Methods and containers for preserving corneal endothelial cells

Abstract

This invention provides a method for preserving corneal endothelial cells with high cell viability. The method comprises: a substrate with a base area of ​​at least about 0.7 cm². 2 The process of preserving corneal endothelial cells and / or corneal endothelial-like cells in a container. Through this invention, corneal endothelial cells can be preserved with a high cell viability rate. Furthermore, the corneal endothelial cells preserved in this way retain the functions of normal corneal endothelial cells and can be used as therapeutic cells for corneal endothelial diseases, etc.

Description

Technical Field

[0001] This invention relates to a technique for preserving corneal endothelial cells. Background Technology

[0002] Visual information is recognized as follows: light transmitted from the cornea, the transparent tissue at the front of the eyeball, reaches the retina, exciting the nerve cells in the retina. The resulting electrical signals travel via the optic nerve to the visual cortex of the brain, where they are perceived. For good vision, it is crucial that the cornea is transparent. This transparency is maintained by utilizing the pump and barrier functions of the corneal endothelial cells to keep the water content constant.

[0003] Human corneal endothelial cells exist at birth at a density of approximately 3,000 per square millimeter, but their regenerative capacity is limited. Therefore, they cannot maintain function when severely damaged, resulting in loss of corneal transparency. In bullous keratopathy, caused by corneal endothelial malnutrition or dysfunction from various causes, corneal edema and opacity occur, leading to significant vision loss. Currently, allogeneic corneal transplantation using donor corneas is used for bullous keratopathy. However, due to surgical invasiveness, rejection reactions, and donor shortages, current corneal transplantation methods face many challenges that need to be addressed.

[0004] To overcome these problems, a minimally invasive cell transplantation therapy has been developed in recent years as a treatment for corneal endothelial diseases (Non-Patent Literature 1). The culture of corneal endothelial cells is challenging, with issues such as non-proliferation (Non-Patent Literature 2 and 3), fibroblastization (Non-Patent Literature 4), and cell senescence (Non-Patent Literature 5) when using conventional methods. Methods for culturing corneal endothelial cells are under intense research, and practical clinical application has become possible (Non-Patent Literature 1).

[0005] Existing technical documents

[0006] Non-patent literature 1: Kinoshita S, Koizumi N, Ueno M, et al. Injection of cultured cells with a ROCK inhibitor for bullous keratopathy. N Engl J Med 2018; 378:995-1003.

[0007] Non-Patent Document 2: Okumura N, Ueno M, Koizumi N, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci 2009; 50: 3680-3687.

[0008] Non-Patent Document 3: Nakahara M, Okumura N, Kay EP, et al. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium. PLoS One 2013; 8: e69009.

[0009] Non-Patent Document 4: Okumura N, Kay EP, Nakahara M, Hamuro J, Kinoshita S, Koizumi N. Inhibition of TGF-beta Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine. PLoS One 2013; 8: e58000.

[0010] Non-Patent Document 5: Hongo A, Okumura N, Nakahara M, Kay EP, Koizumi N. The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells. Invest Ophthalmol Vis Sci 2017; 58: 3325-3334. Summary of the Invention

[0011] Solution for solving the problem

[0012] The inventors conducted in-depth research and discovered a method for preserving corneal endothelial cells with a high survival rate, thus completing the invention. Specifically, the inventors discovered that by preserving corneal endothelial cells in a container with a specific bottom area, it is possible to maintain corneal endothelial cells with a high survival rate. The corneal endothelial cells preserved in this way possess the functions of normal corneal endothelial cells. Therefore, in this invention, we provide cells that can be used directly without further substantial processing (ready-to-use) as therapeutic cells.

[0013] Therefore, the present invention provides the following.

[0014] (Project 1)

[0015] A method for preserving corneal endothelial cells and / or corneal endothelioid cells, comprising: in a basin with a base area of ​​at least about 0.7 cm² 2 The process of storing the corneal endothelial cells and / or corneal endothelial-like cells in a container.

[0016] (Project 2)

[0017] According to the method described in Project 1, the liquid level of the liquid used to preserve the cells is above approximately 0.75 mm.

[0018] (Project 3)

[0019] According to the method described in Project 1 or 2, the bottom area of ​​the container is approximately 0.7 to approximately 4 cm². 2 .

[0020] (Project 4)

[0021] According to the method described in any one of items 1 to 3, the bottom area of ​​the container is approximately 1.5 to approximately 3 cm². 2 .

[0022] (Project 5)

[0023] According to the method described in any one of items 1 to 4, the bottom area of ​​the container is approximately 1.8 to approximately 2 cm². 2 .

[0024] (Project 6)

[0025] The method according to any one of items 1 to 5, wherein the container has not undergone surface treatment for adhesion culture.

[0026] (Project 7)

[0027] The method according to any one of items 1 to 6, wherein the container is a low-adhesion surface container or an untreated surface container.

[0028] (Project 8)

[0029] The method according to any one of items 1 to 7, wherein the container is made of polystyrene, polypropylene, or glass.

[0030] (Project 9)

[0031] The method described in any one of items 1 to 8, wherein the container is selected from the group consisting of a 24-well plate, a tubular bottle, a syringe, and a culture dish.

[0032] (Project 10)

[0033] The method according to any one of items 1 to 9, wherein the cells are preserved at a temperature of about 12°C to about 42°C.

[0034] (Project 11)

[0035] The method according to any one of items 1 to 10, wherein the cells are preserved at a temperature of about 17°C to about 39°C.

[0036] (Project 12)

[0037] The method according to any one of items 1 to 11, wherein the cells are preserved at a temperature of about 27°C to about 37°C.

[0038] (Project 13)

[0039] The method according to any one of items 1 to 12, wherein the cells are preserved at a temperature of about 37°C.

[0040] (Project 14)

[0041] A container for storing corneal endothelial cells and / or corneal endothelial-like cells, having a base area of ​​at least about 0.7 cm². 2 .

[0042] (Project 15)

[0043] The container according to Item 14 has a structure that allows the liquid level to be at least 0.75 mm high.

[0044] (Project 16)

[0045] The container described in item 14 or 15 is for storing corneal endothelial cells and / or corneal endothelioid cells, with a base area of ​​approximately 0.7 to approximately 4 cm². 2 .

[0046] (Project 17)

[0047] The container according to any one of items 14 to 16, wherein the base area of ​​the container is about 1.5 to about 3 cm². 2 .

[0048] (Project 18)

[0049] The container according to any one of items 14 to 17, wherein the base area of ​​the container is about 1.8 to about 2 cm². 2 .

[0050] (Project 19)

[0051] The container according to any one of items 14 to 18, wherein the container has not undergone surface treatment for adhesion culture.

[0052] (Project 20)

[0053] The container according to any one of items 14 to 19, wherein the container is a low-adhesion surface container or an untreated surface container.

[0054] (Project 21)

[0055] The container according to any one of items 14 to 20, wherein the container is made of polystyrene, polypropylene, or glass.

[0056] (Project 22)

[0057] The container according to any one of items 14 to 21, wherein the container is selected from the group consisting of a 24-well plate, a tubular bottle, a syringe, or a petri dish.

[0058] (Project 23)

[0059] A corneal endothelial cell or corneal endothelioid cell, which is preserved by the method described in any one of items 1 to 13.

[0060] (Project 24)

[0061] A composition for treating or preventing corneal endothelial disorders, diseases, or symptoms, comprising the corneal endothelial cells and / or corneal endothelial-like cells described in item 23.

[0062] (Project 25)

[0063] The composition according to item 24 also contains a ROCK inhibitor.

[0064] (Project 26)

[0065] The composition according to item 25 is characterized in that it is administered in combination with a ROCK inhibitor.

[0066] (Project 27)

[0067] The composition according to item 25 or 26, wherein the ROCK inhibitor is Y-27632.

[0068] (Project 28)

[0069] A cell-containing container comprising: (A) corneal endothelial cells and / or corneal endothelioid cells; and (B) a container for storing the corneal endothelial cells and / or corneal endothelioid cells, said cell-containing container having a base area of ​​at least about 0.7 cm². 2 .

[0070] (Project 29)

[0071] The cell-containing container according to item 28 also includes the features described in any one or more of items 15 to 22.

[0072] (Project 30)

[0073] The cell-containing container described in Item 28 or 29 also contains a ROCK inhibitor.

[0074] The present invention further provides the following items.

[0075] (Project 1A)

[0076] A method for preserving corneal endothelial cells and / or corneal endothelioid cells, comprising: in a basin with a base area of ​​at least about 0.7 cm² 2 The process of storing the corneal endothelial cells and / or corneal endothelial-like cells in a container.

[0077] (Project 2A)

[0078] According to the method described in Project 1A, the corneal endothelial cells and corneal endothelioid cells are cells that can be used clinically.

[0079] (Project 3A)

[0080] The method according to Project 1A or 2A is characterized in that the preserved corneal endothelial cells and corneal endothelial-like cells are given directly without further processing or culture.

[0081] (Project 4A)

[0082] According to the method described in any one of items 1A to 3A, the material is stored for at least approximately 6 hours.

[0083] (Project 5A)

[0084] According to the method described in any one of Projects 1A to 4A, the liquid level of the liquid used to preserve the cells is at least 0.75 mm.

[0085] (Project 6A)

[0086] According to the method described in any one of items 1A to 5A, the bottom area of ​​the container is approximately 0.7 to approximately 4 cm². 2 .

[0087] (Project 7A)

[0088] According to the method described in any one of items 1A to 6A, the bottom area of ​​the container is approximately 1.5 to approximately 3 cm². 2 .

[0089] (Project 8A)

[0090] According to the method described in any one of items 1A to 7A, the bottom area of ​​the container is approximately 1.8 to approximately 2 cm². 2 .

[0091] (Project 9A)

[0092] The method according to any one of items 1A to 8A, wherein the container is not subjected to surface treatment for adhesion culture.

[0093] (Project 10A)

[0094] The method according to any one of items 1A to 9A, wherein the container is a low-adhesion surface container or an untreated surface container.

[0095] (Project 11A)

[0096] The method according to any one of items 1A to 10A, wherein the container is made of polystyrene, polypropylene, or glass.

[0097] (Project 12A)

[0098] The method according to any one of items 1A to 11A, wherein the container is selected from the group consisting of a 24-well plate, a tubular bottle, a syringe, and a culture dish.

[0099] (Project 13A)

[0100] The method according to any one of items 1A to 12A, wherein the cells are preserved at a temperature of about 0°C to about 42°C.

[0101] (Project 14A)

[0102] The method according to any one of items 1A to 13A, wherein the cells are preserved at a temperature of about 17°C to about 39°C.

[0103] (Project 15A)

[0104] The method according to any one of items 1A to 14A, wherein the cells are preserved at a temperature of about 27°C to about 37°C.

[0105] (Project 16A)

[0106] The method according to any one of items 1A to 15A, wherein the cells are preserved at a temperature of about 37°C.

[0107] (Project 17A)

[0108] The method according to any one of items 1A to 16A, wherein the corneal endothelial cells and / or corneal endothelioid cells are preserved in a cell suspension.

[0109] (Project 18A)

[0110] The method according to any one of items 1A to 17A, wherein the volume of the suspension is at least about 50 μL.

[0111] (Project 19A)

[0112] The method according to any one of items 1A to 18A, wherein the volume of the suspension is about 100 μL to about 2000 μL.

[0113] (Project 20A)

[0114] The method according to any one of items 1A to 19A, wherein the aforementioned corneal endothelial cells and / or corneal endothelioid cells that have undergone the preservation can be used for cell infusion therapy.

[0115] (Project 21A)

[0116] According to the method described in any one of items 1A to 20A, wherein the cell density of the cells preserved in the container is approximately 2 × 10⁻⁶. 4 10 cells / ml ~ approximately 8 × 10 7 per ml.

[0117] (Project 22A)

[0118] According to the method described in any one of items 1A to 21A, wherein the cell density of the cells preserved in the container is approximately 2 × 10⁻⁶. 6 pcs / ml ~ approximately 4×10 6 per ml.

[0119] (Project 23A)

[0120] A container for storing corneal endothelial cells and / or corneal endothelial-like cells, having a base area of ​​at least about 0.7 cm². 2 .

[0121] (Project 24A)

[0122] The container according to item 23A has a structure that allows a liquid level height of about 0.75 mm or more.

[0123] (Project 25A)

[0124] The container described in item 23A or 24A is for storing corneal endothelial cells and / or corneal endothelioid cells, with a base area of ​​approximately 0.7 to approximately 4 cm². 2 .

[0125] (Project 26A)

[0126] The container according to any one of items 23A to 25A, wherein the base area of ​​the container is about 1.5 to about 3 cm². 2 .

[0127] (Project 27A)

[0128] The container according to any one of items 23A to 26A, wherein the base area of ​​the container is about 1.8 to about 2 cm². 2 .

[0129] (Project 28A)

[0130] The container according to any one of items 23A to 27A, wherein the container has not undergone surface treatment for adhesion culture.

[0131] (Project 29A)

[0132] The container according to any one of items 23A to 28A, wherein the container is a low-adhesion surface container or an untreated surface container.

[0133] (Project 30A)

[0134] The container according to any one of items 23A to 29A, wherein the container is made of polystyrene, polypropylene, or glass.

[0135] (Project 31A)

[0136] The container according to any one of items 23A to 30A, wherein the container is selected from the group consisting of a 24-well plate, a tubular bottle, a syringe, or a petri dish.

[0137] (Project 32A)

[0138] The container according to any one of items 23A to 31A, wherein the container is used to preserve the corneal endothelial cells and / or corneal endothelial-like cells in a cell suspension state.

[0139] (Project 33A)

[0140] The container according to item 32A, wherein the corneal endothelial cells and / or corneal endothelial-like cells are capable of being used for cell infusion therapy.

[0141] (Project 34A)

[0142] The container according to any one of items 23A to 33A, wherein the corneal endothelial cells and / or corneal endothelioid cells are preserved for at least about 6 hours.

[0143] (Project 35A)

[0144] A corneal endothelial cell and / or corneal endothelioid cell, which is preserved by the method described in any one of items 1A to 22A.

[0145] (Project 36A)

[0146] The corneal endothelial cells and / or corneal endothelioid cells as described in Item 35A, wherein the corneal endothelial cells and / or corneal endothelioid cells are preserved for at least about 6 hours.

[0147] (Project 37A)

[0148] A composition for treating or preventing corneal endothelial disorders, diseases, or symptoms, comprising corneal endothelial cells and / or corneal endothelial-like cells as described in item 35A or 36A.

[0149] (Project 38A)

[0150] The composition described in item 37A also contains a ROCK inhibitor.

[0151] (Project 39A)

[0152] The composition according to item 38A is characterized in that it is administered in combination with a ROCK inhibitor.

[0153] (Project 40A)

[0154] The composition according to item 38A or 39A, wherein the ROCK inhibitor is selected from Y-27632, ribosudil, fasudil, or a pharmaceutically permissible salt thereof.

[0155] (Project 41A)

[0156] A cell-containing container comprising: (A) corneal endothelial cells and / or corneal endothelioid cells; and (B) a container for storing the corneal endothelial cells and / or corneal endothelioid cells, said cell-containing container having a base area of ​​at least about 0.7 cm². 2 .

[0157] (Project 42A)

[0158] The cell-containing container according to item 41A further includes the features described in any one or more of items 24A to 34A.

[0159] (Project 43A)

[0160] The cell-containing container as described in Item 41A or 42A also contains a ROCK inhibitor.

[0161] (Project 44A)

[0162] A cell-containing container according to any one of items 41A to 43A, wherein the cell density is approximately 2 × 10⁻⁶. 4 10 cells / ml ~ approximately 8 × 10 7 per ml.

[0163] (Project 45A)

[0164] A cell-containing container according to any one of items 41A to 44A, wherein the number of cells is approximately 2 × 10⁻⁶. 6 pcs / ml ~ approximately 4×10 6 per ml.

[0165] (Project 46A)

[0166] The cell-containing container according to any one of items 41A to 45A, wherein the volume of the suspension of corneal endothelial cells and / or corneal endothelioid cells is at least about 50 μL.

[0167] (Project 47A)

[0168] According to the method described in Project 46A, the volume of the suspension is approximately 300 μl to approximately 600 μl.

[0169] (Project 48A)

[0170] A cell preparation comprising: corneal endothelial cells and / or corneal endothelioid cells, and a container for storing the corneal endothelial cells and / or corneal endothelioid cells, the container having a base area of ​​at least about 0.7 cm².2 .

[0171] (Project 49A)

[0172] The cell preparation according to item 48A, wherein the container has the features described in any one or more of items 24A to 34A.

[0173] (Project 50A)

[0174] According to the cell preparations described in Item 48A or 49A, the corneal endothelial cells and / or corneal endothelioid cells are preserved for at least about 6 hours.

[0175] (Project 51A)

[0176] The cell preparation according to any one of items 48A to 50A, wherein the corneal endothelial cells and / or corneal endothelial-like cells are stored in suspension.

[0177] (Project 52A)

[0178] The cell preparation according to any one of items 48A to 51A, wherein the corneal endothelial cells and / or corneal endothelial-like cells preserved in suspension are capable of being used for cell infusion therapy.

[0179] (Project 53A)

[0180] A method for treating or preventing corneal endothelial disorders, diseases, or symptoms in a subject, comprising the steps of administering to the subject an effective amount of corneal endothelial cells and / or corneal endothelioid cells preserved by the method described in any one of items 1A to 22A.

[0181] (Project 54A)

[0182] According to the method described in Project 53A, the corneal endothelial cells and / or corneal endothelioid cells are preserved for at least about 6 hours.

[0183] (Project 55A)

[0184] According to the method described in Project 53A, the corneal endothelial cells and / or corneal endothelioid cells are preserved in a cell suspension.

[0185] (Project 56A)

[0186] According to the method described in Project 55A, approximately 20 μL to approximately 500 μL of the cell suspension is administered.

[0187] (Project 57A)

[0188] According to the method described in Project 55A, approximately 200 μL to approximately 300 μL of the cell suspension is administered.

[0189] (Project 58A)

[0190] The method according to any one of items 53A to 57A, wherein the corneal endothelial cells or corneal endothelioid cells are administered in combination with an effective amount of ROCK inhibitor.

[0191] (Project 59A)

[0192] According to the method described in Project 58A, the ROCK inhibitor is selected from Y-27632, risudil, fasudil, or a pharmaceutically permissible salt thereof.

[0193] (Project 60A)

[0194] According to the method described in any one of items 53A to 59A, approximately 40,000 to approximately 4 million cells are administered.

[0195] (Project 61A)

[0196] The method described in any of Projects 53A to 60A, wherein approximately 400,000 to approximately 1,000,000 cells are administered.

[0197] (Project 62A)

[0198] A corneal endothelial cell or corneal endothelioid cell preserved by the method described in any one of items 1A to 22A, for disposal or prevention of corneal endothelial impairment, disease or symptoms in a subject.

[0199] (Project 63A)

[0200] The use of preserved corneal endothelial cells or corneal endothelioid cells, as described by any one of items 1A to 22A, in the manufacture of a medicament for the treatment or prevention of corneal endothelial impairment, disease, or symptoms in a subject.

[0201] In this invention, one or more of the above-described features are intended to be provided in further combinations based on the explicit combinations. Further embodiments and advantages of this invention will be apparent to those skilled in the art upon reading and understanding the following detailed description.

[0202] The effects of the invention

[0203] This invention enables the preservation of corneal endothelial cells with a high cell viability. Furthermore, the corneal endothelial cells preserved in this way retain the functions of normal corneal endothelial cells and can be used as therapeutic cells for corneal endothelial diseases, etc. Additionally, this invention provides a cell preparation that can be readily supplied. Attached Figure Description

[0204] Figure 1 : Figure 1 A represents an outline of the exemplary steps for preserving corneal endothelial cells. Figure 1 B represents phase-contrast microscopy images of corneal endothelial cells after they were recovered by gentle pipetting in 24-well (cell culture plate), 24-well (ultra-low adhesion) plate, 48-well (suspension culture) plate, and 96-well (ultra-low adhesion) plate.

[0205] Figure 2 : Figure 2 A represents the ratio of live to dead corneal endothelial cells preserved in 24-well plates (ultra-low adhesion), 48-well plates (suspension culture), 96-well plates (ultra-low adhesion, flat bottom), 96-well plates (ultra-low adhesion, round bottom), 15 ml conical tubes (ultra-low adhesion), and 2 ml cryotubes (CryoVial). Figure 2 B represents the ratio of live to dead corneal endothelial cells preserved at 37°C in a 24-well plate (ultra-low adhesion), 4°C in a 24-well plate (ultra-low adhesion), and 37°C in a 24-well plate (cell culture plate). Figure 2 C represents the ratio of live to dead corneal endothelial cells preserved in 24-well plates (ultra-low adhesion), 24-well plates (untreated), and 10 ml glass tubular vials. Figure 2 D represents the ratio of live to dead corneal endothelial cells preserved in 24-well plates (ultra-low adhesion) at preservation solution volumes of 300 μl, 1000 μl, 1500 μl, and 2500 μl. The white portion of the bar represents dead cells, and the black portion represents live cells. Figure 2 E represents phase-contrast microscopy images of corneal endothelial cells preserved in 24-well (ultra-low adhesion) seeded onto culture plates 3 hours and 2 weeks later.

[0206] Figure 3 : Figure 3 A represents a slit-lamp image of a rabbit model in which corneal endothelial cells preserved in a 24-well plate (ultra-low adhesion) are injected into the anterior chamber. Figure 3 In Figure A, images from left to right are shown on days 1, 7, and 14 after injection, while images from top to bottom are shown as control eyes (no injected cells), eyes injected with cells stored for 24 hours, eyes injected with cells stored for 48 hours, and eyes injected with cells stored for 72 hours. Figure 3B represents Scheimpflug images taken on day 14 after injection.

[0207] Figure 4 : Figure 4 A indicates that corneal endothelial cells preserved in a 24-well plate (ultra-low adhesion) were injected into a rabbit model of the anterior chamber, and pentacam was used on day 14 post-injection. TM A color image of the corneal thickness obtained. Figure 4 B represents the central corneal thickness after the injection of preserved corneal endothelial cells. Figure 4 C represents the corneal volume after the injection of preserved corneal endothelial cells. Figure 4 D represents the cell density of the eye on day 14 after the injection of preserved corneal endothelial cells. Figure 4 E represents a fluorescence microscope image of a rabbit cornea stained with immunofluorescence. Blue indicates DAPI staining (cell nuclei). Green, from left to right, represents Na+. + / K + -ATPase, ZO-1, N-cadherin.

[0208] Figure 5 This indicates the state of preservation by suspending 500,000 corneal endothelial cells cultured in a 1ml syringe in 300μl of cell preservation solution.

[0209] Figure 6 This is a phase-contrast micrograph showing corneal endothelial cells preserved in a 1ml syringe for 72 hours. Cells are deposited on the side of the syringe.

[0210] Figure 7 This is a phase-contrast microscopy image taken after corneal endothelial cells have been preserved in a 1ml syringe for 72 hours, followed by a gentle tap. The cells float to the side of the syringe.

[0211] Figure 8 This is a phase-contrast micrograph showing the results after corneal endothelial cells were preserved in a 1ml syringe for 72 hours, gently tapped, and the syringe was gently rinsed with cell preservation solution. No cell adhesion was observed on the side of the syringe.

[0212] Figure 9 This indicates the ratio of live to dead corneal endothelial cells stored at 12℃, 17℃, 22℃, 27℃, 32℃, 35℃, 37℃, 39℃, and 42℃. The white part of the bar represents dead cells, and the black part represents live cells.

[0213] Figure 10Photographs are shown for (1) a tubular bottle (Maruemu Corporation, Osaka, 0501-02), (2) a tubular bottle (2ml, 16mm in diameter, 33mm in height, IRAS treated, Iwata Glass Industrial Co., Ltd., Osaka, lot: 181024), and (3) a tubular bottle (2ml, 16mm in diameter, 33mm in height, IRAS treated and silica coated (SiO2 coated), IwataGlass Industrial Co., Ltd., Osaka, lot: 181102).

[0214] Figure 11 This indicates the ratio of live to dead corneal endothelial cells preserved in (1) tubular bottles (Maruemu Corporation, Osaka, 0501-02), (2) tubular bottles (2ml, 16mm diameter, 33mm height, IRAS treated, Iwata Glass Industrial Co., Ltd., Osaka), and (3) tubular bottles (2ml, 16mm diameter, 33mm height, IRAS treated and silica coated (SiO2 coated), Iwata Glass Industrial Co., Ltd., Osaka). The white part of the bar represents dead cells, and the black part represents live cells. Detailed Implementation

[0215] The present invention will now be described. Throughout this specification, singular expressions, unless otherwise specified, should be understood to include their plural forms as well. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include their plural forms as well, unless otherwise specified. Furthermore, the terms used in this specification, unless otherwise specified, should be understood to be used in the sense commonly understood in the art. 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 invention pertains. In case of conflict, this specification (including definitions) shall prevail. In this specification, "about" means the value following it ±10%.

[0216] (definition)

[0217] In this specification, "corneal endothelial cells" is used in its common sense as used in the art. The cornea is one of the layered tissues that make up the eye; it is transparent and located closest to the outside. In the human body, the cornea consists of five layers from the outermost layer (body surface): corneal epithelium, Bowman's membrane, lamina propria, Desmere membrane (corneal endothelial basement membrane), and corneal endothelium. Unless otherwise specified, the portion other than the epithelium and endothelium is sometimes referred to as the "corneal parenchyma," and this is also the term used in this specification. In this specification, "HCEC" (human corneal endothelial cells) is an abbreviation for human corneal endothelial cells.

[0218] In this specification, “corneal endothelial-like cells” refers to cells differentiated from stem cells, such as iPS cells, and possessing the same functional properties as corneal endothelial cells. Methods for differentiating stem cells, such as ES cells and iPS cells, 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). In a typical example, simply put, iPS cells are seeded on 35 mm matrix gel-coated plates (cornering plates divided into 12 plates) on day 0 using a 1:12 dilution of cell dissociation buffer (Life Technologies). The iPS cells are then allowed to proliferate in culture medium (mTeSR1; STEMCELL Technologies Inc.) for 4 days. On day 4, the mTeSR1 medium was replaced with Smad inhibitor medium, which was prepared by adding 500 ng / mL human recombinant head protein (R&D Systems, Minneapolis, MN, USA) and 10 μM SB431542 (MilliporeSigma) to a basal medium of 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).On day 6, the Smad inhibitor medium was replaced with corneal medium, which was prepared as a basal medium of 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mL M glutamine (STEMCELL Technologies, inc.), 0.1 mM β-mercaptoethanol (Millipore Sigma), and 8 ng / mL βFGF (Millipore Sigma) containing 0.1 × B27 nutrients (Life Technologies), 10 ng / mL recombinant human platelet-derived growth factor-BB (PDGF-BB; PeproTech, Rocky Hill, NJ, USA), and 10 ng / mL recombinant human Dickkopf-associated protein-2 (DKK-2; R&D Systems). On day 7, differentiating CECs were transferred to new streptavidin-coated plates (35 mm) and allowed to proliferate in corneal medium for 13 days. Differentiated CECs were recovered on day 20. The above examples are typical, and those skilled in the art can also use other methods 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). In addition, those skilled in the art can appropriately adjust the conditions of methods known in the art to produce corneal endothelial-like cells.

[0219] "Corneal endothelial cells" and "corneal endothelioid cells" can also contain magnetic materials (e.g., iron). For example, when corneal endothelial cells containing magnetic materials are injected into the anterior chamber, magnetic forces can be used to promote proximity and adhesion to the inner side of the cornea (e.g., Desmere membrane) (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). "Magnetic materials" refer to substances that can be magnetized by a magnetic field, such as iron, cobalt, nickel, ferrite, etc.

[0220] In this specification, "adhesion culture" refers to culturing cells by causing them to adhere to a container. Examples of containers used for adhesion culture include those that have undergone cell culture (TC) treatment.

[0221] In this specification, "low adhesion" means that cells hardly adhere to the container during cell culture or preservation. Low-adhesion surface containers include those coated with hydrogel (e.g., via covalent bonding) and / or coated with silica (SiO2). Other examples include glass containers with IRAS-treated surfaces. IRAS-treated tubular vials are available from Iwata Glass Industrial Co., Ltd. (Osaka). IRAS treatment suppresses the leaching of alkali from borosilicate glass. Treatment methods other than IRAS treatment can also be used in this invention as long as alkali leaching is suppressed. For example, coating treatments using LIPIDURE (registered trademark)-CM5206, LIPIDURE (registered trademark)-CR2001, LIPIDURE (registered trademark)-CR3001, LIPIDURE (registered trademark)-PC, and LIPIDURE (registered trademark)-NH01 provided by Nippon Oil (https: / / www.nof.co.jp / business / life / product04.html). Low-adhesion surface containers are sometimes commercially available as suspension culture containers. Suspension culture containers, provided they have undergone surface treatments that prevent cell adhesion, are considered low-adhesion surface containers in this specification. In addition, silicon coating, silicon processing (silica film coating), and sulfide treatment can also be used.

[0222] In this specification, "untreated surface" means without surface treatment; "untreated surface container" refers to a container that, like a low-adhesion surface container, allows cells to be cultured or preserved without adhering to the container. Untreated surface containers are sometimes also commercially available as suspension (floating) culture containers; suspension (floating) culture containers that allow cells to be cultured or preserved without adhering to the container without surface treatment can also be considered untreated surface containers.

[0223] In this specification, "preservation" of cells means the holding of cells in a container for a certain period of time (typically at least 6 hours, but not limited to) for any purpose (e.g., cell infusion therapy, or transportation for that purpose), meaning maintaining cell function in a container without causing cell proliferation. Preservation is different from "culture" for the purpose of cell proliferation. Furthermore, preservation does not mean transferring cells to a syringe or other container shortly before administration; nor does it mean temporarily holding them in a container for use preparation prior to administration.

[0224] In this specification, "a container containing cells" means a container containing cells (representing corneal endothelial cells or corneal endothelial-like cells) and representatively possesses the features disclosed in this specification.

[0225] (Preferred Implementation)

[0226] The following description describes preferred embodiments, but these embodiments are illustrative of the invention and should be understood as not limiting the scope of the invention to these preferred embodiments. Those skilled in the art should also understand that changes, modifications, etc., can be easily made within the scope of the invention by referring to the following preferred embodiments. Any combination of these embodiments can be suitably combined by those skilled in the art.

[0227] (Save method)

[0228] In one application, the present invention provides a method for preserving corneal endothelial cells and / or corneal endothelioid cells, as well as various techniques (e.g., including containers) required for the preservation method; the method includes the step of preserving the corneal endothelial cells and / or corneal endothelioid cells in a container having a specific bottom area.

[0229] In one embodiment, the bottom area of ​​the container used in the method of the present invention may be at least about 0.7 cm². 2 It can typically be about 0.7 to about 4 cm. 2 I don't want to be bound by theory, but the base area is less than 0.7 cm². 2 In the case of containers (e.g., 12-well plates), high cell viability cannot be achieved; while in containers with a bottom area greater than 4 cm², high cell viability cannot be achieved. 2In the case of containers (e.g., 48-well plates), when a cell suspension suitable for corneal endothelial cell infusion therapy or a liquid for cell preservation (e.g., about 300 μl) is added to the container, the liquid level becomes too low to be suitable for preservation. In one embodiment, the height of the liquid for cell preservation is preferably about 0.5 mm or more, about 0.6 mm or more, about 0.7 mm or more, about 0.75 mm or more, about 0.8 mm or more, about 0.9 mm or more, about 1.0 mm or more, about 1.2 mm or more, about 1.4 mm or more, about 1.6 mm or more, about 1.8 mm or more, or about 2 mm or more. In another embodiment, the liquid level for cell preservation can be about 1.0 mm or less, about 1.5 mm or less, about 1.8 mm or less, about 2 mm or less, about 3 mm or less, about 4 mm or less, about 5 mm or less, about 6 mm or less, about 7 mm or less, about 8 mm or less, about 9 mm or less, about 10 mm or less, about 15 mm or less, or about 20 mm or less. In some embodiments, the bottom area of ​​the container used in the method of the present invention can be about 0.7 to about 3.5 cm². 2 Approximately 0.7 to 3 cm 2 Approximately 0.7 to 2.5 cm 2 Approximately 0.7 to 2.0 cm 2 Approximately 1 to 4 cm 2 Approximately 1 to 3.5 cm 2 Approximately 1 to 3 cm 2 Approximately 1 to 2.5 cm 2 Approximately 1 to 2 cm 2 Approximately 1.5 to 3 cm 2 Approximately 1.5 to 4 cm 2 Approximately 1.6 to 2.2m 2 or approximately 1.8 to approximately 2m 2 When the amount of corneal endothelial cell suspension stored exceeds the amount usable in corneal endothelial cell infusion therapy, there is no particular upper limit to the bottom area of ​​the container. For example, within the range of bottom areas, an upper limit could be set at approximately 4.5 cm². 2 Approximately 5cm 2 Approximately 5.5cm 2 Approximately 6cm 2 Approximately 7cm 2 Approximately 8cm 2 Approximately 9cm 2 Approximately 10cm 2 Approximately 15cm 2 Approximately 20cm 2 Approximately 25cm 2 Approximately 30cm 2 Approximately 40cm 2 Approximately 50cm2 Approximately 60cm 2 Approximately 70cm 2 Approximately 80cm 2 Approximately 90cm 2 Approximately 100cm 2 The preferred bottom area of ​​the container is approximately 1.5 to approximately 3 cm². 2 More preferably about 2cm 2 In a specific embodiment, the bottom area of ​​the container is approximately 1.88 cm². 2 .

[0230] Corneal endothelial cells and corneal endothelioid cells preserved using the method of this invention are cells that can be used in clinical applications such as cell infusion therapy. The preservation method of this invention maintains high viability and cell function, and the corneal endothelial cells and corneal endothelioid cells can be maintained in a cell suspension state (including a state where cells do not adhere to the bottom of the container and can be easily dispersed by gentle pipetting), a so-called "ready-to-use" state. Therefore, corneal endothelial cells and corneal endothelioid cells preserved using the method of this invention can be directly administered without further processing (such as peeling / suspending cells from the preservation container using pharmaceutical treatment, separating specific cells from cell clusters using instruments, etc.) or culture. Therefore, this invention provides cells, containers, and cell preparations contained in containers that can be administered without further processing or with only minimal operation, and can be used as cell preparations.

[0231] In a specific embodiment, cells preserved by the method of the present invention can be directly administered to the subject without undergoing cell culture or static incubation for more than 24 hours, 18 hours, 12 hours, preferably more than 6 hours (or any other time unit) for the purpose of proliferation and / or redifferentiation.

[0232] Therefore, in this context, the present invention provides a method for preserving a cell preparation containing corneal endothelial cells and / or corneal endothelioid cells, the method comprising: a step of preserving the cell preparation containing corneal endothelial cells and / or corneal endothelioid cells in a container having a specific bottom area; and also providing a cell preparation preserved by the preservation method or capable of being preserved, and a method for disposal or prevention using the preserved cell preparation.

[0233] In some embodiments, corneal endothelial cells and / or corneal endothelioid cells can be preserved for at least about 1 hour, at least about 2 hours, at least 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. While not intended to be limiting, the preservation method of the present invention is intended for the preservation of cell preparations delivered to medical institutions without a cell culture center (CPC); therefore, the preservation time can take into account the time spent on delivery, the waiting time until delivery, etc., preserving corneal endothelial cells and / or corneal endothelioid cells for, for example, at least about 3 hours, at least about 6 hours, or longer depending on the delivery distance. In specific embodiments, the preservation method of the present invention can preserve cells for a maximum of about 72 hours, a maximum of about 96 hours, or a maximum of about 120 hours. While not bound by theory, the preservation method of the present invention has shown a survival rate of approximately 80% after approximately 72 hours of preservation; approximately 57% after approximately 96 hours (data not shown); and even after approximately 120 hours of preservation, a survival rate of at least approximately 30% can be predicted. In some embodiments, corneal endothelial cells and / or corneal endothelioid cells can be preserved in a cell suspension state for the aforementioned time. Surprisingly, as shown in the examples, corneal endothelial cells and / or corneal endothelioid cells can be preserved in a suspension state for a certain period of time (e.g., approximately 6 hours) rather than in an adherent state. While not bound by theory, when cells are preserved in a suspension state, the preserved cells show reduced tightness of adhesion compared to cells cultured in an adherent state or cells cultured / preserved while forming aggregates. Tightness of adhesion can be measured using indicators such as ZO-1. In some embodiments, the preservation method of the present invention does not involve the use of a scaffold (a substrate, etc., that promotes cell adhesion or aggregate formation).

[0234] In some embodiments, preservation may also be accompanied by transportation. The preservation method of the present invention can withstand vibrations during transportation. Transportation can be land transportation, air transportation, or any other type of transportation.

[0235] The cell density of the cells preserved in the method of this invention is typically about 2 × 10⁻⁶. 4 Cells / ml or higher. While it's desirable not to be bound by theory, in cases used for cell infusion, too low a cell density will not yield therapeutic effects; conversely, too high a cell density increases cell weight, potentially accelerating cell death during preservation. Therefore, typically, a cell density of approximately 2 × 10⁻⁶ cells / ml is recommended. 4 10 cells / ml ~ approximately 8 × 10 7 The appropriate range is within the range of cells / ml, preferably about 2×10. 4 10 cells / ml ~ approximately 8 × 10 7 10 cells / ml, more preferably about 2×105 10 cells / ml ~ approximately 8 × 10 6 1 x 10⁻⁶ / ml, more preferably about 1 × 10⁻⁶ / 6 10 cells / ml ~ approximately 8 × 10 6 pcs / ml, optimally approximately 2×10 6 pcs / ml ~ approximately 4×10 6 Cells / ml. Anyone skilled in the art can determine the appropriate cell density based on the intended use.

[0236] The method of the present invention can preserve corneal endothelial cells while maintaining a high cell count and high cell viability from the start of preservation. For example, when all cells at the start of preservation are set to 100%, the method of the present invention can achieve a total cell count of at least about 30% and a viable cell ratio (viable cells / viable cells + dead cells) of about 70% or more after preservation, preferably about 50% and a viable cell ratio of about 80% or more, and most preferably about 70% and a viable cell ratio of about 90% or more after preservation. In a specific embodiment, the cell viability (the ratio of viable cells to the total cells at the start of preservation) of the method of the present invention is preferably about 30% or more, more preferably about 60% or more, further preferably about 80% or more, and most preferably about 90% or more. In a further specific embodiment, based on the above-mentioned cell viability, the viable cell ratio (viable cells / viable cells + dead cells) is preferably about 70% or more, more preferably about 80% or more, and most preferably about 90% or more.

[0237] The bottom area refers to the area of ​​the surface referred to as the bottom surface of the container. The method of this invention also attempts to preserve the syringe in a horizontally laid-down position; however, in this case, the bottom area of ​​the container refers to the area of ​​the gravity-bearing surface of the cylindrical curved surface in contact with the cell suspension. Furthermore, the same applies when at least a portion of the bottom surface is bent or tilted, in addition to the syringe being laid horizontally; the area of ​​the gravity-bearing surface in contact with the cell suspension is used as the bottom area. While it is desirable not to be bound by theory, it has been found in this invention that it is important to consider the effects of placing cells on a gravity-bearing localized surface, and that defining the bottom area based on this can improve cell preservation.

[0238] In this invention, the storage temperature can be any range, as long as it does not freeze or denature the cells, such as approximately 0°C to approximately 50°C. This is because cell viability decreases whether the temperature is too high or too low. Those skilled in the art can appropriately determine the preferred storage temperature. In some embodiments, the storage temperature can be approximately 0°C, approximately 1°C, approximately 2°C, approximately 3°C, approximately 4°C, approximately 5°C, approximately 6°C, approximately 7°C, approximately 8°C, approximately 9°C, approximately 10°C, approximately 11°C, approximately 12°C, approximately 13°C, approximately 14°C, approximately 15°C, approximately 16°C, approximately 17°C, approximately 18°C, approximately 19°C, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, or approximately 24°C. The storage temperature range is approximately 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C. The preferred storage temperature range is approximately 12°C to approximately 42°C, more preferably approximately 17°C to approximately 39°C, and even more preferably approximately 27°C to approximately 37°C. In a preferred embodiment, the storage temperature can be room temperature or approximately 37°C. In the most preferred embodiment, the storage temperature can be about 37°C, but it is not limited to this. Those skilled in the art will understand that temperature fluctuations of a few degrees Celsius (e.g., ±1°C, ±2°C, ±3°C, ±4°C, ±5°C, etc.) are permissible during storage / transport. Preferably, the temperature variation is based on 37°C and is ±3°C.

[0239] Corneal endothelial cells can be derived from mammals (humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, horses, sheep, monkeys, etc.), preferably from primates, and especially preferably from humans.

[0240] In some embodiments, the container is not surface-treated for adhesion culture. In some embodiments, the container is a low-adhesion surface container or an untreated surface container. In some embodiments, the container is made of polystyrene, polypropylene, or glass. In specific embodiments, examples of containers include, but are not limited to, 24-well plates, tubular bottles, syringes, and culture dishes.

[0241] The container provided in this invention can preserve cells without causing cell adhesion, or even if cell adhesion occurs but it does not affect the standards for use as a cell preparation, it can be made of either oxygen-permeable or non-oxygen-permeable materials. Examples of oxygen-permeable materials include polyethylene and any other oxygen-permeable materials. Alternatively, oxygen-permeable materials can be processed using methods known in the art to make them permeable.

[0242] In this invention, the preservation solution for corneal endothelial cells or corneal endothelioid cells can be any known preservation solution available in the art, or a newly provided composition, provided that it is suitable for preservation. Examples of usable preservation solutions include, but are not limited to, OptiMEM-I (registered trademark) (Thermo Fisher Scientific), MEM, DMEM, M199, corneal endothelial cell preservation solution (preservation solution used in this specification, Generation and Feasibility Assessment of a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction. Okumura N, Kakutani K, Inoue R, Matsumoto D, Shimada T, Nakahara M, Kiyanagi Y, Itoh T, Koizumi N. PLoS One. 2016 Jun 29; 11(6): e0158427. doi: 10.1371 / journal.pone.0158427.eCollection2016.PMID: 27355373).

[0243] In some embodiments, the liquid volume during storage is 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, which can be appropriately varied depending on the purpose. As a product specification, it can be, for example, ± about 5%, ± about 10%, ± about 15%, ± about 20%, ± about 25%, ± about 50% of a reference volume (e.g., 300 μl). For example, the volume of liquid for preservation can be at least about 50 μl, such as 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, in cell infusion therapy, when intended to be injected into both eyes, the product specification can be 2, 3, or 4 times the administered amount. Even when injection into both eyes is not desired, to prevent administration failure, the product specification can also be 2, 3, or 4 times the administered amount. The range of liquid volumes can be a suitable combination of the above values. In some embodiments, corneal endothelial cells and / or corneal endothelial-like cells can be preserved in the form of a cell suspension. The above-mentioned liquid volume can be the volume of the cell suspension.

[0244] (container)

[0245] In another aspect, the present invention provides a container with a specific bottom area for storing corneal endothelial cells or corneal endothelial-like cells. The specific features of the container can be achieved using any of the embodiments described above, particularly (the storage method). It is desirable not to be bound by theory, but rather to provide a container with a specific bottom area, processed to suit the storage of corneal endothelial cells or corneal endothelial-like cells, thereby enabling the provision of ready-to-use cell preparations in the present invention.

[0246] (A container containing cells)

[0247] In another embodiment, the present invention is a container containing cells, comprising: corneal endothelial cells or corneal endothelial-like cells; and a container for storing the corneal endothelial cells or corneal endothelial-like cells; the container used herein is a container with a specific bottom area. Specific features of the container may be achieved through any of the embodiments described above, particularly (the storage method).

[0248] In some embodiments, the container of the present invention may contain a Rho kinase (ROCK) inhibitor. The ROCK inhibitor used herein may be any of the embodiments described in this specification.

[0249] (corneal endothelial cells)

[0250] In another embodiment, the present invention provides corneal endothelial cells or corneal endothelioid cells preserved by the above-described method. In some embodiments, the corneal endothelial cells or corneal endothelioid cells of the present invention can be used to treat or prevent corneal endothelial disorders, diseases, or symptoms in a subject.

[0251] In a further embodiment, the present invention provides a composition for treating or preventing corneal endothelial disorders, diseases, or symptoms, comprising corneal endothelial cells or corneal endothelial-like cells preserved by the methods described above.

[0252] In some implementations, the corneal endothelial impairment, disease, or symptom is selected from the group consisting of Freund's keratinocyte dystrophy, post-corneal transplant impairment, corneal endotheliitis, trauma, ophthalmic surgery, post-laser ophthalmic surgery impairment, aging, posterior polymorphic dystrophy (PPD), congenital hereditary corneal endothelial dystrophy (CHED), idiopathic corneal endothelial impairment, and cytomegalovirus corneal endotheliitis.

[0253] In some embodiments, the compositions of the present invention may contain a Rho kinase (ROCK) inhibitor, or may be administered in combination with a ROCK inhibitor. The corneal endothelial cells or corneal endothelioid cells of the present invention may also be administered in combination with a ROCK inhibitor. Examples of ROCK inhibitors include: US Patent 4,678,783, Japanese Patent No. 3,421,217, International Publication Nos. 95 / 28387, 99 / 20620, 99 / 61403, 02 / 076976, 02 / 076977, 2002 / 083175, 02 / 100833, 03 / 059913, 03 / 062227, 2004 / 009555, 2004 / 022541, and 2... The compounds disclosed in International Publication No. 004 / 108724, International Publication No. 2005 / 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, and International Publication No. 2007 / 026664, etc. These compounds can be manufactured by the methods described in the respective invention documents. Specific examples include 1-(5-isoquinolinylsulfonyl)piperazine or its salts (e.g., fasudil (1-(5-isoquinolinylsulfonyl)piperazine)), (+)-cis-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane ((R)-(+)-cis-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) or its salts (e.g., Y-27632 ((R)-(+)-cis-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) etc.), and commercially available products (Wako Pure Chemical Industries, Ltd., Asahi Kasei Pharma Corporation, etc.) are also preferred.

[0254] In some embodiments, examples of usable ROCK inhibitors include Y-27632 ((+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane), ripasudil (4-fluoro-5-{[(2S)-2-methyl-1,4-diazepam-1-yl]sulfonyl}isoquinoline), fasudil (1-(5-isoquinolineylsulfonyl)piperazine), and pharmaceutically permissible salts thereof, but are not limited thereto. For example, US4678783, Japanese Patent 3421217, WO99 / 20620, WO99 / 61403, WO02 / 076976, WO02 / 076977, WO02 / 100833, WO Other ROCK inhibitors, including compounds disclosed in WO2004 / 009555, WO2004 / 022541, WO2004 / 108724, WO2005 / 003101, WO2005 / 039564, WO2005 / 034866, WO2005 / 037197, WO2005 / 037198, WO2005 / 035501, WO2005 / 035503, WO2005 / 035506, WO2005 / 080394, WO2005 / 103050, WO2006 / 057270, WO2007 / 026664, etc. (but not limited to these).

[0255] (Cellular preparations)

[0256] In a further development, the present invention provides a cell preparation comprising corneal endothelial cells and / or corneal endothelial-like cells, and a container. A cell preparation is provided in a container suitable for storing the corneal endothelial cells and / or corneal endothelial-like cells, the container having a base area of ​​at least about 0.7 cm². 2The corneal endothelial cells and / or corneal endothelioid cells in the formulation are maintained, and the number of viable cells is hardly reduced. Therefore, the cell formulation of the present invention is a so-called ready-to-use formulation that can be directly administered. Such a formulation can be used for cell infusion therapy. In some embodiments, the cell formulation of the present invention can be used to treat or prevent corneal endothelial disorders, diseases, or symptoms in a subject. Examples of corneal endothelial disorders, diseases, or symptoms include, but are not limited to, Flexner's keratitis, post-corneal transplant disorder, keratitis, trauma, post-ophthalmic surgery disorder, post-laser ophthalmic surgery disorder, aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus keratitis.

[0257] (Treatment methods)

[0258] In a further embodiment, the present invention provides a method for treating or preventing corneal endothelial disorders, diseases, or symptoms in a subject, comprising the steps of administering corneal endothelial cells or corneal endothelial-like cells preserved by the method of the present invention to the subject.

[0259] The therapeutic effect is reduced when the number of cells administered is too small, therefore it can be at least about 40,000. In cases where the corneal endothelial impairment, disease, or symptom is localized, a smaller number than usual may be used, for example, at least 40,000, at least about 100,000, and preferably at least about 200,000. In some embodiments, the number of cells administered can be from about 40,000 to about 4,000,000, preferably from about 100,000 to about 2,000,000, more preferably from about 200,000 to about 1,400,000, and most preferably from about 400,000 to about 1,000,000.

[0260] The volume of liquid given can be appropriately set taking into account suitable viscosity, the allowable amount that can be given to the given site (e.g., the anterior chamber), etc. In some embodiments, a volume of liquid (cell suspension) of about 20 μL to about 500 μL, preferably about 30 μL to about 400 μL, more preferably about 50 μL to about 400 μL, and most preferably about 200 μL to about 300 μL can be given.

[0261] (application)

[0262] In a further embodiment, the present invention provides the use of corneal endothelial cells or corneal endothelioid cells preserved by the method of the present invention in the manufacture of a medicament for the treatment or prevention of corneal endothelial impairment, disease, or symptoms in a subject.

[0263] The present invention has been described above with reference to preferred embodiments for easy understanding. The invention will now be described based on examples. The above description and the following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Therefore, the scope of the invention is not limited to the specific embodiments and examples described in this specification, but only to the scope of the claims.

[0264] Example

[0265] The present invention will be described in more detail below based on embodiments. It will be understood that, in addition to the reagents specifically illustrated, reagents available from Sigma-Aldrich, BASF Japan Ltd., etc., may also be used in this embodiment. Human tissue was processed based on the ethical guidelines of the Declaration of Helsinki. Human donor corneas were supplied by SightLife. TM Provided by (Seattle, WA). For donations of eyes for research purposes, corneas were collected in accordance with the Uniform Cadaver Donation Act (UAGA) of the state, with written consent obtained from the close relatives of the deceased donor. Experiments with rabbits were conducted based on guidelines approved by the Doshisha University Animal Experimentation Committee (Authorization No. A18003).

[0266] (Example 1: Selection of materials and sizes for containers in cell preservation)

[0267] (Materials and methods)

[0268] (Culture of corneal endothelial cells)

[0269] Five donor corneas were obtained from donors aged 40 and over with the disease. All corneas were stored at 4°C in Optisol (ChironVision, Irvine, CA) for no more than 14 days prior to use. Human corneal endothelial cells (HCECs) were cultured as follows. Briefly, the Desmere membrane containing the corneal endothelium was mechanically detached from the donor cornea and digested by incubation at 37°C in 1 mg / mL collagenase A (Roche Applied Science, Penzberg, Germany) for 12 hours. After washing with OptiMEM-I (Life Technologies Corp., Carlsbad, CA), the HCECs were seeded into one well of a 48-well plate coated with a laminin E8 fragment (iMatrix-511; Nippi, Incorporated, Tokyo).

[0270] The culture medium was prepared as follows. In short, OptiMEM-I supplemented with 8% bovine serum peptide (FBS), 5 ng / mL epidermal growth factor (EGF; Thermo Fisher Scientific), 20 μg / mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), 200 mg / L calcium chloride, 0.08% chondroitin sulfate (Sigma-Aldrich), and 50 μg / mL gentamicin (Thermo Fisher Scientific) was acclimated to NIH-3T3 for 24 hours. The acclimated medium was then recovered and filtered through a 0.22 μm filter unit (EMD Millipore Corporation, Billerica, MA) for use as the HCEC medium.

[0271] HCECs were cultured at 37°C under a humidified atmosphere containing 5% CO2, with the culture medium changed three times a week. In the passage culture of HCECs, cells were trypsinized for 5 minutes at 37°C using TrypLE Select Enzyme (10X) (Thermo Fisher Scientific) and seeded at a 1:2 ratio. In this experiment, HCECs that had been passaged for 9 generations starting from passage 5 were used.

[0272] Use without Ca 2+ Mg 2+ HCECs were washed with phosphate-buffered saline (PBS) and digested with TrypLE Select Enzyme (10X) at 37°C for 15 minutes. After being removed from the culture plate, the cells were washed twice, centrifuged at 280G for 3 minutes, and resuspended in OptiMEM-I. HCECs were then cultured in serum-free medium (cell therapy medium provided by Cell Science Research Institute (Miyagi), Co., Ltd.) at 1.0 × 10⁶ cells / mL. 6Cells were stored at a density of 300 μl at 4°C or 37°C in suspension for 72 hours. To screen for the size and material used for HCEC preservation, the following cell culture plates, tubes, and flasks were used: 24-well plates (ultra-low adhesion) (Corning Inc., Corning, New York), 24-well plates (cell culture) (Corning Inc., Corning), 24-well plates (untreated) (AGC TECHNOGLASS Co., Ltd., Shizuoka), 48-well plates (suspension culture) (Sumitomo Bakelite Co., Ltd.), 96-well plates (ultra-low adhesion, round bottom) (Corning Inc.), 96-well plates (ultra-low adhesion, flat bottom) (Corning Inc.), 15 ml conical tubes (ultra-low adhesion) (Sumitomo Bakelite Co., Ltd., Tokyo), 2 ml cryovials (Corning Inc., Corning), and 10 ml glass flasks (Maruemu Corporation., Osaka) (Table 1).

[0273] [Table 1]

[0274]

[0275] After 72 hours of storage, HCECs were slowly recovered from cell culture plates, test tubes, or tubular flasks by pipetting, and centrifuged at 280G for 3 minutes at 1.0×10⁻⁶. 6 Cells were resuspended in cell therapy medium at a density of 600 μl. Cell viability was calculated by staining dead cells with 0.5% trypan blue (NACALAITESQUE, INC. Kyoto). As a control, HCECs recovered by trypsin digestion with TrypLE (trademark) Select Enzyme (10X) were centrifuged at 280 G for 3 minutes and resuspended in cell therapy medium. Cell viability was then evaluated immediately without preservation in cell suspension.

[0276] (result)

[0277] (The effect of cell suspension preservation conditions on cell viability)

[0278] HCECs were cultured in CTV in suspension form at a concentration of 1.0 × 10⁻⁶ cells. 6 Cells were stored at a density of 300 cells / 300 μl for 72 hours. Cells were collected by gentle pipetting, and cell viability was evaluated. Figure 1A). Phase-contrast images showed that 80% to 90% of the area on the adhesion culture plates was covered by HCECs after smooth pipetting for cell recovery. On the other hand, almost no cells were observed in any of the 24-well, 48-well, and 96-well plates used for suspension cell culture. Figure 1 B). Surface treatments used for adhesion culture are not suitable for preserving HCECs in suspension form. Therefore, in this example, the effect of the size and shape of commercially available culture plates and tubes on cell viability was evaluated. In the control group, immediately after trypsin digestion and collection from the culture plate, the ratio of viable cells to dead cells was 90.3% and 9.7%, respectively; in 24-well plates (ultra-low adhesion), the ratios were 82.6% and 10.1% (relative to the original number of cells preserved). However, the proportion of viable cells was significantly reduced compared to the controls in 48-well plates (suspension culture), 96-well plates (ultra-low adhesion, flat bottom), 96-well plates (ultra-low adhesion, round bottom), 15 ml conical tubes (ultra-low adhesion), and 2 ml cryovials (p < 0.01). Figure 2 A). Perhaps due to cell adhesion to the bottom or wall of the culture plate or test tube, the number of cells collected was significantly reduced after preservation in 48-well plates (suspension culture), 15 ml conical tubes (ultra-low adhesion), and 2 ml cryovials. The inventors further evaluated the effect of temperature on cell viability, showing that 95.1% of cells survived at 37°C; in 24-well plates (ultra-low adhesion) at 4°C, almost no cells were collected after preservation due to significant cell death. Figure 2 B). Other culture plates or tubes were also evaluated without surface treatments for adhesion culture. Similar to the ultra-low adhesion in 24-well plates, untreated 24-well plates and 10 ml glass tubular vials (approximately 1.9 cm) were also evaluated. 2 Similarly, the underground area is approximately 2.7 cm². 2 In this study, a cell viability rate of over 90% was maintained. Figure 2 C); volumes in between did not significantly alter cell viability. Figure 2 D).

[0279] HCECK cells preserved in suspension in 24-well plates (ultra-low adhesion) were seeded onto culture plates after 72 hours of storage at 37°C. Representative phase-contrast images showed that, like the control, the preserved HCECs adhered to the culture plates 3 hours after seeding without significant cell death. After 2 weeks, the preserved HCECs formed the same polygonal monolayer sheet-like structure as the control. Figure 2 E).

[0280] (Example 2: Injection of RCEC into a rabbit corneal endothelial compensation disorder model)

[0281] (Materials and methods)

[0282] (Rabbit CEC culture)

[0283] In this embodiment, 10 rabbit eyes purchased from Funakoshi Co., Ltd. (Tokyo) were used. Rabbit CECs (RCECs) were cultured as described above. Briefly, the stripped Desmee membrane containing RCECs was incubated at 37°C in 0.6 U / mL Accutase (NACALAI TESQUE, INC., Kyoto) for 15 minutes, and the recovered RCECs were inoculated onto culture plates coated with FNC Coating Mix (registered trademark) (Athena Environmental Sciences, Inc., Baltimore, MD). The RCECs were cultured in Durbecco's modified Eagle's medium (Life Technologies Corp., Carlsbad, CA) supplemented with 10% peptide bovine serum (FBS), 50 U / mL penicillin, 50 μg / mL streptomycin, and 2 ng / mL fibroblast growth factor 2 (Life Technologies Corp.). Cultured RCECs that had undergone three passages from generation 1 were used.

[0284] (Injection of RCEC into a rabbit corneal endothelial compensation disorder model)

[0285] The right eye of 12 Japanese White Rabbits was used in the experiment, while the left eye was not used to avoid blindness. The rabbit corneal endothelial compensation model was created as described above. In short, to deepen the anterior chamber depth, the lens was removed one week prior, and the corneal endothelium was mechanically removed using a 20G silicone needle (Soft Tapered Needle, Inami & Co., Ltd., Tokyo). The Desmay membrane was stained with 0.1% trypan blue to confirm complete detachment of the corneal endothelium from the Desmay membrane.

[0286] The cultured RCECs were washed with PBS, digested with 0.05% Trypsin-EDTA (Life Technologies) at 37°C for 5 minutes, and then neutralized with culture medium. The RCECs were washed three times and cultured in 24-well plates (ultra-low adhesion) at a concentration of 1.0 × 10⁻⁶. 6 CTV cells were stored at a density of 1 cell / 300 μl in suspension. After storage for 24, 48, or 72 hours, RCEC (1.0 × 10⁻⁶ cells / 300 μl) was prepared for injection into rabbit eyes. 6600 μl of CTV and 100 μM of Y-27632 were added to RCEC (1.0 × 10⁻⁶). 6 The mixture of 5.0 × 10⁻⁶ CTV (200 μM Y-27632 (WakoPure Chemical Industries, Ltd.) / 300 μL CTV) was slowly mixed with the ROCK inhibitor (200 μM Y-27632 / 300 μL CTV). The mixture was then injected into the anterior chamber of the corneal endothelial compensation model using a 26G syringe. 5 RCECs and 300 μl CTV containing 100 μM Y-27632 were used. Rabbits were under general anesthesia and kept in an endothelial-faced position for 3 hours. As a control, CTV containing Y-27632 (final concentration: 100 μM) was injected into the anterior chamber of the corneal endothelial compensation model. Three rabbit eyes were used for each group (control, cell preservation at 24, 48, and 72 hours).

[0287] The anterior chamber of the eye was evaluated using a slit-lamp test for 14 days. Sham images were obtained using a Pentacam (registered trademark) (OCULUS Optikgerate GmbH, Wetzlar, Germany), and the corneal volume (7 mm diameter) was also measured using Sham images. The central corneal thickness was measured using an ultrasonic pachymeter (SP-2000; Tomey, Nagoya). Corneal thickness that could not be measured due to severe edema was considered to be the instrument's maximum reading of 1200 μm.

[0288] (Immunofluorescence and actin staining)

[0289] Rabbit corneas were collected and immobilized with 4% formaldehyde for 10 minutes at room temperature. The samples were then incubated at 37°C with 1% bovine serum albumin for 45 minutes, followed by incubation with a sodium-targeting agent. + / K +Antibodies against ATPase (1:300, Upstate Biotechnology, Lake Placid, NY), ZO-1 (1:300, Life Technologies Corp., Carlsbad, CA), and N-cadherin (1:300, BD Biosciences, San Jose, CA) were incubated overnight at 4°C. After washing the samples three times with PBS, they were incubated for 60 minutes at room temperature with Alexa Fluor 488-labeled goat anti-mouse (1:1000, Life Technologies). After incubation, the samples were stained with actin for 60 minutes at room temperature using Alexa Fluor 488-labeled phalloidin (1:400, Life Technologies). Cell nuclei were stained with DAPI (Dojin Chemical Research Institute, Kumamoto). The samples were observed under a fluorescence microscope (TCS SP2AOBS; Leica Microsystems, Wetzlar, Germany).

[0290] (Statistical Analysis)

[0291] Statistical significance (p-value) in comparisons of multiple sample groups was calculated using the Kruskal-Wallistest test. Results are expressed as mean ± standard deviation. A p-value less than 0.05 was considered statistically significant.

[0292] (result)

[0293] (Preserving the feasibility of implementing RCEC cell therapy in rabbit models)

[0294] Recurrent corneal epithelial cells (RCECs) were collected and cultured, and stored in 24-well plates (ultra-low adhesion) at 37°C for 24, 48, or 72 hours in their cultured form. Before injection into the rabbit model, Y-27632 was added to the RCECs, which were then injected into the anterior chamber of the rabbit corneal endothelial compensation model. In slit-lamp experiments, corneal transparency was observed within 7 days in eyes where RCECs were injected after 24, 48, or 72 hours of storage. Figure 3 A). In the control eye, the entire cornea is opaque due to corneal endothelial decompensation. In the Sham images, injection of RCEC preserved for 24, 48, and 72 hours showed successful regeneration of the anatomically normal cornea, while the control eye exhibited severe corneal edema. Figure 3 B).

[0295] Color images of corneal thickness obtained using Pentacam (trademark) show that RCEC stored for 24 and 48 hours regenerated normal corneal thickness from the center to the periphery 14 days after injection. On the other hand, in eyes injected with RCEC stored for 72 hours, the cornea was transparent but thickened in slit-lamp tests. Figure 3 (A and 4A). Ultrasonic pachymetry showed that in eyes injected with RCEC stored for 24 and 48 hours, the central corneal thickness was approximately 400 μm (normal range) after 10 days. However, in eyes injected with RCEC stored for 72 hours, the reduction in central corneal thickness was less compared to eyes injected with RCEC stored for 24 or 48 hours, and significantly higher throughout the 14 days (p < 0.01). Figure 4 B). Similar to the central corneal thickness, the corneal volume determined by Scheimpflug imaging was smaller in eyes injected with RCEC stored for 24 or 48 hours compared to eyes injected with RCEC stored for 72 hours. Figure 4 C). Regarding the cell density of regenerated corneal endothelium evaluated using actin and DAPI, it was 2465 cells / mm² in eyes injected with RCEC preserved for 24 hours. 2 In the eyes of RCEC, which has been injected and stored for 48 hours, the number is 2368 / mm. 2 However, the cell density of eyes injected with RCEC preserved for 72 hours was 1548 cells / mm². 2 Compared to eyes injected with RCEC preserved for 24 or 48 hours, the values ​​were significantly lower (p < 0.05). Figure 4 D).

[0296] Immunofluorescence staining confirmed that the functional marker Na was expressed in the lateral membranes of all regenerated CECs in the eyes of RCECs stored for 24, 48, and 72 hours. + / K + -ATPase (a marker of pump function), ZO-1 (a tight-binding marker), and N-cadherin (an adhesion-binding marker). Actin staining showed that regenerated corneal endothelium formed polygonal monolayer sheet-like structures. On the other hand, in the control eyes, due to corneal endothelial decompensation, almost no cells expressing functional markers associated with fibrosis were observed. Figure 4 E).

[0297] (Example 3: Preservation of corneal endothelial cells using a 1ml syringe)

[0298] (Materials and methods)

[0299] HCECs were cultured in CTV in suspension form at a concentration of 1.0 × 10⁻⁶ cells. 6 Cells at a density of 300 μl were stored for 72 hours in a 1 ml syringe (disposable syringe, manufacturer's name: 08B2X10007000001, Misawa Medical Industry Co., Ltd., Ibaraki Prefecture). The syringe was held horizontally and stored at 37°C for 72 hours. After storage, the syringe was shaken with a fingertip, and the cells were expelled from the syringe using a 26G needle. Cell viability was then evaluated. To assess whether cells adhered to the inside of the syringe during storage, the syringe was shaken with a fingertip to expel the cells after storage. The inside of the syringe was then gently washed with cell preservation solution, and the condition of the syringe's inner surface was observed.

[0300] (Calculation of base area)

[0301] In all experiments, the liquid volume was kept constant at 300 μl, and the base area was adjusted by changing the position of the piston. The inner diameter of the 1 ml syringe used was 6 mm. With the piston tip (rubber part) positioned 10 mm from the tip of the syringe and the syringe laid horizontally, the base area, according to the definition in this manual, is (6 × 3.14 ÷ 2) × 10 = 94.2 mm². 2 =0.942cm 2 The amount of air at this time is approximately 0 μl.

[0302] With the piston tip (rubber part) positioned 20mm from the tip of the syringe and the syringe laid horizontally, the base area, as defined in this instruction manual, is (6 × 3.14 ÷ 2) × 20 = 188.4 mm². 2 =1.884cm 2 The amount of air at this time is approximately 300 μl.

[0303] With the piston tip (rubber part) positioned 20mm from the tip of the syringe and the syringe placed longitudinally, the base area is 6×6×3.14≒28mm². 2 ≒ Approximately 0.28cm 2 .

[0304] (result)

[0305] 500,000 corneal endothelial cells cultured in a 1ml syringe were suspended in 300μl of cell preservation solution for preservation. Figure 5 After storing corneal endothelial cells in a syringe for 72 hours, cell deposits were observed on the side of the syringe. Figure 5 (Bottom left). After a light tap, the cells floated up from the side of the syringe, indicating that the cells did not adhere to the inside of the syringe during preservation. Figure 5 (Lower middle). After 72 hours of preservation, the syringe was gently tapped and then rinsed with cell preservation solution. No cells were found adhering to the side of the syringe. Figure 5 lower right).

[0306] Compared to when all cells were set to 100% at the start of preservation (viable cell rate: 90.3%, dead cell rate: 9.7%), after 72 hours of preservation in the syringe, the viable cell rate became 70.9% and the dead cell rate was 8.2%, approximately 90% of the cells survived, which is the same as at the start of preservation. Figure 6 On the other hand, when preserving cells in a syringe, if air is added and the syringe is positioned horizontally, the bottom area inside the syringe is set to approximately 2 cm². 2 At that time, the cell recovery rate was high, but the syringe contained no air, and the bottom area was set to approximately 1 cm². 2 At that time, the number of recovered cells decreased to about half. Also, with air added to the syringe, when the syringe was stood upright, the base area was approximately 0.28 cm². 2 However, the number of cells recovered was significantly reduced. Figure 7 These indicate that when cells are preserved inside a syringe, the bottom area is approximately 2 cm². 2 This difference improves cell recovery and survival rates.

[0307] (Example 4: Detailed study of the optimal temperature for cell preservation)

[0308] (Materials and methods)

[0309] (Culture of corneal endothelial cells)

[0310] Five donor corneas were obtained from donors aged 40 and over with the disease. All corneas were stored at 4°C in Optisol (Chiron Vision, Irvine, CA) for no more than 14 days before use. Human corneal endothelial cells (HCECs) were cultured as follows. Briefly, the Desme membrane containing the corneal endothelium was mechanically detached from the donor cornea and digested by incubation at 37°C in 1 mg / mL collagenase A (Roche Applied Science, Penzberg, Germany) for 12 hours. After washing with OptiMEM-I (Life Technologies Corp., Carlsbad, CA), the HCECs were seeded into one well of a 48-well plate coated with a laminin E8 fragment (iMatrix-511; Nippi, Incorporated, Tokyo).

[0311] The culture medium was prepared as follows. In short, OptiMEM-I supplemented with 8% bovine serum peptide (FBS), 5 ng / mL epidermal growth factor (EGF; Thermo Fisher Scientific), 20 μg / mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), 200 mg / L calcium chloride, 0.08% chondroitin sulfate (Sigma-Aldrich), and 50 μg / mL gentamicin (Thermo Fisher Scientific) was acclimated to NIH-3T3 for 24 hours. The acclimated medium was then recovered and filtered through a 0.22 μm filter unit (EMD Millipore Corporation, Billerica, MA) for use as the HCEC medium.

[0312] HCECs were cultured at 37°C under a humidified atmosphere containing 5% CO2, with the culture medium changed three times a week. In the passage culture of HCECs, cells were trypsinized for 5 minutes at 37°C using TrypLE SelectEnzyme (10X) (Thermo Fisher Scientific) and seeded at a 1:2 ratio. In this experiment, HCECs passaged for 9 generations starting from passage 5 were used.

[0313] For HCEC, use a method that does not contain Ca 2+ Mg 2+ Cells were washed with phosphate-buffered saline (PBS) and treated with TrypLE Select Enzyme (10X) at 37°C for 15 minutes. After being recovered from the culture plate, cells were washed twice, centrifuged at 280G for 3 minutes, and resuspended in OptiMEM-I. HCECs were then cultured in serum-free medium (cell therapy medium provided by Cell Science & Technology Institute, Inc.) at 1.0 × 10⁶ cells / mL. 6 Cells were stored in suspension at a density of 300 μl / cell in 24-well plates (untreated) (AGC TECHNO GLASS Co., Ltd., Shizuoka) for 72 hours. To investigate the optimal temperature for HCEC preservation, preservation was performed at nine different temperatures: 12°C, 17°C, 22°C, 27°C, 32°C, 35°C, 37°C, 39°C, and 42°C.

[0314] After 72 hours of storage, HCECs were slowly recovered from cell culture plates by pipetting and centrifuged at 280G for 3 minutes at 1.0×10⁻⁶. 6Cells were resuspended in cell therapy medium at a density of 600 μl. Cell viability was calculated by staining dead cells with 0.5% trypan blue (NACALAI TESQUE, INC. Kyoto). As a control, HCECs recovered by trypsin digestion with TrypLE Select Enzyme (10X) were centrifuged at 280 G for 3 minutes and resuspended in cell therapy medium. Cell viability was then evaluated immediately without preservation in cell suspension form.

[0315] (result)

[0316] (The effect of temperature conditions on cell viability during cell suspension preservation)

[0317] HCECs were cultured in CTV in suspension form at 1.0 × 10⁻⁶ cells. 6 Cells were stored at a density of 300 cells / 300 μl for 72 hours. Cells were collected by gentle pipetting, and cell viability was evaluated. Figure 9 ).

[0318] Under nine temperature conditions, the highest cell viability was observed at 37°C. Compared to when all cells were at 100% at the start of storage (92.4% viable cells, 7.6% dead cells), after 72 hours of storage, the viable cell rate increased to 95.4% and the dead cell rate to 4.9%, showing the same cell viability as at the start of storage. Furthermore, at temperatures below 37°C, the cell viability decreased proportionally to the decrease in temperature. Storage at 12°C, 17°C, and 22°C showed significantly lower viable cell rates compared to the start of storage, but still maintained a viability of over 30%. Similarly, at 39°C and 42°C, the cell viability decreased proportionally to the increase in temperature, showing significantly lower viable cell rates compared to the start of storage, but still maintained a viability of over 30%. Thus, it is preferable to store the product at a temperature of 12°C to 42°C, which can maintain a survival rate of 30% or more; more preferably at a temperature of 17°C to 39°C, which can maintain a survival rate of 60% or more; even more preferably at a temperature of 27°C to 37°C, which can maintain a survival rate of 80% or more; and most preferably at a temperature of 37°C, which can maintain a survival rate of 90% or more, the same as the control.

[0319] (Example 5: Study on cell preservation using glass tubular bottles)

[0320] (Materials and methods)

[0321] HCECs were cultured in CTV in suspension form at a concentration of 1.0 × 10⁻⁶ cells. 6Cells at a density of 300 μl were stored in glass vials for 72 hours. Three types of glass vials were used: vials with a standard glass surface (Maruemu Corporation, Osaka, 0501-02), vials treated with low adsorption (IRAS treatment) (Iwata Glass Industrial Co., Ltd., Osaka, lot: 181024), and vials with a silica surface treatment (IRAS treatment and SiO2 coating) (Iwata Glass Industrial Co., Ltd., Osaka, lot: 181102). Figure 10 The base area of ​​these tubular bottles is approximately 2 cm². 2 .

[0322] After 72 hours of storage, the contents were slowly recovered from the tubular bottle by pipetting and centrifuged at 280G for 3 minutes at a concentration of 1.0 × 10⁻⁶. 6 Cells were resuspended in cell therapy medium at a density of 600 μl. Cell viability was calculated by staining dead cells with 0.5% trypan blue (NACALAI TESQUE, INC. Kyoto). As a control, HCECs recovered by trypsin digestion with TrypLE Select Enzyme (10X) were centrifuged at 280 G for 3 minutes and resuspended in cell therapy medium. Cell viability was then evaluated immediately without preservation in suspension.

[0323] (result)

[0324] HCECs were cultured in CTV in suspension form at 1.0 × 10⁻⁶ cells. 6 Cells were stored at a density of 300 cells / 300 μl for 72 hours. Cells were collected by gentle pipetting, and cell viability was evaluated. Figure 11 ).

[0325] In the tubular flasks (Maruemu Corporation., Osaka, 0501-02), cell adhesion to the flasks was observed. On the other hand, in the tubular flasks (Iwata Glass Industrial Co., Ltd., Osaka, 181024) and (Iwata Glass Industrial Co., Ltd., Osaka, 181102), cells did not adhere to the flasks and could be recovered by gentle pipetting. With all cells at the start of preservation set at 100%, the control showed a viable cell rate of 92.4% and a dead cell rate of 7.6%. In contrast, the cells preserved in the tubular flasks (Maruemu Corporation., Osaka, 0501-02) showed a viable cell rate of 38.9% and a dead cell rate of 7.4%, significantly lower than the viable cell count at the start of preservation. Cells preserved in tubular vials (Iwata Glass Industrial Co., Ltd., Osaka, 181024) showed a viable cell rate of 90.7% and a dead cell rate of 7.8%; cells preserved in tubular vials (Iwata Glass Industrial Co., Ltd., Osaka, 181102) showed a viable cell rate of 84.6% and a dead cell rate of 8.4%, exhibiting the same cell viability as at the start of preservation. Thus, all glass tubular vials showed a cell viability of 30%, but those with a low-adhesion surface treatment showed a higher cell viability.

[0326] (Example 6: Manufacturing and transportation examples of cell-containing products)

[0327] Corneal endothelial cells cultured from donor corneas, or corneal endothelial cells differentiated from iPS cells, ES cells, neural crest cells, etc., or cells with the same function as corneal endothelial cells, are recovered from culture dishes through enzymatic treatment. The recovered cells are resuspended at a ratio of approximately 500,000 to approximately 1,000,000 cells per 300 μl of cell infusion solution, and approximately 500 to approximately 800 μl of the cell suspension is stored in tubular bottles (Iwata Glass Industrial Co., Ltd., Osaka, lot: 181024) or tubular bottles (Iwata Glass Industrial Co., Ltd., Osaka, lot: 181102). The tubular bottles are sealed with rubber stoppers and aluminum (primary containers). The tubular bottles are further stored in secondary containers to ensure airtightness and leak-proofness. The secondary containers are stored in an incubator maintained at 37°C. The secondary container is placed inside an outer container that absorbs external impacts to maintain a temperature of 37°C during transport to medical facilities.

[0328] As described above, the present invention has been illustrated by means of preferred embodiments, but it is to be understood that the scope of the invention should be interpreted only by means of the scope of the claims. The contents of all patents, patent applications, and documents referenced in this specification are, in the same manner as those specifically described herein, and should be construed as incorporated herein by reference. This application claims the benefit of priority to Japanese Patent Application No. 2018-187754, filed October 2, 2018, and Japanese Patent Application No. 2018-247970, filed December 28, 2018, the contents of which are incorporated herein by reference.

[0329] Industrial availability

[0330] A method for preserving corneal endothelial cells is provided. This invention enables the preservation of corneal endothelial cells with a high cell viability. The corneal endothelial cells preserved in this way retain the functions of normal corneal endothelial cells and can be used as therapeutic cells for corneal endothelial diseases, thus enabling their application in pharmaceuticals and other fields.

Claims

1. A method for preserving corneal endothelial cells and / or corneal endothelioid cells, comprising: in a basin with a base area of ​​at least 0.7 cm² 2 The process of storing the corneal endothelial cells and / or corneal endothelioid cells in a container, wherein, The corneal endothelial cells and / or corneal endothelioid cells are preserved in a cell suspension, wherein the container has not undergone surface treatment for adhesion culture, and It must be stored for at least 6 hours.

2. The method according to claim 1, wherein, The corneal endothelial cells and corneal endothelioid cells mentioned are cells that can be used clinically.

3. The method according to claim 1, characterized in that, Preserved corneal endothelial cells and corneal endothelioid cells can be given directly without further processing or culture.

4. The method according to claim 1, wherein, Store for 24-72 hours.

5. The method according to claim 1, wherein, The liquid level used to preserve the cells is above 0.75 mm.

6. The method according to claim 1, wherein, The bottom area of ​​the container is 0.7–4 cm². 2 .

7. The method according to claim 1, wherein, The bottom area of ​​the container is 1.5–3 cm². 2 .

8. The method according to claim 1, wherein, The bottom area of ​​the container is 1.8–2 cm². 2 .

9. The method according to claim 1, wherein, The container is a low-adhesion surface container or a surface-untreated container.

10. The method according to claim 1, wherein, The container is made of polystyrene, polypropylene, or glass.

11. The method according to claim 1, wherein, The container is selected from the group consisting of a 24-well plate, a tubular bottle, a syringe, and a petri dish.

12. The method according to claim 1, wherein, The cells were preserved at temperatures ranging from 0°C to 42°C.

13. The method according to claim 1, wherein, The cells were preserved at temperatures ranging from 17°C to 39°C.

14. The method according to claim 1, wherein, The cells were preserved at a temperature of 27°C to 37°C.

15. The method according to claim 1, wherein, The cells were preserved at 37°C.

16. The method according to claim 1, wherein, The volume of the suspension is at least 50 μL.

17. The method according to claim 1, wherein, The volume of the suspension is 100 μL to 2000 μL.

18. The method according to claim 1, wherein, The corneal endothelial cells and / or corneal endothelioid cells that have undergone the preservation process can be used for cell infusion therapy.

19. The method according to claim 1, wherein, The cell density of the cells stored in the container is 2 × 10⁻⁶. 4 8 x 10 cells / ml ~ 8 x 10 7 pcs / ml 20. The method according to claim 1, wherein, The cell density of the cells stored in the container is 2 × 10⁻⁶. 6 pcs / ml ~ 4×10 6 pcs / ml 21. A cell preparation comprising: corneal endothelial cells and / or corneal endothelioid cells, and a container for storing the corneal endothelial cells and / or corneal endothelioid cells, the container having a base area of ​​at least 0.7 cm². 2 ,in, The container was not subjected to surface treatment for adhesion culture, wherein the corneal endothelial cells and / or corneal endothelial-like cells were stored in suspension, and The corneal endothelial cells and / or corneal endothelioid cells are preserved for at least 6 hours.

22. The cell preparation according to claim 21, wherein, The corneal endothelial cells and / or corneal endothelioid cells were preserved for 24 to 72 hours.

Images