the corneal endothelial cells and / or corneal endothelial-like cells in the presence of a ROCK inhibitor.

21. 2. The method of claim 1, wherein the freezing step comprises decreasing the temperature at a first rate to a first target temperature and decreasing the temperature from the first target temperature to a second target temperature at a second rate, the first rate being less than 1° C. per minute and slower than the second rate.

22. 22. The method of claim 21, wherein the freezing step further comprises reducing the temperature to the first target temperature and then maintaining the first target temperature.

23. 22. The method of claim 21, wherein the first target temperature is a temperature within a range of about -20°C to about -5°C.

24. 22. The method of claim 21, wherein the first target temperature is a temperature in the range of about -15°C to about -10°C.

25. 22. The method of claim 21, wherein the second target temperature is a temperature of about -20°C or less.

26. 22. The method of claim 21, wherein the second target temperature is a temperature within a range of about -196°C to about -80°C.

27. 22. The method of claim 21, wherein the first rate is a rate of about 0.5°C to about 0.05°C per minute.

28. 22. The method of claim 21, wherein the first rate is a rate of about 0.3°C to about 0.1°C per minute.

29. 22. The method of claim 21, wherein the second rate is a rate of about 0.5 to about 5° C. per minute.

30. 22. The method of claim 21, wherein the second rate is a rate of about 1 to about 3 degrees Celsius per minute.

31. A method for producing a frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells, comprising: a step of mixing the corneal endothelial cells and / or corneal endothelial-like cells in an unfrozen state with a pharmaceutically acceptable component as needed, and freezing the cells to produce a frozen preparation, the step including at least one step of decreasing the temperature at a rate of less than 1°C per minute when changing the temperature from a non-freezing temperature to a target freezing temperature; If necessary, maintaining the frozen preparation of the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state. A method comprising:

32. 32. The method of claim 31, further comprising one or more features as recited in the methods of any one or more of claims 2 to 30.

33. A frozen preparation of corneal endothelial cells and / or corneal endothelial-like cells produced by the method according to claim 1.

34. The corneal endothelial cells and / or corneal endothelial-like cells are about 1×10 ５ ~Approx. 3×10 ６ 34. The frozen formulation of claim 33, comprising:

35. 34. The frozen formulation of claim 33, wherein the volume of the frozen formulation is from about 50 μL to about 600 μL.

36. The frozen formulation according to claim 33, wherein the frozen formulation is administered in an amount of about 50 μL to about 350 μL per administration.

37. A device for preserving corneal endothelial cells and / or corneal endothelial-like cells, a storage and preservation unit that stores a container that stores the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control unit that issues a command to control the temperature of the corneal endothelial cells and / or corneal endothelium-like cells in the container stored in the storage / preservation unit; a temperature control unit that can control the temperature in the storage / preservation unit based on a command from the temperature control unit; Including, The temperature control unit can instruct the device to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a freezing target temperature, and can instruct the device to maintain the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, as necessary.

38. A program encoding a method for causing a computer to implement the program so that corneal endothelial cells and / or corneal endothelial-like cells can be stored in an apparatus, the apparatus comprising: a storage / storage unit that stores a container for storing the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control unit that issues a command to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container stored in the storage / storage unit; and a temperature adjustment unit that can adjust the temperature in the storage / storage unit based on a command from the temperature control unit, The program causes the temperature control unit to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and, if necessary, causes the corneal endothelial cells and / or corneal endothelial-like cells to be maintained in a frozen state.

39. A recording medium storing a program encoding a method for causing a computer to implement the program so as to be able to preserve corneal endothelial cells and / or corneal endothelial-like cells in an apparatus, the apparatus comprising: a storage / storage unit that stores a container for storing the corneal endothelial cells and / or corneal endothelial-like cells; a temperature control unit that issues a command to control the temperature of the corneal endothelial cells and / or corneal endothelial-like cells in the container stored in the storage / storage unit; and a temperature adjustment unit that can adjust the temperature in the storage / storage unit based on a command from the temperature control unit, The program causes the temperature control unit to control the temperature to include at least one step of changing the temperature at a rate of less than 1°C per minute when lowering the temperature from a non-freezing temperature to a target freezing temperature, and, if necessary, causes the corneal endothelial cells and / or corneal endothelial-like cells to be maintained in a frozen state.

40. A frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells.

41. A frozen formulation that can be administered directly to the eye after thawing, comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells.

42. A frozen preparation comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells, frozen under slow freezing conditions.

43. A frozen preparation comprising less than 7% DMSO, corneal endothelial cells and / or corneal endothelial-like cells, and a physiological saline component in a frozen state.

44. A frozen preparation comprising less than 7% DMSO, corneal endothelial cells and / or corneal endothelial-like cells, and medium components in a frozen state.

45. A frozen cell preparation with long-term post-thaw stability, comprising less than 7% DMSO and corneal endothelial cells and / or corneal endothelial-like cells.

46. 41. The frozen formulation of claim 40, comprising about 5% or less DMSO.

47. 41. The frozen formulation of claim 40, comprising about 2% or less DMSO.

48. 41. The frozen formulation of claim 40, which is DMSO-free.

49. 41. The frozen formulation of claim 40, further comprising a ROCK inhibitor.

50. 50. The frozen formulation of claim 49, wherein the ROCK inhibitor is Y-27632.

51. A frozen preparation comprising a ROCK inhibitor and corneal endothelial cells and / or corneal endothelial-like cells in a frozen state.

52. A frozen preparation comprising a ROCK inhibitor, corneal endothelial cells and / or corneal endothelial-like cells, and a physiological saline component in a frozen state.

53. A frozen preparation comprising a ROCK inhibitor, corneal endothelial cells and / or corneal endothelial-like cells, and medium components in a frozen state.

54. A frozen cell preparation having long-term post-thaw stability, the preparation comprising corneal endothelial cells and / or corneal endothelial-like cells and a ROCK inhibitor.

55. 55. The preparation according to claim 54, wherein the viability of the corneal endothelial cells and / or corneal endothelial-like cells is at least 80% at room temperature for at least 6 hours after thawing.

56. A frozen preparation that does not inhibit the engraftment and in vivo survival of corneal endothelial cells and / or corneal endothelial-like cells when administered after thawing, said preparation comprising corneal endothelial cells and / or corneal endothelial-like cells and a ROCK inhibitor in a state frozen under slow freezing conditions.

57. A frozen preparation comprising less than 7% DMSO, a ROCK inhibitor, and corneal endothelial cells and / or corneal endothelial-like cells, frozen under slow freezing conditions.

58. The formulation of any one of claims 51 to 57, wherein the ROCK inhibitor is Y-27632.

59. 57. The formulation of any one of claims 51 to 56, comprising less than about 7% DMSO.

60. 57. The formulation of any one of claims 51 to 56, comprising about 5% or less DMSO.

61. 57. The formulation of any one of claims 51 to 56, comprising about 2% or less DMSO.

62. 57. The formulation of any one of claims 51 to 56, which is DMSO-free.

63. 53. The formulation of any one of claims 31, 33 to 52, wherein the formulation comprises the cells in a state frozen under slow freezing conditions.

64. 54. The formulation of any one of claims 31, 33 to 53, wherein the formulation is frozen by reducing the temperature from a non-freezing temperature at a rate of less than 1°C per minute.

65. The preparation according to any one of claims 40 to 57, wherein the corneal endothelial cells and / or corneal endothelial-like cells are used in cell injection therapy.

66. 58. The formulation of any one of claims 40 to 57, wherein the frozen formulation is administered without further processing or culturing after thawing.

67. The corneal endothelial cells and / or corneal endothelial-like cells are about 1×10 ５ ~Approx. 3×10 ６ 58. The formulation of any one of claims 40 to 57, comprising:

68. 58. The formulation of any one of claims 40 to 57, wherein the volume of the frozen formulation is from about 50 μL to about 600 μL.

69. The formulation according to any one of claims 40 to 57, wherein the formulation is administered in an amount of about 50 μL to about 350 μL per dose.

70. A frozen preparation kit comprising a container for storing a frozen preparation containing corneal endothelial cells and / or corneal endothelial-like cells in a frozen state, and a container for storing the container while maintaining the container in a frozen state.

71. The frozen formulation kit according to claim 70, wherein the frozen formulation is a formulation according to any one of claims 31 and 33 to 57.

72. A frozen formulation kit comprising the formulation according to any one of claims 40 to 57, a container for containing the formulation, and a container for containing the container while maintaining the formulation in a frozen state.

73. A frozen formulation kit comprising a container and a container for accommodating the container, A kit, wherein the container is adapted to contain the formulation according to any one of claims 40 to 57, and the container is adapted to maintain the formulation in a frozen state.

74. A frozen preparation kit comprising: a container for storing a frozen preparation containing a ROCK inhibitor and corneal endothelial cells and / or corneal endothelial-like cells in a frozen state; and a container for storing the container while maintaining the container in a frozen state.

75. Use of a kit comprising a container and a container for housing the container, 58. Use wherein the container is adapted to contain the formulation according to any one of claims 40 to 57, and the container is adapted to maintain the formulation in a frozen state.

76. A method for transporting and / or storing the formulation according to any one of claims 40 to 57, comprising: placing the formulation in a container of a kit comprising a container and a receptacle for receiving the container; maintaining the formulation in the kit in a frozen state; A method comprising:

77. A method for performing corneal endothelial cell injection therapy, comprising: providing corneal endothelial cells and / or corneal endothelial-like cells suitable for the cell injection therapy; a freezing step comprising at least one step of lowering the temperature of the corneal endothelial cells and / or corneal endothelial-like cells from a non-freezing temperature at a rate of less than 1°C per minute; maintaining the corneal endothelial cells and / or corneal endothelial-like cells in a frozen state and transporting them to the injection therapy as needed; thawing the corneal endothelial cells and / or corneal endothelial-like cells; and administering the corneal endothelial cells and / or corneal endothelial-like cells to a subject A method comprising: