Adhesion promoter for corneal endothelial cell

Abstract

To provide means capable of efficiently proliferating a corneal endothelial cell, and means for stably supplying corneal endothelium preparation.SOLUTION: There are provided an adhesion promoter for a corneal endothelial cell which contains an Rho kinase inhibitor, culture solution of a corneal endothelial cell which contains an Rho kinase inhibitor, cornea preservation solution which contains an Rho kinase inhibitor, a manufacturing method of a corneal endothelium preparation which includes a process of culturing a corneal endothelial cell by using the culture solution, and a corneal endothelium protective agent which contains an Rho kinase inhibitor. The adhesion promoter for a corneal endothelial cell of the present invention promotes adhesion of the corneal endothelial cell, and enables formation of a corneal endothelial cell layer having a satisfactory cell morphology and high cell density.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a corneal endothelial cell adhesion promoter, which is used for the adhesion, maintenance or preservation of corneal endothelial cells. [Background technology]

[0002] Visual information is perceived when light enters the cornea, the transparent tissue at the front of the eyeball, reaches the retina, excitates the retinal nerve cells, and the generated electrical signals are transmitted via the optic nerve to the visual cortex of the cerebrum. A transparent cornea is necessary to have good vision. The transparency of the cornea is maintained by keeping the water content constant through the pumping and barrier functions of the corneal endothelial cells.

[0003] At birth, human corneal endothelial cells are present at a density of approximately 3,000 cells per square millimeter, but once damaged, they are unable to regenerate. In bullous keratopathy, which occurs due to corneal endothelial degeneration or corneal endothelial dysfunction caused by various factors, the cornea becomes edematous and cloudy, resulting in a significant decrease in vision. Currently, full-thickness keratoplasty is performed to transplant all three layers of the cornea: epithelium, stroma, and endothelium. However, there is a shortage of donor corneas in Japan, and while there are approximately 5,500 patients waiting for corneal transplants, only about 2,700 corneal transplants are performed annually in the country.

[0004] In recent years, the idea of ​​"part transplantation," in which only damaged tissue is transplanted, has been attracting attention in order to reduce the risk of rejection and postoperative complications and to obtain better visual function. Among corneal transplants, epithelial transplantation, in which only the corneal epithelium is transplanted, cultured oral mucosa epithelial transplantation, in which oral mucosa is transplanted instead of the corneal epithelium, and deep lamellar keratoplasty, which is a transplant of stromal tissue, have been performed. A method of transplanting only the corneal endothelium has also been considered. For transplantation of the corneal endothelium, a corneal endothelium-like sheet consisting of a corneal endothelium layer cultured on a collagen layer is known (see Patent Documents 1 and 2). However, corneal endothelial cells, especially human-derived corneal endothelial cells, are limited in the number of corneal donors and are difficult to culture in a test tube, and it takes time and money to obtain the number of cultured cells required for transplantation.

[0005] Human embryonic stem (ES) cells have both high self-renewal and pluripotency, and have been attracting attention from the viewpoint of medical applications, but they have a practical problem in that cell death easily occurs when cells are dispersed during the culture process, resulting in a significant loss of cell number. In recent years, it has been discovered that cell death that occurs when human ES cells are cultured is caused by activation of Rho kinase (ROCK), and that cell death is significantly suppressed by inhibiting ROCK. It has been reported that the use of ROCK inhibitors such as Y-27632 has made it possible to mass-culture human ES cells and produce cerebral cells (Non-Patent Document 1).

[0006] Rho kinase (ROCK) inhibitors are known to have various actions. For example, Patent Document 3 discloses that Rho kinase inhibitors promote corneal neurite formation and are used as corneal sensitivity recovery agents. Patent Document 4 discloses that Rho kinase inhibitors promote axonal extension of retinal ganglion cells and are used for the treatment of visual dysfunction. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2004-24852 A [Patent Document 2] JP 2005-229869 A [Patent Document 3] WO2005 / 118582 Brochure [Patent Document 4] WO2002 / 083175 Pamphlet [Non-patent literature]

[0008] [Non-Patent Document 1] Nat Biotechnol. 2007, 25, 681 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a means for enabling efficient adhesion of corneal endothelial cells and a means for stable supply of a corneal endothelial preparation. [Means for solving the problem]

[0010] In view of the above problems, the present inventors have conducted intensive research and found that by culturing corneal endothelial cells in the presence of a Rho kinase inhibitor, adhesion of the cells to a culture substrate is dramatically promoted, thereby completing the present invention.

[0011] [1] An adhesion promoter for corneal endothelial cells comprising a Rho kinase inhibitor. [2] The promoter according to the above [1], wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof. [3] The promoter according to [1] above, wherein the corneal endothelial cells are of human origin. [4] The promoter according to any one of [1] to [3] above, which is a preparation for preventing or treating corneal endothelial damage. [5] The enhancer according to [4] above, which is in the form of an intracameral injection or an ocular irrigation solution. [6] A culture medium for corneal endothelial cells containing a Rho kinase inhibitor. [7] The culture medium according to [6] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof. [8] The culture medium according to [6] above, wherein the corneal endothelial cells are of human origin. [9] A corneal storage solution containing a Rho kinase inhibitor.

[10] The preservation solution according to [9] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[11] The preservation solution according to [9], wherein the cornea is of human origin.

[12] A method for producing a corneal endothelial preparation, comprising a step of culturing corneal endothelial cells using the culture medium according to any one of [6] to [8] above.

[13] The method of producing according to

[12] above, wherein the corneal endothelial cells are of human origin.

[14] Use of a Rho kinase inhibitor for producing an agent for promoting adhesion of corneal endothelial cells.

[15] The use according to

[14] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[16] The use according to

[14] above, wherein the corneal endothelial cells are of human origin.

[17] The use according to any one of

[14] to

[16] above, wherein the corneal endothelial cell adhesion promoter is a preparation for preventing or treating corneal endothelial disorders.

[18] The method according to any one of the above [1], wherein the corneal endothelial cell adhesion promoter is in the form of an intracameral injection or an ocular irrigation solution. 7) Use as described above.

[19] Use of a Rho kinase inhibitor for producing a culture medium for corneal endothelial cells.

[20] The use according to

[19] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[21] The use according to

[19] above, wherein the corneal endothelial cells are of human origin.

[22] Use of a Rho kinase inhibitor for producing a corneal preservation solution.

[23] The use according to

[22] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[24] The use according to

[22] above, wherein the cornea is of human origin.

[25] A method for promoting adhesion of corneal endothelial cells, comprising contacting an effective amount of a Rho kinase inhibitor with a subject or corneal endothelial cells in need of promotion of adhesion of corneal endothelial cells.

[26] The method of promoting Rho kinase according to

[25] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[27] The method for promoting corneal endothelial cells according to

[25] above, wherein the corneal endothelial cells are of human origin.

[28] The method for promoting corneal endothelial cell proliferation according to

[25] above, which is intended to prevent or treat corneal endothelial damage.

[29] The method for promoting ocular hyperplasia according to

[25] above, wherein an effective amount of a Rho kinase inhibitor is administered in the form of an intracameral injection or an ocular irrigation solution.

[30] A method for culturing corneal endothelial cells, comprising the step of culturing corneal endothelial cells in a culture medium containing a Rho kinase inhibitor.

[31] The culture method according to

[30] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[32] The culture method described in

[30] above, wherein the corneal endothelial cells are of human origin.

[33] A method for protecting a cornea, comprising the step of maintaining a corneal slice or corneal endothelial cells in a corneal protective solution containing a Rho kinase inhibitor.

[34] The method for protecting according to the above

[33] , wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[35] The method for protecting a cornea according to

[33] above, wherein the cornea is of human origin.

[36] A method for producing a corneal endothelial preparation, comprising a step of culturing corneal endothelial cells in a culture medium containing a Rho kinase inhibitor.

[37] The method according to

[36] above, wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof.

[38] The method of producing according to

[36] above, wherein the corneal endothelial cells are of human origin.

[39] A method for culturing corneal endothelial cells using a culture medium containing a Rho kinase inhibitor; and A step of transplanting the corneal endothelial preparation obtained by the culturing step into a subject in need of corneal endothelial transplantation. 13. A method for treating bullous keratopathy, corneal edema, or corneal leukoplakia, comprising:

[40] Rho kinase inhibitor (+)-trans-4-(1-aminoethyl)-1-( The method for treatment according to the above

[39] , wherein the compound is at least one selected from the group consisting of 4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[41] The method of treatment described in

[39] above, wherein the corneal endothelial cells are of human origin. Effect of the Invention

[0012] The corneal endothelial cell adhesion promoter of the present invention contains a Rho kinase inhibitor as an active ingredient. The corneal endothelial cell adhesion promoter of the present invention promotes the adhesion of corneal endothelial cells and enables the formation of a corneal endothelial cell layer having good cell morphology and high cell density. This makes it useful as a therapeutic or preventive agent for diseases accompanied by corneal endothelial disorders, such as bullous keratopathy and corneal endotheliitis. The corneal endothelial cell adhesion promoter of the present invention is useful as a corneal endothelial protective agent in the treatment or prevention of diseases accompanied by corneal endothelial disorders. Furthermore, the corneal endothelial cell adhesion promoter of the present invention can be used as a corneal endothelial protective agent in the treatment or prevention of corneal endothelial disorders associated with intraocular surgery such as cataract surgery and vitreous surgery, corneal endothelial disorders caused by increased intraocular pressure (particularly glaucoma attacks), or corneal endothelial disorders caused by oxygen deficiency due to contact lens wear. Since the culture medium or corneal preservation solution of the present invention contains a Rho kinase inhibitor, it is possible to satisfactorily culture, maintain or preserve corneal endothelial cells, and it is possible to stably supply, maintain or preserve corneal endothelial preparations. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a phase contrast micrograph of primary cultured rabbit corneal endothelial cells (24 hours after the start of culture, magnification: 100 times). [Diagram 2] 2 is a graph showing the results of investigating the proliferation of primary cultured rabbit corneal endothelial cells by MTS assay. The vertical axis represents the relative value to the absorbance of the Rock inhibitor (-) group on Day 1. [Diagram 3]3 is a graph showing the adhesion of primary cultured rabbit corneal endothelial cells 24 hours after the start of culture. The vertical axis shows the relative value to the absorbance of the Rock inhibitor (-) group 24 hours after the start of culture. [Figure 4] 4 is a phase contrast micrograph of primary cultured monkey corneal endothelial cells on day 3 after the start of culture (magnification: 100x). [Diagram 5] FIG. 5 is a phase contrast micrograph of primary cultured human corneal endothelial cells (day 7, magnification: 100x). [Figure 6] FIG. 6 shows photographs of primary cultured monkey corneal endothelial cells (A: ROCK inhibitor(-), B: ROCK inhibitor(+), magnification: 20x) and a graph (C) showing the total area of ​​the cultured cells. [Figure 7] FIG. 7 is a graph showing the viable cell count of primary cultured monkey corneal endothelial cells. [Figure 8] FIG. 8 is a graph examining the effect of Y-27632 on subculture of monkey corneal endothelial cells. [Figure 9] FIG. 9 is a set of phase-contrast micrographs (magnification: 100×) showing the effect of Y-27632 on the cell morphology during subculture of monkey corneal endothelial cells. [Figure 10] FIG. 10 is a set of micrographs (phalloidin fluorescent staining, magnification: 200 times) showing the effect of Y-27632 on the cell morphology during subculture of monkey corneal endothelial cells. [Figure 11] FIG. 11 shows micrographs (anti-Ki67 antibody staining, magnification: 200x) examining the effect of Y-27632 on the cell cycle of monkey corneal endothelial cells. [Figure 12] Figure 12 shows the results of flow cytometry investigating the effect of Y-27632 on the cell cycle of monkey corneal endothelial cells. Figure 12A shows the first day of subculture of the ROCK inhibitor(-) group, B shows the second day of subculture of the ROCK inhibitor(-) group, C shows the first day of subculture of the ROCK inhibitor(+) group, and D shows the second day of subculture of the ROCK inhibitor(+) group. [Figure 13]FIG. 13 is a graph showing the results of flow cytometry in FIG. 12 in terms of the anti-Ki67 antibody positive cell rate. [Figure 14] FIG. 14 is a graph showing the effect of Y-27632 on apoptosis in monkey corneal endothelial cells. [Figure 15] FIG. 15 is a graph showing the effect of Y-27632 on apoptosis of monkey corneal endothelial cells in terms of the number of dead cells. [Figure 16] FIG. 16 is a graph showing the effect of Y-27632 on the density of cultured monkey corneal endothelial cells. [Figure 17] 17 is a graph showing the adhesion of cultured monkey corneal endothelial cells 24 hours after subculture. The vertical axis shows the relative value of the luminescence intensity of the fasudil group to that of the control group 24 hours after the start of subculture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The corneal endothelial cell adhesion promoter of the present invention (hereinafter sometimes abbreviated as "the adhesion promoter of the present invention") contains a Rho kinase inhibitor as an active ingredient.

[0015] In the present invention, Rho kinase refers to a serine / threonine kinase that is activated in association with the activation of Rho. For example, ROKα (ROCKII: Leung, T. et al., J. Biol. Chem., 270, 29051-29054, 1995), p160ROCK (ROKβ, ROCK-I) : Ishizaki, T. et al., The EMBO J., 15(8), 1885-1893, 1996) and other proteins having serine / threonine kinase activity.

[0016] In the present invention, one type of Rho kinase inhibitor may be contained alone, or several types may be contained in combination as necessary.

[0017] Rho kinase inhibitors are described in the following literature: US4678783, Patent 3421217, WO99 / 20620, WO99 / 61403, WO02 / 076976, WO02 / 076977, WO02 / 100833, WO03 / 059913, WO03 / 062227, WO2004 / 009555, WO2004 / 022541, WO2004 / 108724, WO2005 / 003101, WO2005 Examples of the compounds include those disclosed in WO2005 / 039564, WO2005 / 034866, WO2005 / 037197, WO2005 / 037198, WO2005 / 035501, WO2005 / 035503, WO2005 / 035506, WO2005 / 080394, WO2005 / 103050, WO2006 / 057270, WO2007 / 026664, etc. Such compounds can be produced by the methods described in the respective documents. Specific examples include 1-(5-isoquinolinesulfonyl)homopiperazine (fasudil) and (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane (Y-27632), and commercially available products of these compounds can also be suitably used.

[0018] Among them, (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and their pharmacologically acceptable salts are preferably used because they are particularly excellent in promoting adhesion of corneal endothelial cells. As the salt of the compound, a pharma- ceutically acceptable acid addition salt is preferable, and examples of such an acid include inorganic acids such as hydrochloric acid, hydrobromic acid, and sulfuric acid, and organic acids such as methanesulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, tartaric acid, and salicylic acid. (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane dihydrochloride and 1-(5-isoquinolinesulfonyl)homopiperazine hydrochloride are more preferable.

[0019] In the present invention, "promotion of adhesion of corneal endothelial cells" includes, for example, both promotion of adhesion between corneal endothelial cells and promotion of adhesion between corneal endothelial cells and a culture substrate.

[0020] The adhesion promoter of the present invention exhibits an adhesion-promoting effect on corneal endothelial cells isolated from corneal tissue derived from mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.) or isolated and passaged corneal endothelial cells. Since the adhesion promoter of the present invention is particularly excellent in the adhesion-promoting effect on corneal endothelial cells derived from humans, which are considered to be difficult to culture and passage, it is preferable to target corneal endothelial cells derived from humans.

[0021] Corneal endothelial cells play a role in maintaining the transparency of the cornea. When the density of these endothelial cells decreases beyond a certain limit, swelling occurs in the cornea, the transparency of the cornea cannot be maintained, and corneal endothelial disorder is caused. The adhesion promoter of the present invention promotes the adhesion of corneal endothelial cells, enables the formation of a corneal endothelial cell layer with good cell morphology and high cell density, and further has an apoptosis inhibitory effect on corneal endothelial cells, and is therefore useful as a therapeutic or preventive agent for diseases accompanied by corneal endothelial disorder, such as bullous keratopathy and corneal endotheliitis. In addition, the adhesion promoter of the present invention is useful as a therapeutic or preventive agent for corneal endothelial disorder associated with intraocular surgery such as cataract surgery and vitreous surgery, corneal endothelial disorder caused by increased intraocular pressure (particularly glaucoma attacks), or corneal endothelial disorder caused by oxygen deficiency due to wearing contact lenses.

[0022] The adhesion promoter of the present invention is not particularly limited as long as it is in a dosage form suitable for local administration to the eye, but is preferably formulated in the form of an intracameral injection or ocular irrigation solution. These can be prepared using conventional techniques commonly used in the art. When the adhesion promoter is locally administered to the eye in the form of an intracameral injection or ocular irrigation solution, the Rho kinase inhibitor comes into contact with corneal endothelial cells in vivo, promoting adhesion of the corneal endothelial cells.

[0023] For example, when the adhesion promoter of the present invention is used as an intracameral injection solution or an ocular irrigation solution, stabilizers (e.g., sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.), solubilizing agents (e.g., glycerin, propylene glycol, macrogol, polyoxyethylene hydrogenated castor oil, etc.), suspending agents (e.g., polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, etc.), emulsifiers (e.g., polyvinylpyrrolidone, soybean lecithin, egg yolk lecithin, polyoxyethylene hydrogenated castor oil, polysorbate 80, etc.), buffers (e.g., phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamic acid, epsilon aminocaproic acid, etc.), thickeners (e.g., methylcellulose, hydrochloric acid, etc.), etc., may be used. Additives that can be added include water-soluble cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and carboxymethyl cellulose, sodium chondroitin sulfate, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, macrogol, etc., preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, paraoxybenzoic acid esters, sodium edetate, boric acid, etc.), isotonicity agents (e.g., sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.), pH adjusters (e.g., hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.), and refreshing agents (e.g., l-menthol, d-camphor, d-borneol, peppermint oil, etc.). The amount of these additives to be added varies depending on the type and use of the additive, but it is sufficient to add a concentration that can achieve the purpose of the additive.

[0024] The amount of the active ingredient in the adhesion promoter of the present invention is usually 0.00001 to 1 w / v%, preferably 0.00001 to 0.1 w / v%, and more preferably 0.0001 to 0.01 w / v%. The dosage and frequency of administration vary depending on symptoms, age, body weight, and dosage form. On the other hand, when used as an intracameral injection, for example, a preparation containing 0.0001 to 0.1 w / v%, preferably 0.0001 to 0.01 w / v%, of the active ingredient can be administered several times a day, preferably 1 to 2 times, more preferably once, at 0.01 to 0.1 mL per administration.

[0025] The adhesion promoter of the present invention can also be added to a culture medium when corneal endothelial cells are cultured in a test tube. By adding a Rho kinase inhibitor to the culture medium and continuing the culture, the Rho kinase inhibitor comes into contact with the corneal endothelial cells outside the body, promoting the adhesion of the corneal endothelial cells.

[0026] The present invention provides a culture medium for corneal endothelial cells containing a Rho kinase inhibitor. The Rho kinase inhibitor contained in the culture medium of the present invention is as described above.

[0027] The culture medium of the present invention may contain a medium commonly used for culturing endothelial cells (e.g., DMEM (GIBCO BRL), serum (e.g., fetal bovine serum (FBS)), growth factors (e.g., b-FGF), antibiotics, etc. (e.g., penicillin, streptomycin), etc.

[0028] The concentration of the Rho kinase inhibitor in the culture medium of the present invention is usually 1 to 100 μM, preferably 5 to 20 μM, and more preferably 10 μM.

[0029] The culture medium of the present invention prevents cell shedding by enhancing adhesion of corneal endothelial cells and enables the formation of a corneal endothelial cell layer having good cell morphology and high cell density, and is therefore preferably used in the method for producing a corneal endothelial preparation of the present invention described below. The culture medium of the present invention is also used for maintaining corneal endothelial cells.

[0030] The present invention provides a corneal preservation solution containing a Rho kinase inhibitor. The Rho kinase inhibitor contained in the corneal preservation solution of the present invention is as described above. The corneal preservation solution is a liquid agent for preserving a corneal fragment extracted from a donor until it is transplanted into a recipient.

[0031] Examples of the corneal preservation solution of the present invention include preservation solutions commonly used in corneal transplantation (corneal scleral fragment preservation solution (Optisol GS: registered trademark), eyeball preservation solution for corneal transplantation (EPII: registered trademark)), saline, phosphate buffered saline (PBS), etc., which contain a Rho kinase inhibitor.

[0032] The concentration of the Rho kinase inhibitor in the corneal preservation solution of the present invention is usually 1 to 100 μM, preferably 5 to 20 μM, and more preferably 10 μM.

[0033] The corneal preservation solution of the present invention prevents cell shedding by enhancing adhesion of corneal endothelial cells, and enables the formation of a corneal endothelial cell layer with good cell morphology and high cell density, and is therefore used as a preservation solution for the cornea to be used in organ transplantation, etc. The preservation solution of the present invention is also used as a preservation solution for cryopreserving corneal endothelial cells. For cryopreservation, glycerol, dimethyl sulfoxide, propylene glycol, acetamide, etc. may be further added to the preservation solution of the present invention.

[0034] The present invention provides a method for producing a corneal endothelial preparation, which comprises a step of culturing corneal endothelial cells using the culture medium of the present invention.

[0035] The corneal endothelial preparation is characterized by comprising a substrate and a corneal endothelial cell layer cultured in vitro on the substrate.

[0036] In the present invention, the substrate is not particularly limited as long as it can support a cultured corneal endothelial cell layer and maintain its shape in the living body for at least 3 days after transplantation. Moreover, the substrate may serve as a scaffold when corneal endothelial cells are cultured in a test tube, or may only serve to support the corneal endothelial cell layer after culture. Preferably, the substrate serves as a scaffold that is used for culturing corneal endothelial cells and can be directly used for transplantation after completion of culture.

[0037] Examples of the substrate include naturally occurring polymeric materials such as collagen, gelatin, and cellulose; synthetic polymeric materials such as polystyrene, polyester, polycarbonate, and poly(N-isopropylacrylamide); biodegradable polymeric materials such as polylactic acid and polyglycolic acid; hydroxyapatite; and amniotic membrane.

[0038] The shape of the substrate is not particularly limited as long as it supports a corneal endothelial cell layer and is suitable for transplantation, but is preferably in the form of a sheet. When the preparation of the present invention is in the form of a sheet, it can be cut to a size that matches the application site at the time of transplantation. It is also possible to insert the sheet through the wound after rolling it into a small shape. A preferred example is a circular shape that covers about 80% of the area of ​​the damaged corneal endothelium. It is also preferable to make an incision in the periphery of the circle so that it can be closely attached to the application site.

[0039] In a preferred embodiment, the substrate is collagen. As the collagen, the collagen sheet described in JP-A-2004-24852 can be suitably used. Such a collagen sheet can be prepared, for example, from amniotic membrane according to the method described in JP-A-2004-24852.

[0040] The corneal endothelial cell layer preferably has at least one of the following characteristics, more preferably has two or more of the following characteristics, and even more preferably has all of the following characteristics. (1) The cell layer has a single-layer structure, which is one of the characteristics of the corneal endothelial cell layer in vivo. (2) The cell density in the cell layer is about 1,000 to about 4,000 cells / mm 2 In particular, when the recipient is an adult, the number of cells per mm is about 2,000 to 3,000. 2 It is preferable that: (3) The planar shape of the cells constituting the cell layer is approximately hexagonal. This is one of the characteristics of cells constituting the corneal endothelial cell layer in the living body. The preparation of the present invention is similar to the corneal endothelial cell layer in the living body, and exhibits the same functions as a natural corneal endothelial cell layer, while also being able to exhibit proliferation ability in the living body. (4) Cells are regularly aligned in the cell layer. In the corneal endothelial cell layer of a living body, the cells that make up the layer are regularly aligned, which is believed to maintain the normal function and high transparency of the corneal endothelial cells and allow the cornea to properly regulate moisture. Therefore, by providing such morphological characteristics, the preparation of the present invention is expected to exhibit the same function as the corneal endothelial cell layer in a living body.

[0041] The production method of the present invention comprises a step of culturing corneal endothelial cells using the culture medium of the present invention, and can be carried out, for example, by the following method. <1> Harvesting and in vitro culture of corneal endothelial cells Corneal endothelial cells are harvested from the cornea of ​​the recipient or a suitable donor in a conventional manner. Considering the transplant conditions in the present invention, allogeneic corneal endothelial cells may be prepared. For example, the Descemet's membrane and endothelial cell layer of the corneal tissue are peeled off from the corneal stroma, transferred to a culture dish, and treated with dispase or the like. This causes the corneal endothelial cells to fall off from the Descemet's membrane. The remaining corneal endothelial cells can be removed by pipetting or the like. After removing the Descemet's membrane, the corneal endothelial cells are cultured in the culture medium of the present invention. As the culture medium, for example, commercially available DMEM (Dulbecco's Modified Eagle's Medium) to which FBS (fetal bovine serum), b-FGF (basic-fibroblast growth factor), and antibiotics such as penicillin and streptomycin are appropriately added, and further Y-27632 or fasudil can be added, can be used. It is preferable to use a culture vessel (culture dish) whose surface is coated with type I collagen, type IV collagen, fibronectin, laminin, or the extracellular matrix of bovine corneal endothelial cells. Alternatively, a normal culture vessel treated with a commercially available coating agent such as FNC coating mix (registered trademark) can be used. By using such a coating in combination with the culture medium of the present invention, adhesion of the corneal endothelial cells to the surface of the culture vessel is promoted, and good proliferation is achieved.

[0042] The temperature conditions for culturing the corneal endothelial cells are not particularly limited as long as the corneal endothelial cells grow, but are, for example, about 25 to about 45° C., preferably about 30 to about 40° C. in consideration of proliferation efficiency, and more preferably about 37° C. The culture method is performed in a normal cell culture incubator under humidified conditions in an environment with a CO2 concentration of about 5 to 10%.

[0043] <2> Subculture Subculture can be performed after the corneal endothelial cells subjected to culture have proliferated. Subculture is preferably performed when the cells become subconfluent or confluent. Subculture can be performed as follows. First, the cells are detached from the surface of the culture vessel by treatment with trypsin-EDTA or the like, and then the cells are recovered. The culture medium of the present invention is added to the recovered cells to prepare a cell suspension. Centrifugation is preferably performed when recovering the cells or after recovery. A cell suspension with a high cell density can be prepared by such centrifugation. A preferred cell density is about 1 to 2 × 10 6The centrifugation conditions here are, for example, 500 rpm (30 g) to 1000 rpm (70 g) and 1 to 10 minutes.

[0044] The cell suspension is seeded in a culture vessel in the same manner as in the above initial culture, and is subjected to culture. The dilution ratio during subculture varies depending on the state of the cells, but is about 1:2 to 1:4, preferably about 1:3. Subculture can be performed under the same culture conditions as the above initial culture. The culture time varies depending on the state of the cells used, but is, for example, 7 to 30 days. The above subculture can be performed multiple times as necessary. By using the culture medium of the present invention, it is possible to shorten the culture period by enhancing cell adhesion in the early stage of culture.

[0045] <3> Preparation of corneal endothelial cell layer The cell suspension is seeded on a substrate such as a collagen sheet and cultured. At this time, the number of cells seeded is adjusted so that a cell layer with a desired cell density is formed in the finally produced corneal endothelial preparation. Specifically, the cell density is adjusted to about 1,000 to about 4,000 cells / mm 2 The cells are seeded so that a cell layer of the above is formed. The culture can be carried out under the same conditions as the initial culture described above. The culture time varies depending on the state of the cells used, but is, for example, 3 to 30 days.

[0046] By carrying out the culture as described above, a corneal endothelial preparation is obtained in which a corneal endothelial cell layer cultured in vitro is formed on the substrate.

[0047] In the present invention, the corneal endothelial preparation may contain the culture medium of the present invention in order to maintain the corneal endothelial cells. Furthermore, the corneal endothelial preparation may contain the corneal preservation solution of the present invention until it is used for transplantation. The present invention also provides a combination of the corneal endothelial preparation and the culture medium or preservation solution of the present invention. .

[0048] The corneal endothelial preparation obtained by the production method of the present invention can be used as a graft in the treatment of diseases requiring corneal endothelial transplantation, such as bullous keratopathy, corneal edema, and corneal leukoplakia, particularly bullous keratopathy caused by corneal endothelial damage resulting from corneal dystrophy, trauma, or intraocular surgery.

[0049] The adhesion promoter and corneal endothelial preparation of the present invention can be administered to mammals (for example, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.). EXAMPLES

[0050] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. When using experimental animals, the International Guiding Principles for Biomedical Research involving Animals shall be followed. The experiment was conducted in accordance with the Law Concerning the Welfare and Management of Animals and the standards for the care and management of laboratory animals. The experiment was also conducted in accordance with the Guidelines of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmic and Vision Research.

[0051] Example 1 Effects of ROCK inhibitors on rabbit corneal endothelial cell cultures Descemet's membrane with corneal endothelial cells attached was isolated from the corneal tissue of rabbits immediately after euthanasia. The Descemet's membrane was then soaked in 1.2 U / ml dispase II (Roche Applied Science) and then washed with 1.2 U / ml dispase II (Roche Applied Science). After incubation at 37°C and 5% CO2 for 45 minutes, the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged, and the ROCK inhibitor(+) group was cultured in a corneal endothelial medium containing 10 μM Y-27632. The inhibitor(-) group was cultured at the same concentration using a medium for corneal endothelium without Y-27632. The cells were mixed to a uniform density and seeded on a 96-well plate at a density of approximately 2000 cells per well. As a medium for corneal endothelium, a culture medium (DMEM, Gibco Invitrogen) supplemented with 1% fetal bovine serum and 2 mg / ml bFGF (Gibco Invitrogen) was used. The plate was pretreated with FNC coating mix (Athena ES) for 10 minutes. The culture medium was replaced 72 hours after the start of culture, and from 72 hours onwards, the culture medium was replaced with a ROCK inhibitor (+ In both the ROCK inhibitor(-) and ROCK inhibitor(-) groups, normal corneal endothelial culture medium was used without the addition of Y-27632. The primary cultured rabbit corneal endothelial cells were cultured for 5 days using a phase contrast microscope. A phase contrast microscope photograph of the primary cultured rabbit corneal endothelial cells 24 hours after the start of culture is shown in FIG. 1. From the first to third days after the start of culture, the primary cultured rabbit corneal endothelial cells were cultured every 24 hours using CellTiter 96 (registered trademark) AQueous One Solution Cell Proliferation (Promega). The cell number was measured by the MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (Figure 2).

[0052] In the ROCK inhibitor(+) group (Fig. 1B), cell adhesion was significantly enhanced by approximately 2.8 times 24 hours after cell seeding (day 1) compared with the ROCK inhibitor(-) group (Fig. 1A) (Fig. 2 , 3). On the third day, when the cells reached confluence, no significant difference was observed between the two groups (Figure 2), which demonstrated that Y-27632 has the effect of enhancing cell adhesion early after subculture in primary cultured rabbit corneal endothelial cells. Similar results were also obtained in studies using subcultured rabbit corneal endothelial cells, suggesting that Y-27632 acts on cell adhesion early after subculture in both primary and subculture cultures.

[0053] Example 2 Examination of the effect of Y-27632 on monkey corneal endothelial cell culture Corneal endothelial cells were attached from corneal tissue extracted from cynomolgus monkeys euthanized for another purpose. In the ROCK inhibitor(+) group, Descemet's membrane was treated with 10 μM Y-2. The ROCK inhibitor(-) group was incubated in a corneal endothelium medium containing Y-27632 for 10 minutes at 37°C and 5% CO2. The Descemet's membrane was incubated in the same medium as in Example 1 for 10 minutes under the same conditions. The same cell culture medium as in Example 1 was used as the medium for corneal endothelium. After incubation, the Descemet's membrane was incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes. After incubation, the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and mixed with Y-27632(+) and Y-27632(-) corneal endothelial medium to the same concentration, and seeded on a 12-well plate at a density of approximately 20,000 cells per well. The plate was pretreated with FNC coating mix (Athena ES) for 10 minutes. The culture medium was replaced 72 hours after the start of culture, and from 72 hours onwards, both the ROCK inhibitor(+) group and the ROCK inhibitor(-) group were cultured using normal corneal endothelial medium without Y-27632.

[0054] As shown in Figure 4, in primary cultured cynomolgus monkey corneal endothelial cells 24 hours after the start of culture, clearly more cells were attached to the plate in the ROCK inhibitor(+) (Figure 4B) than in the ROCK inhibitor(-) (Figure 4A). The number of days until confluence was reached was 3 days for the ROCK inhibitor(+) and 7 days for the ROCK inhibitor(-). This shows that Y-27632 is a useful drug that enhances cell adhesion early after the start of culture and enables cell culture, even in monkey corneal endothelial cells, which are considered to be more difficult to culture in vitro than rabbit cells.

[0055] Example 3 Examination of the effect of Y-27632 on human corneal endothelial cell culture The peripheral corneal tissue was obtained from a US eye bank and the central 7 mm diameter portion was used for corneal transplantation. Descemet's membrane with corneal endothelial cells attached was isolated, and in the ROCK inhibitor(+) group, the Descemet's membrane was treated with corneal endothelial cells supplemented with 10 μM Y-27632. The ROCK inhibitor(-) group was placed in corneal endothelial medium without Y-27632 and incubated for 10 minutes at 37°C and 5% CO2. The Descemet's membrane was incubated for 45 minutes with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2, and then the membrane was removed by pipetting. Corneal endothelial cells were mechanically isolated by a mixing operation. After centrifugation, the isolated corneal endothelial cells were mixed with Y-27632(+) and Y-27632(-) corneal endothelial medium to the same concentration, and seeded at a density of approximately 10,000 cells per well in a 48-well plate that had been pretreated with FNC coating mix (Athena ES). Since the number of human corneal endothelial cells obtained from peripheral corneal tissue is extremely small, one well of cells was cultured from one donor cornea. The donor corneas used in this study were 69 years old for the ROCK inhibitor(+) group, and 51 years old for the ROCK inhibitor(-) group, and 7 days after the donor's death before the start of culture. It is considered that the effects of age and the time elapsed from the donor's death until the start of cell culture are almost equal. The culture medium was replaced 72 hours after the start of culture, and after 72 hours, ROCK inhibitor(+) In both the control group and the ROCK inhibitor(-) group, a normal corneal endothelium culture medium was used without the addition of Y-27632. To investigate the effects on cell adhesion and cell morphology early after the start of culture, cells were observed using a phase-contrast microscope and photographed using a digital camera after the start of culture (Figure 5).

[0056] As shown in Figure 5, on the 7th day after the start of culture, the ROCK inhibitor(+) group had homogeneous polygonal cells. In contrast, in the ROCK inhibitor(-) group, a confluent, dense monolayer of cells similar to normal corneal endothelial cells was formed (Fig. 5B). Only endothelial cells with a fibroblast-like extended morphology were found to have attached in island-like formation (Figure 5A). These results suggest that in primary cultures of human corneal endothelium, which are known to be extremely difficult to culture, it is possible to form a corneal endothelial cell layer with good cell morphology and high cell density by adding Y-27632, which has the effect of enhancing cell adhesion early after the start of culture, to the culture medium.

[0057] Formulation Example 1 Intracameral injection containing a Rho kinase inhibitor The intracameral injection solution shown below is prepared in a conventional manner. Y-27632 10mg Sodium dihydrogen phosphate 0.1g Sodium chloride 0.9g Sodium hydroxide (appropriate amount) Appropriate amount of sterile purified water Total volume 100mL (pH 7) Y-27632 manufactured by Wako Pure Chemical Industries, Ltd. is used.

[0058] Formulation Example 2 Ocular irrigation solution containing a Rho kinase inhibitor The eye irrigation solution shown below is prepared by a conventional method. Y-27632 1.0mg Opeguard MA appropriate amount Total volume 100mL Opeguard MA is manufactured by Senju Pharmaceutical, and Y-27632 is manufactured by Wako Pure Chemical Industries.

[0059] Formulation Example 3 Culture medium for preparing corneal endothelial sheet containing a Rho kinase inhibitor The culture medium shown below is prepared by a conventional method. Y-27632 0.5mg FBS 10mL Penicillin-streptomycin solution 1mL FGF basic 200ng DMEM appropriate amount Total volume 100mL FBS is manufactured by Invitrogen, penicillin-streptomycin solution is manufactured by Nacalai Tesque (containing 5000 u / mL penicillin and 5000 μg / mL streptomycin), FGF basic is manufactured by Invitrogen, Y-27632 is manufactured by Wako Pure Chemical Industries, and DMEM is manufactured by Invitrogen.

[0060] Formulation Example 4 Corneal preservation solution containing a Rho kinase inhibitor Prepare the preservation solution shown below in a conventional manner. Y-27632 0.2mg Optisol-GS appropriate amount Total volume 100mL Optisol-GS is manufactured by Bausch & Lomb, and Y-27632 is manufactured by Wako Pure Chemical Industries.

[0061] Example 4 Examination of the effect of Y-27632 on primary culture of monkey corneal endothelial cells Descemet's membrane with corneal endothelial cells attached thereto was isolated from the corneal tissue excised from the euthanized cynomolgus monkey. As in Example 2, in the ROCK inhibitor(+) group, Descemet's membrane was treated with 10 μM The cells were then placed in a corneal endothelial medium containing Y-27632 and incubated at 37°C and 5% CO2 for 10 minutes. In the ROCK inhibitor(-) group, no Y-27632 was added. The Descemet's membrane was placed in a medium for corneal endothelium and incubated for 10 minutes under the same conditions. The same cell culture medium as in Example 1 was used as the medium for corneal endothelium. After incubation, the Descemet's membrane was incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 4 hours. After 5 minutes of incubation, the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and mixed with Y-27632(+) and Y-27632(-) corneal endothelial medium to the same concentration, and seeded on a 96-well plate at a density of about 2000 cells per well. The culture medium was replaced 72 hours after the start of culture, and from 72 hours onwards, both the ROCK inhibitor(+) group and the ROCK inhibitor(-) group were cultured using normal corneal endothelial medium without Y-27632. On the 10th day of culture, the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and stained with toluidine blue (Figures 6A and B). The total area of ​​the cells was measured and analyzed using Image J (National Institutes of Health) (Figure 6C). In primary cultured corneal endothelial cells from cynomolgus monkeys on the 10th day after the start of culture, ROCK inhibitor Compared with (-) (Fig. 6A), ROCK inhibitor(+) (Fig. 6B) reduced adhesion in the early stage of culture. By promoting the cell culture, the total area of ​​the cultured cells was significantly increased by approximately 1.6 times (Figure 6C). This indicates that Y-27632 is a useful drug for primary culture of monkey corneal endothelial cells, which are considered difficult to culture in vitro.

[0062] Example 5 Optimal concentration of Y-27632 for monkey corneal endothelial cells Monkey corneal endothelial cells (primary culture) collected in the same manner as in Example 2 were placed in corneal endothelial medium containing 1, 10, 33, and 100 μM Y-27632, and in corneal endothelial medium not containing Y-27632, respectively, and stirred to the same concentration, and seeded on a 96-well plate at a density of about 2000 cells per well. 24 hours after the start of culture, CellTiter-Glo (registered trademark ) (Promega) was used to measure the number of viable cells (Figure 7). When corneal endothelial cells were cultured in a medium containing 10 μM Y-27632 24 hours after the start of culture, the number of cells that adhered to the plate was significantly greater than when the cells were cultured in a corneal endothelial medium that did not contain Y-27632 or in a corneal endothelial medium that contained Y-27632 at other concentrations (Figure 7).This demonstrated that Y-27632 at a concentration of 10 μM is the most effective in monkey corneal endothelial cells.

[0063] Example 6 Examination of the effect of Y-27632 on subculture of monkey corneal endothelial cells Descemet's membrane with attached corneal endothelial cells was isolated from the corneal tissue excised from euthanized cynomolgus monkeys. The Descemet's membrane was then soaked in 1.2 U / ml Dispase II (Roche Applied Science ) at 37°C and 5% CO2 for 45 minutes, and then the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and then seeded on a medium for corneal endothelium. Corneal endothelial cells that had reached confluence were incubated with 0.05% trypsin at 37°C and 5% CO2 for 10 minutes for subculture. After 4-6 subcultures, the corneal endothelial cells were placed in a 96-well plate at 1 / 2, 1 / 6, or 1 / 8 dilutions, and in the ROCK inhibitor(+) group, 10 μM Y-27632 was added. In the ROCK inhibitor(-) group, the medium for corneal endothelium was cultured without Y-27632. Subculture was continued using a fresh medium for corneal endothelium. 24 hours after subculture, the cell count was measured using CellTiter-Glo (registered trademark) (Promega) in the same manner as in Example 5 (FIG. 8). When 24 hours after subculture, cultured cynomolgus monkey corneal endothelial cells were subcultured in a medium containing Y-27632, the plate number was increased compared to a medium for corneal endothelium that did not contain Y-27632. The number of cells that adhered to the matrix was significantly higher (Figure 8). This demonstrated that Y-27632 is effective not only in primary culture but also in subculture of monkey corneal endothelial cells.

[0064] Example 7 Examination of the effect of Y-27632 on cell morphology during subculture of monkey corneal endothelial cells Macaque corneal endothelial cells subcultured in the same manner as in Example 6 were subcultured on a slide glass so as to be diluted to 1 / 4. After 24 hours of subculture, the cell morphology was observed using a phase contrast microscope (FIGS. 9A and 9B). Furthermore, the corneal endothelial cells on the slide glass were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then the actin fibers were stained with phalloidin fluorescent stain (FIGS. 10A and 10B). In cynomolgus monkey cultured corneal endothelial cells 24 hours after subculture, when subcultured in a medium containing Y-27632 (Fig. 9B), adhesion of the cells to the slide glass was enhanced, flattening to a corneal endothelial cell-like shape was promoted, and cell clustering was also enhanced, compared to a medium for corneal endothelium not containing Y-27632 (Fig. 9A). In addition, when subcultured in a medium containing Y-27632 (Fig. 10B), actin stress fibers were clearly observed in phalloidin fluorescent staining, compared to a medium for corneal endothelium not containing Y-27632 (Fig. 10A), and it was found that Y-27632 promotes cytoskeleton formation.

[0065] Example 8 Effects of Y-27632 on the cell cycle of monkey corneal endothelial cells Macaca fascicularis corneal endothelial cells were subcultured in the same manner as in Example 6 and subcultured on a slide glass at a dilution of 1 / 4. In the ROCK inhibitor(-) group, the medium for corneal endothelium containing 32 was cultured with Y-27632. A medium for corneal endothelium without addition of any reagent was used. On days 1, 2, and 14 of subculture, the corneal endothelial cells on the slide glass were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then immunostained using anti-Ki67 antibody (Figure 11). Similarly, cynomolgus monkey corneal endothelial cells were subcultured in the ROCK inhibitor(+) and ROCK inhibitor(-) groups at a dilution of 1 / 4, and on days 1 and 2 of subculture, they were recovered using 0.05% trypsin, and flow cytometry was performed using anti-Ki67 antibody to examine the cell cycle (Figures 12 and 13). Compared with the ROCK inhibitor(-) group, the ROCK inhibitor(+) group had more Ki67-positive cells on both the first and second days of subculture, whereas on the 14th day, when the cells reached almost confluence, the number of positive cells in the ROCK inhibitor(+) group was less than that in the ROCK inhibitor(-) group (Fig. 11). Flow cytometry also showed more Ki67-positive cells in the ROCK inhibitor(+) group than in the ROCK inhibitor(-) group on both the first and second days of subculture (Figs. 12 and 13). Since the Ki67 antigen is found in the nuclei of cells in the G1 and S to M phases of the cell proliferation cycle, Y-27632 has the effect of promoting the cell cycle early after subculture of cynomolgus monkey corneal endothelial cells, and it was shown to be a useful drug for efficient cell culture.

[0066] Example 9 Effects of Y-27632 on apoptosis in monkey corneal endothelial cells Macaca fascicularis corneal endothelial cells were subcultured in the same manner as in Example 6, and subcultured to a dilution of 1 / 4. In the ROCK inhibitor(+) group, corneal endothelial cells were cultured in a 1 / 4 dilution. In the ROCK inhibitor(-) group, the corneal endothelial medium was used as the medium for endothelium without Y-27632. On the first day of subculture, all cells, including those in the medium, were harvested using 0.05% trypsin, and then analyzed by flow cytometry using Annexin V and PI (propidium iodide). and examined apoptosis (Figures 14 and 15). A significant decrease in the apoptotic cell rate in the total cells on the first day of subculture was observed in the ROCK inhibitor(+) group compared to the ROCK inhibitor(-) group (Figure 14). 4 The number of apoptotic cells per cell was significantly higher in the ROCK i group than in the ROCK inhibitor(-) group. A significant decrease was observed in the inhibitor(+) group (FIG. 15). This demonstrated that Y-27632 has the effect of suppressing apoptosis during subculture of cynomolgus monkey corneal endothelial cells.

[0067] Example 10 Effects of Y-27632 on the density of cultured monkey corneal endothelial cells Descemet's membrane with attached corneal endothelial cells was isolated from the corneal tissue excised from euthanized cynomolgus monkeys. The Descemet's membrane was then soaked in 1.2 U / ml Dispase II (Roche Applied Science ) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and then seeded in a medium for corneal endothelium. Corneal endothelial cells that had reached confluence were incubated with 0.05% trypsin at 37°C and 5% CO2 for 10 minutes for subculture. A corneal endothelial medium containing 10 μM Y-27632 was used for the ROCK inhibitor(+) group, while a corneal endothelial medium without Y-27632 was used for the ROCK inhibitor(-) group. The culture medium was replaced 72 hours after the start of culture, and from 72 hours onwards, the ROCK inhibitor(+) group In both the control and ROCK inhibitor(-) groups, normal corneal endothelial medium without Y-27632 was used. After each subculture, the cells were photographed using a phase-contrast microscope, and the cell density was measured to examine the effect of Y-27632 on the density of cultured monkey corneal endothelial cells. After seven passages, the ROCK inhibitor(- A higher cell density was observed in the ROCK inhibitor(+) group compared to the ROCK inhibitor(+) group (Figure 16). Therefore, it is believed that the use of Y-27632 will enable the cultivation of corneal endothelial cells at high density, and that this will be useful in the future for regenerative medicine such as the transplantation of cultured corneal endothelial sheets.

[0068] Example 11 Examination of the effect of fasudil on monkey corneal endothelial cell cultures Descemet's membrane with attached corneal endothelial cells was isolated from the corneal tissue excised from euthanized cynomolgus monkeys. The Descemet's membrane was then soaked in 1.2 U / ml Dispase II (Roche Applied Science After incubation with 100% CO2 at 37°C for 45 minutes, the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and then subcultured in a medium for corneal endothelium. The same cell culture medium as in Example 1 was used as the medium for corneal endothelium. During subculture, the cultured monkey corneal endothelial cells were harvested by 0.05% trypsin treatment, separated, and centrifuged. In the fasudil group, 10 μM fasudil (SIGMA-ALDRICH) was added. The corneal endothelial medium was used, and for the control group, corneal endothelial medium without fasudil was used, and the cells were stirred to the same concentration and seeded on a 96-well plate at a density of about 2000 cells per well. 24 hours after the start of culture, the number of adherent cells was measured using CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay (Promega) (Figure 17). Fasudil, a ROCK inhibitor, was also found to have the effect of promoting cell adhesion of corneal endothelial cells.

[0069] Formulation Example 5 Intracameral injection containing a Rho kinase inhibitor The intracameral injection solution shown below is prepared in a conventional manner. Fasudil 10mg Sodium dihydrogen phosphate 0.1g Sodium chloride 0.9g Sodium hydroxide (appropriate amount) Appropriate amount of sterile purified water Total volume 100mL (pH 7)

[0070] Formulation Example 6 Culture medium for preparing corneal endothelial sheet containing a Rho kinase inhibitor The culture medium shown below is prepared by a conventional method. Fasudil 0.5mg FBS 10mL Penicillin-streptomycin solution 1mL FGF basic 200ng DMEM appropriate amount Total volume 100mL FBS is manufactured by Invitrogen, penicillin-streptomycin solution is manufactured by Nacalai Tesque (containing 5000 u / mL penicillin and 5000 μg / mL streptomycin), FGF basic is manufactured by Invitrogen, and DMEM is manufactured by Invitrogen.

[0071] This application is based on Japanese Patent Application No. 2007-223141 (filing date: August 29, 2007) and Japanese Patent Application No. 2008-016088 (filing date: January 28, 2008), the contents of which are incorporated in their entirety herein.

Claims

[Claim 1] The invention described in the examples.

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