Cell inoculant and substrate for cell transplantation

Abstract

The purpose of the present invention is to provide a cell inoculant and a substrate for cell transplantation, which can be subcultured without causing cell transformation and can be efficiently implanted into cells. Provided are an inoculant and a transplantation base material for promoting the implantation of cultured corneal endothelial cells or corneal endothelial alternative cells, each of which contains a phenolphthalein derivative. The inoculant and the substrate for transplantation can promote adhesion of cells during passage operation and implantation of cells during transplantation.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202280039468.4, application date July 7, 2022, and invention name “Cell inoculant and cell transplantation substrate”. Technical Field

[0002] The invention relates to a cell inoculant and a base material for cell transplantation, in particular to an inoculant and a base material for transplantation of corneal endothelial cells or corneal endothelial replacement cells. Background Art

[0003] Due to corneal endothelial damage such as corneal endothelial cell reduction, the function of corneal endothelial cells is impaired and corneal stromal edema occurs. This causes a decrease in corneal transparency and decreased vision. This condition is called bullous keratopathy. On the other hand, it is known that human corneal endothelial cells have almost no regeneration ability once damaged. Therefore, when corneal endothelial cells are reduced due to certain injuries, corneal transplantation is the only effective treatment. In fact, bullous keratopathy due to corneal endothelial dysfunction accounts for about half of corneal transplant cases.

[0004] Currently, patients with corneal endothelial damage are treated with full-thickness corneal transplants that transplant all three layers of the cornea, namely the epithelium, stroma and endothelium. Although full-thickness corneal transplants are a mature technology, there is currently a shortage of corneas in Japan and there is a problem of rejection. In order to solve these problems, "partial transplants" that only transplant damaged tissue are becoming popular. It is known that there are deep lamellar keratoplasty (DLKP) that retains the corneal endothelium and transplants only the epithelium and stroma of the donor, and corneal endothelial transplants that only transplant a part of the cornea including the endothelium. However, for example, in corneal endothelial transplants, the source of the transplant material is still the corneal endothelium itself. Given the limited number of corneal donors, the problem of insufficient donors cannot be overcome, as with full-thickness corneal transplants. In addition, corneal endothelial cells are difficult to culture, and the time and cost required to prepare a sufficient number of cultured cells for transplantation are high.

[0005] In order to solve the problem of insufficient donors, attempts are being made to create cells that become corneal endothelial cells or replace corneal endothelial cells that have the same functions as corneal endothelial cells.

[0006] For example, methods for inducing cells that replace corneal endothelial cells from iPS cells or ES cells have been reported. The induction method and cell culture method for corneal endothelial cells (in fact, since corneal endothelial cell-specific surface markers have not yet been established, cells with properties close to corneal endothelial cells, so-called corneal endothelial-like cells) from iPS cells or ES cells are reviewed in Non-Patent Document 1. In addition, the inventors of the present application have previously established a method for producing cells that replace corneal endothelial cells with the same functions as corneal endothelial cells (Patent Documents 1 to 3), and named the corneal endothelial-like cells obtained by this method "corneal endothelial replacement cells". Clinical research on corneal endothelial replacement cells from iPS cells for bullous keratopathy has also begun.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: WO / 2013 / 051722

[0010] Patent Document 2: WO / 2016 / 093359

[0011] Patent Document 3: WO / 2019 / 142833

[0012] Non-patent literature

[0013] Non-patent literature 1: Hatou S, Shimmura S; Inflamm Regen. 2019; 39: 19. Summary of the invention

[0014] Technical problem to be solved by the invention

[0015] In order to mass-produce target cells, especially corneal endothelial replacement cells induced from iPS cells or ES cells, and convert them into final products, in the manufacturing process, it is necessary not only to proliferate cells, but also to engraft cells as efficiently as possible in the subculture operation (the operation of collecting cells from a cell culture container and re-seeding them in a new cell culture container) across each step. In addition, in order to perform subculture operations without causing cell transformation, the components contained in the culture medium during the subculture operation are important. In addition, the operation of applying a cell suspension to the anterior chamber of the eye and uniformly implanting it on the posterior surface of the cornea (endothelial surface) is an in vivo subculture operation. Like the culture medium during cell inoculation, the components contained in the transplantation substrate are also important.

[0016] In the passage operation of the expansion culture step of iPS cells, a method of temporarily adding a ROCK inhibitor represented by Y-27632 to the culture medium is used. However, the inventors of the present application found that when a ROCK inhibitor is used in the differentiation induction step from iPS cells to corneal endothelial replacement cells, the formation of the cytoskeleton and tight junctions is inhibited, resulting in a transformation that is considered to be an epithelial-mesenchymal transition (EMT). When EMT occurs, the function of corneal epithelial cells as epithelial cells (e.g., barrier function) is reduced.

[0017] Therefore, an object of the present invention is to provide a cell seeding agent and a substrate for cell transplantation, which can be operated by subculturing without causing cell transformation and can efficiently implant cells.

[0018] Solutions to technical problems

[0019] In view of the above problems, the inventors of the present application conducted in-depth research and found that during the passaging operation when inducing corneal endothelial cells or corneal endothelial replacement cells from iPS cells or ES cells, the implantation of cells can be effectively promoted by including phenolphthalein derivatives even in the absence of ROCK inhibitors, and that the phenolphthalein derivatives in the cell transplantation substrate can also promote uniform implantation on the posterior surface of the cornea, resulting in an improved therapeutic effect of the transplanted cells, thereby completing the present invention.

[0020] That is, the present invention provides the following contents.

[0021] [1] An inoculant for promoting implantation of cultured corneal endothelial cells or corneal endothelial substitute cells, comprising a phenolphthalein derivative.

[0022] [2] An inoculant for promoting implantation of corneal endothelial replacement cells, comprising a phenolphthalein derivative.

[0023] [3] The inoculation agent according to [2] above, wherein the corneal endothelial replacement cells are derived from stem cells.

[0024] [4] The inoculation agent according to [3] above, wherein the stem cells are pluripotent stem cells.

[0025] [5] The inoculation agent according to [4] above, wherein the pluripotent stem cells are derived from iPS cells (including cells that have been given additional functions by gene editing or gene introduction).

[0026] [6] An inoculant according to any one of [1] to [5] above, wherein the phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

[0027] [7] The inoculant according to any one of [1] to [6] above, wherein the phenolphthalein derivative is phenol red.

[0028] [8] The inoculant according to [7] above, wherein the phenol red concentration is 1 to 20 mg / L.

[0029] [9] The inoculant according to any one of [1] to [6] above, wherein the phenolphthalein derivative is cresol red.

[0030]

[10] The inoculant according to [9] above, wherein the concentration of cresol red is 1 to 20 mg / L.

[0031]

[11] The inoculant according to any one of [1] to

[10] above, which does not contain a ROCK inhibitor.

[0032]

[12] Use of phenolphthalein derivatives in the manufacture of an inoculant for promoting implantation of cultured corneal endothelial cells or corneal endothelial replacement cells.

[0033]

[13] Use of phenolphthalein derivatives in the manufacture of inoculants for promoting implantation of corneal endothelial replacement cells.

[0034]

[14] The use according to

[12] or

[13] above, wherein the corneal endothelial replacement cells are derived from stem cells.

[0035]

[15] The use according to

[14] above, wherein the stem cells are pluripotent stem cells.

[0036]

[16] The use according to

[15] above, wherein the pluripotent stem cells are derived from iPS cells (including cells that have been endowed with additional functions by gene editing or gene introduction).

[0037]

[17] The use according to any one of

[12] to

[17] above, wherein the phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

[0038]

[18] The use according to any one of

[12] to

[17] above, wherein the phenolphthalein derivative is phenol red.

[0039]

[19] The use according to any one of

[12] to

[17] above, wherein the phenolphthalein derivative is cresol red.

[0040]

[20] A phenolphthalein derivative for use in promoting the engraftment of corneal endothelial cells or corneal endothelial replacement cells when seeding and culturing the cells.

[0041]

[21] A phenolphthalein derivative for use in promoting the engraftment of corneal endothelial replacement cells when seeding the cells.

[0042]

[22] The derivative according to

[20] or

[21] above, wherein the corneal endothelial replacement cells are derived from stem cells.

[0043]

[23] The derivative according to

[22] above, wherein the stem cells are pluripotent stem cells.

[0044]

[24] The derivative according to

[23] above, wherein the pluripotent stem cells are derived from iPS cells (including cells that have been given additional functions by gene editing or gene introduction).

[0045]

[25] A derivative according to any one of

[20] to

[24] above, wherein the phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

[0046]

[26] The derivative according to any one of

[20] to

[25] above, wherein the phenolphthalein derivative is phenol red.

[0047]

[27] The derivative according to any one of

[20] to

[25] above, wherein the phenolphthalein derivative is cresol red.

[0048]

[28] A method for promoting cell implantation, characterized in that corneal endothelial cells or corneal endothelial replacement cells are inoculated and cultured in the presence of a phenolphthalein derivative.

[0049]

[29] A method for promoting cell implantation, characterized in that corneal endothelial replacement cells are inoculated in the presence of a phenolphthalein derivative.

[0050]

[30] The method according to

[28] or

[29] above, wherein the corneal endothelial replacement cells are derived from stem cells.

[0051]

[31] The method according to

[30] above, wherein the stem cells are pluripotent stem cells.

[0052]

[32] The method according to

[31] above, wherein the pluripotent stem cells are derived from iPS cells, including cells that have been endowed with additional functions by gene editing or gene introduction.

[0053]

[33] The method according to any one of

[28] to

[32] above, wherein the phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

[0054]

[34] The method according to any one of

[28] to

[33] above, wherein the phenolphthalein derivative is phenol red.

[0055]

[35] The method according to any one of

[28] to

[33] above, wherein the phenolphthalein derivative is cresol red.

[0056]

[36] A cell suspension comprising the inoculant according to any one of [2] to

[11] above and corneal endothelial replacement cells.

[0057]

[37] The cell suspension according to

[36] , wherein the cell concentration is 1.0 to 20×10 4 cells / mL.

[0058]

[38] The cell suspension according to

[36] or

[37] , characterized in that the 3 cells / cm 2 Above and less than 1.04×10 4 cells / cm 2 Inoculate at an inoculation density of .

[0059]

[39] A transplantation substrate for culturing corneal endothelial cells or corneal endothelial replacement cells, comprising a phenolphthalein derivative.

[0060]

[40] A base material for transplantation of corneal endothelial replacement cells, comprising a phenolphthalein derivative.

[0061]

[41] The substrate according to

[39] or

[40] above, wherein the corneal endothelial replacement cells are derived from stem cells.

[0062]

[42] The substrate according to

[41] above, wherein the stem cells are pluripotent stem cells.

[0063]

[43] The substrate according to

[42] above, wherein the pluripotent stem cells are derived from iPS cells, including cells that have been given additional functions by gene editing or gene introduction.

[0064]

[44] A substrate according to any one of

[39] to

[43] above, wherein the phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), phenolphthalein, bromophenol blue, isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

[0065]

[45] The substrate according to any one of

[39] to

[44] above, wherein the phenolphthalein derivative is phenol red.

[0066]

[46] The substrate according to

[45] above, wherein the phenol red concentration is 1 to 20 mg / L.

[0067]

[47] The substrate according to any one of

[39] to

[44] above, wherein the phenolphthalein derivative is cresol red.

[0068]

[48] ​​The substrate according to

[47] above, wherein the concentration of cresol red is 1 to 20 mg / L.

[0069]

[49] The substrate according to any one of

[39] to

[48] above, which does not contain a ROCK inhibitor.

[0070]

[50] A cell suspension comprising the substrate according to any one of

[39] to

[49] above and corneal endothelial replacement cells.

[0071]

[51] The cell suspension according to

[50] above, wherein the cell suspension is used for transplantation into the anterior chamber.

[0072]

[52] The cell suspension according to

[51] , wherein the cell concentration is 0.5 to 10×10 6 cells / mL.

[0073] Effects of the Invention

[0074] According to the present invention, subculture operation can be performed without causing cell transformation, and cells can be effectively implanted. Corneal endothelial cells and corneal endothelial replacement cells can be mass-produced and cultured, and the implantation rate of transplanted cells can be further improved, thereby improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1The graph shows the results of observing the tight junction formation with and without the use of a ROCK inhibitor (Y-27632) during the passaging operation in the differentiation induction step of corneal endothelial replacement cells derived from iPS cells. The use of the ROCK inhibitor suppressed the formation of tight junctions.

[0076] Figure 2 The figure shows the results of studying the influence of phenolphthalein derivatives (phenol red) on the passage operation in the differentiation induction step of corneal endothelial replacement cells from iPS cells. The upper part (A) is a microscope photograph of the state of cells cultured on the 3rd, 5th and 7th days in the presence or absence of phenol red, and the lower part (B) is a graph showing the results of studying the number and survival rate of cells collected on the 14th day of culture in the presence or absence of phenol red.

[0077] Figure 3 This is a graph showing the results of measuring corneal thickness when corneal endothelial replacement cells derived from iPS cells were transplanted in the presence or absence of phenol red using a bullous keratopathy model in cynomolgus macaques (Macaca fascicularis). Transplantation in the presence of phenol red reduced corneal thickness and was found to improve corneal edema.

[0078] Figure 4 A diagram showing the results of observing the distribution of corneal endothelial replacement cells in the anterior chamber of the eye when corneal endothelial replacement cells derived from iPS cells were transplanted in the presence or absence of phenol red using a cynomolgus monkey bullous keratopathy model. DETAILED DESCRIPTION

[0079] The present invention is described below. Unless otherwise specified, the terms used in this specification have the meanings commonly used in the art.

[0080] 1. Cell inoculants and cell transplantation substrates

[0081] The present invention provides a novel cell inoculant. In cell culture technology, the cell inoculant refers to a liquid (solution, suspension, etc.) that is mixed with cells when cells are inoculated into a new culture container during a subculture operation.

[0082] More specifically, the present invention provides a cell inoculant used in the step of inducing corneal endothelial replacement cells from iPS cells or ES cells, which contains a phenolphthalein derivative. Preferably, the cell inoculant is an inoculant for promoting cell implantation. Preferably, it is an inoculant for promoting implantation of corneal endothelial replacement cells containing a phenolphthalein derivative. In this specification, these are sometimes collectively referred to and abbreviated as the cell inoculant of the present invention.

[0083] In addition, the present invention also provides a novel transplantation matrix. The transplantation matrix refers to a liquid (solution, suspension, etc.) mixed with cells when the cells are administered into the human body or animal body.

[0084] More specifically, the present invention provides a transplantation substrate for culturing corneal endothelial cells or corneal endothelial replacement cells, which contains a phenolphthalein derivative. Preferably, it is a transplantation substrate for corneal endothelial replacement cells containing a phenolphthalein derivative. In this specification, these are sometimes collectively referred to and abbreviated as the cell transplantation substrate of the present invention.

[0085] The cell inoculant of the present invention is suitable for culturing corneal endothelial cells or corneal endothelial substitute cells, preferably for the passaging operation of corneal endothelial substitute cells (the operation of collecting cells from a cell culture container and re-seeding them into a new cell culture container). The cell transplantation substrate of the present invention is suitable for use when transplanting cultured corneal endothelial cells or corneal endothelial substitute cells (preferably corneal endothelial substitute cells) into the anterior chamber of the eye.

[0086] (cell)

[0087] In the cell seeding agent or cell transplantation substrate of the present invention, the target cells are cultured corneal endothelial cells or corneal endothelial replacement cells, preferably corneal endothelial replacement cells, more preferably corneal endothelial replacement cells derived from stem cells. Here, the stem cells are preferably pluripotent stem cells, more preferably iPS cells.

[0088] The cultured corneal endothelial cells may be primary cultured cells or cell lines. However, since primary culture of corneal endothelial cells is difficult, it is preferable to use a cell line.

[0089] In the present invention, corneal endothelial replacement cells are cells induced from stem cells such as iPS cells, ES cells, or cells endowed with additional functions by gene editing or gene introduction into these cells, which can treat corneal endothelial dysfunction and replace corneal endothelial cells. In other words, corneal endothelial replacement cells have the same physiological functions as corneal endothelial cells.

[0090] Stem cells refer to cells that can be cultured in vitro and differentiated into a variety of cell lineages that constitute the body, specifically embryonic stem cells (ES cells), pluripotent stem cells (EG cells) from fetal primordial germ cells, pluripotent stem cells (GS cells) from testicles, induced pluripotent stem cells (induced pluripotent stem cells; iPS cells) from somatic cells, and human adult stem cells (tissue stem cells). As iPS cells, cells from any warm-blooded animal (preferably mammals) can be used. As mammals, for example, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, monkeys, humans, etc. can be listed. Preferably, iPS cells from humans can be used.

[0091] Specifically, iPS cells include, for example, cells obtained by introducing multiple genes into somatic cells such as skin cells and having acquired the same pluripotency as ES cells. For example, iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene, C-Myc gene and Sox2 gene, and iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene (Nat Biotechnol 2008; 26: 101-106). In addition, technical improvements are being made in methods for producing iPS cells, such as methods for further reducing the number of introduced genes (Nature. 2008 Jul 31; 454(7204): 646-50), methods for using low molecular weight compounds (Cell Stem Cell. 2009 Jan 9; 4(1): 16-9, Cell Stem Cell. 2009 Nov 6; 5(5): 491-503), and methods for replacing genes with transcription factor proteins (Cell Stem Cell. 2009 May 8; 4(5): 381-4). However, regardless of the production method, as long as the basic characteristics of the iPS cells produced (i.e., pluripotency) are the same, they can all serve as a source of corneal endothelial replacement cells used in the present invention.

[0092] Specifically, as iPS cells, 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 836B3, FF-I14s03, FF-I01s04, MH09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs 09, QHJI14s03, QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all iPS Academia Japan Company or Tokyo University iPS Research Foundation), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), and Toe (JCRB1338 strain), Lollipop (JCRB1336 strain) (Senior Medical Center, Institute of Basic Medical Sciences, Department of Intractable Diseases and Disease Resources, JBRC Cell Bank), UTA-1 strain and UTA-1-SF-2-2 strain (both from Tokyo University), 21526, 21528, 21530, 21531, 31536, 31538 strains (all from FUJIFILM Cellular Dynamics), etc.

[0093] The corneal endothelial replacement cells used as the target in the cell inoculation agent or cell transplantation substrate of the present invention can be, for example, corneal endothelial-like cells developed by the inventors of the present application (Patent Documents 1 to 3), and can be manufactured and prepared by the methods described in Patent Documents 1 to 3. Preferably, the corneal endothelial replacement cells (Corneal Endothelial Cell Substitute from iPS cells; CECSi cells) are derived from iPS cells (including cells endowed with additional functions by gene editing or gene introduction), characterized in that they have corneal endothelial cell-like properties and functions, and the gene expression level of NR3C2 (nuclear receptor subfamily 3, group C, member 2) is enhanced (Patent Document 3).

[0094] Specific examples of corneal endothelial cell-like properties and functions possessed by corneal endothelial replacement cells include the following characteristics (i) to (iv). The cells possess at least one of these characteristics, preferably two, more preferably three, and most preferably all four.

[0095] (i) Cell-cell adhesion is composed of N-cadherin.

[0096] (ii) Tight junctions are formed between cells.

[0097] (iii) Na,K-ATPase α1 subunit is expressed on the cell membrane.

[0098] (iv) Expression of transcription factor PITX2 was observed in the cell nucleus.

[0099] Whether or not cell-cell adhesion is constituted by N-cadherin can be confirmed by immunostaining for N-cadherin.

[0100] Whether tight junctions are formed between cells can be confirmed by immunostaining ZO-1 to observe the presence of ZO-1, a protein constituting tight junctions, or by directly observing the structure using an electron microscope.

[0101] Whether or not Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by immunostaining ZO-1 and Na,K-ATPase α1 subunit to co-stain the two.

[0102] Whether the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining of PITX2.

[0103] In one embodiment of the present invention, the corneal endothelial replacement cells used as the object in the cell inoculant or cell transplantation substrate of the present invention are characterized by having a CD24 positive phenotype. CD24 is a highly glycosylated small mucin-like glycosylphosphatidyl-inositol (GPI) binding cell surface protein. CD24 is highly expressed in hematopoietic cells such as B cells, T cells, neutrophils, eosinophils, dendritic cells and macrophages, and non-hematopoietic cells such as nerve cells, ganglion cells, epithelial cells, keratinocytes, muscle cells, pancreatic cells and epithelial stem cells. In recent years, it has been reported that CD24 has the function of avoiding macrophage phagocytosis. Therefore, the cell inoculant or cell transplantation substrate of the present invention containing corneal endothelial replacement cells with a CD24 positive phenotype can be expected to reduce rejection during transplantation. On the other hand, human corneal endothelial cells are reported to be CD24 negative (Japanese Patent Table No. 2019-510509, Japanese Patent Laid-Open No. 2020-073602).

[0104] The presence or absence of a CD24-positive phenotype can be confirmed by immunostaining for CD24 antigen expressed on the cell surface.

[0105] Each immunostaining is routinely performed in the art, and the reagents, equipment, etc. used can be purchased commercially or prepared according to previous reports.

[0106] In the above-mentioned subculturing operation in the step of inducing differentiation of corneal endothelial replacement cells, stem cells such as iPS cells, ES cells, or cells endowed with additional functions by gene editing or gene introduction are added to the cell inoculum and are usually cultured at a rate of 1.0 to 20×10 4 cells / mL, preferably 2.5 to 10×10 4 The cell suspension was suspended at a concentration of 1.04×10 3 cells / cm 2 Above and less than 1.04×10 4 cells / cm 2 , preferably 1.04×10 3 cells / cm 2 Above and 5.02×10 3 cells / cm 2 The following inoculation density is inoculated into a culture container such as a culture dish.

[0107] During transplantation into the eye, especially into the anterior chamber of the eye, the cultured corneal endothelial cells or corneal endothelial substitute cells, preferably corneal endothelial substitute cells, are added to a cell transplantation substrate and are usually transplanted at 0.5 to 10×10 6 cells / mL, preferably 1.0 to 5×10 6 If the cell concentration and / or seeding density is too high or too low, the cell implantation efficiency will be reduced.

[0108] In the present invention, the cultured corneal endothelial cells or corneal endothelial replacement cells can be a spherical cell mass (hereinafter also referred to as a spheroid) obtained by aggregating and spheroidizing tens to hundreds of cells through suspension culture. Here, "spheroid" includes not only the completely spherical case, but also roughly spherical shapes such as oval and rugby ball shapes. The diameter or area equivalent diameter of the spheroid is preferably in the range of 10 to 200 μm, more preferably in the range of 30 to 150 μm, particularly preferably in the range of 30 to 100 μm, further preferably in the range of 30 to 90 μm, and further preferably in the range of 40 to 80 μm. If the spheroid is too large, the implantation will be biased and cannot be implanted uniformly. If the spheroid is too small, it will be difficult to attach and the implantation efficiency will be reduced. If the size of the spheroid is within this range, the cells can be effectively implanted at the transplant site. "Area equivalent diameter" refers to the diameter of a circle equal to the projected area of ​​the particle (spheroid in this application), also known as the Heywood diameter or equivalent circle diameter. That is, the diameter of a circle having an area equal to the projected area of ​​the spherical body of the substantially spherical shape.

[0109] The cell concentration or seeding density can be determined by sampling a portion of the cell suspension and measuring the number of cells in the sample; in the case of a spheroid suspension, sampling a portion of the spheroid suspension, enzymatically treating the spheroids in the sample to disperse the cells one by one to measure the number of cells, and using the result to determine the cell concentration or seeding density.

[0110] (Phenolphthalein derivatives)

[0111] The cell seeding agent or cell transplantation substrate of the present invention is characterized by containing a phenolphthalein derivative. The phenolphthalein derivative is not particularly limited as long as it has a basic skeleton represented by the following formula and can obtain the desired effect.

[0112]

[0113] In the formula, -X- is -SO2- or -CO-, ring Ar1 and ring Ar2 are the same or different and are an aromatic ring with 6 or 12 carbon atoms (e.g., benzene, naphthalene) which may have 1 to 3 substituents (e.g., a branched alkyl group with 1 to 3 carbon atoms such as methyl, ethyl, propyl, isopropyl, etc.; a halogen atom such as bromine atom, chlorine atom, etc.; an acyl group such as acetyl and isovaleryl, etc.).

[0114] Preferably, -X- is -SO2. Ring Ar1 and ring Ar2 are the same or different and are benzene which may have 1 to 3, preferably 1 or 2, substituents (eg, methyl, isopropyl, bromine atoms).

[0115] The phenolphthalein derivative may be a salt or an ester thereof, or may be an acid adduct thereof.

[0116] As phenolphthalein derivatives, for example, phenolsulfonphthalein (phenol red), phenolphthalein, bromophenol blue, isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronide, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple can be listed. Phenolphthalein derivatives can be prepared by methods known per se. As phenolphthalein derivatives, it is known that phthalic acid and various corresponding phenols can be used for synthesis. If the phenol used is phenol, phenolphthalein can be synthesized. In addition, various phenolphthalein derivatives can be purchased commercially. From the viewpoint of convenience and quality stability, commercial purchase is preferred. As phenolphthalein derivatives, phenol red (also called phenolsulfonphthalein) (left) and cresol red (right) of the following structures are preferred. Phenol red is usually included in the culture medium as a pH indicator with a color change range of pH 6.8 to pH 8.4 (yellow to red), but from the perspective of cell culture, it is an optional additive, and there are also commercially available culture media without phenol red. In addition, it is known that cresol red is a pH indicator with a color change range of pH 7.2 to pH 8.8 (yellow to red). The concentration of phenol red contained in the cell inoculant or cell transplantation substrate of the present invention is usually 1 to 20 mg / L, preferably 1 to 15 mg / L, and more preferably 5 to 10 mg / L. The concentration of cresol red contained in the cell inoculant or cell transplantation substrate of the present invention is usually 1 to 20 mg / L, preferably 1 to 15 mg / L, and more preferably 5 to 10 mg / L during differentiation induction. It is 1 to 16 mg / L during transplantation, preferably 1 to 12 mg / L, and more preferably 4 to 8 mg / L. The concentration of the phenolphthalein derivative other than phenol red and cresol red is usually 1 to 20 mg / L, preferably 1 to 15 mg / L, and more preferably 5 to 10 mg / L.

[0117]

[0118] The "desirable effect" possessed by the phenolphthalein derivative refers to an effect of promoting engraftment of cells after passaging or transplantation.

[0119] Examples of the administration targets of the substrate for cell transplantation of the present invention include mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, and humans, preferably humans.

[0120] The dosage of the cell transplantation substrate of the present invention cannot be generalized according to the weight, age, symptoms, etc. of the subject, but if the body weight is 25 to 300 kg, the age is 16 to 100 years old, and the symptoms are bullous keratopathy, 50 to 200 μL is injected into the anterior chamber of each eye. The cell transplantation substrate of the present invention can suspend cells or cell spheroids and apply them to the anterior chamber as a cell suspension, which will not cause side effects such as increased intraocular pressure or obvious inflammation, and is safe. Diseases suitable for transplantation are diseases that require corneal endothelial transplantation, including but not limited to bullous keratopathy, keratoconus, keratitis, corneal chemical burns, corneal stromal dystrophy, corneal edema, and corneal leukoma.

[0121] 2. Method for producing a cell seeding agent or a cell transplantation substrate

[0122] The cell inoculant or cell transplantation substrate of the present invention contains a medium for dissolving and / or diluting the phenolphthalein derivative and maintaining cell survival.

[0123] There is no particular restriction as the medium of cell inoculant, as long as it has the ability to make the cell proliferation and implantation after inoculation, physiological saline, various physiological buffers (such as PBS, HBSS, etc.), various mediums based on the basal medium for cell culture, etc. can be used. There is no particular restriction as the basal medium, as long as it can be used for the cultivation of animal cells, for example, MEM culture medium, BME culture medium, BGJb culture medium, CMRL 1066 culture medium, Glasgow MEM culture medium, Improved MEM Zinc Option culture medium, IMDM culture medium, Medium 199 culture medium, Eagle MEM culture medium, αMEM culture medium, DMEM culture medium, Ham's culture medium (for example, F10, F12), RPMI 1640 culture medium, Fischer's culture medium and their mixed culture medium, etc. These culture mediums can all be purchased commercially. In addition, these culture mediums can be serum-containing culture medium, serum-free culture medium. It is preferably serum-free culture medium. When the culture medium used in the present invention is a serum-containing culture medium, mammalian serum such as bovine serum and fetal bovine serum can be used, and the concentration of the serum in the culture medium is 0.1 to 20% (v / v), preferably 1 to 10% (v / v).

[0124] There is no particular limitation on the medium used as the transplantation substrate, as long as it is a solution suitable for intraocular administration, especially intracameral administration, and non-irritating isotonic culture fluid or physiological saline, buffer, etc. can be used. Preferably, the same culture fluid as that used to culture corneal endothelial cells or corneal endothelial replacement cells is used. In addition to phenolphthalein derivatives, the transplantation substrate can contain various components as needed. The component is not particularly limited, as long as it is effective for the cultured corneal endothelial cells or corneal endothelial replacement cells administered (transplanted) into the anterior chamber to differentiate into a corneal endothelial cell layer without shedding, and preferably insulin or insulin-like growth factor is added. In addition, retinoic acid, EGF and bFGF are also preferably added components.

[0125] The cell inoculation agent and the cell transplantation substrate of the present invention can be prepared by adding and dissolving the phenolphthalein derivative into the above-mentioned medium. The concentration of the phenolphthalein derivative added to the medium has been described in the above-mentioned "1. Cell inoculation agent and cell transplantation substrate", and is the same as the concentration of the phenolphthalein derivative contained in the cell inoculation agent or cell transplantation substrate of the present invention, and is usually 1 to 20 mg / L, preferably 1 to 15 mg / L, and more preferably 5 to 10 mg / L.

[0126] Example

[0127] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to the following examples. In addition, unless otherwise indicated, all reagents and materials used can be purchased commercially. Unless otherwise indicated, the abbreviations used herein are the same as those commonly used in the art.

[0128] (Materials and methods)

[0129] Reference Example 1: Effects of ROCK inhibitors on tight junction formation

[0130] The effects of ROCK inhibitors on the passage operation during the differentiation induction of corneal endothelial replacement cells from iPS cells were studied. The expression of ZO-1 was studied according to Example 1 and Test Example 2 of Patent Document 3 (however, 10 μM Y-27632 was used instead of fasudil as the ROCK inhibitor). The results are shown in Figure 1 The use of ROCK inhibitors was found to inhibit the formation of tight junctions and lead to a transformation known as epithelial-mesenchymal transition. These results suggest the need to develop a cell seeding agent that does not contain ROCK inhibitors.

[0131] Example 1: Effects of phenolphthalein derivatives on engraftment and survival of cells after inoculation

[0132] (Materials and methods)

[0133] 1. Culture medium for induction of corneal endothelial replacement cells

[0134] DMEM / F12 medium (Nacalai Tesque, #08460-95) was used as the basal medium. N-2MAX medium supplement (100X) (R&D System, #AR009), ascorbic acid (Fuso Pharmaceutical Co., Ltd.; ascorbic acid injection (50 μg / mL)), IGF1 (Orphan Pacific; Mecasermin Injection; Somazon Injection) (20 ng / mL)), KGF (Fuji Film Wako Pure Chemical, #119-00661 (5 ng / mL)), LIF (Sigma-Aldrich, #L5283 (1 ng / mL)), IL6 (Fuji Film Wako Pure Chemical, #119-00661 (5 ng / mL)), and IL-6 (Fuji Film Wako Pure Chemical, #119-00661 (5 ng / mL)) were added to the DMEM / F12 basal medium. Chemical; #099-04631 (1 ng / mL)), epinephrine (Daiichi Sankyo, Bosmin injection (0.5 μg / mL)), dexamethasone (Kyowa CritiCare, Orgadrone injection (38 ng / mL)) and aldosterone (Sigma-Aldrich, #A9477 (720 ng / mL)) were used as additives, and culture dishes or culture flasks coated with laminin 511-E8 fragment (iMatrix511, Nippi) at a concentration of 3 μg / ml were used as scaffold materials.

[0135] 2. Induction of corneal endothelial replacement cells

[0136] The method described in the embodiment of Patent Document 3 was used for implementation. The details are as follows.

[0137] Y-27632 (Wako / 039-24591) was added to the iPS cell culture medium AK03N (Ajinomoto) at a concentration of 10 μM (hereinafter referred to as AK03N+Y medium). A 6-well culture plate (Greiner 657160) was coated with iMatrix511 at a concentration of 6 μg / ml. 4 cells / cm 2 iPS cells (FF-I01s04 strain) were suspended in AK03N+Y medium at a density of 1.5 and seeded into the 6-well plate. 2The cells were cultured in an incubator and the next day, the medium was replaced with AK03N medium without Y-27632. After that, the medium was replaced every 2 to 3 days, and the cells were passaged once a week on a 6-well plate coated with iMatrix511 using TrypLE-Select (Thermo Fisher Scientific) at the same density as above. iPS cells of more than 3 passages and less than 29 passages were used to induce corneal endothelial replacement cells.

[0138] When induction begins, iPS cells are collected from the 6-well plate using TrypLE-Select and suspended in the above-mentioned medium for corneal endothelial replacement cell induction. 35 mm culture dishes or 100 mm culture dishes are pre-coated with iMatrix511 at a concentration of 3 μg / ml and incubated at 2.0 × 10 4 cells / cm 2 iPS cells were seeded at a density of 1.50 μl and incubated at 37°C in a CO 2 Start culturing in an incubator, and then replace the culture medium every 2 to 3 days.

[0139] Example 2: Effect of phenolphthalein derivatives on the passage procedure in the differentiation induction step of corneal endothelial replacement cells from iPS cells

[0140] When induction into corneal endothelial replacement cells was initiated, iPS cells collected using TrypLE-Select (Thermo Fisher Scientific) were suspended in a cell seeding medium supplemented with phenol red or a cell seeding medium without phenol red. 100 mm culture dishes (Sumitomo Bakelite) were pre-coated with iMatrix511-E8 (iMatrix511, Nippi) at a concentration of 3 μg / ml and incubated at 1.0×10 4 cells / cm 2 iPS cells suspended in each cell inoculum were seeded at a density of . Cultivation was started in a CO2 incubator at 37°C, and then the medium for induction of corneal endothelial replacement cells supplemented with phenol red or the medium for induction of corneal endothelial replacement cells without phenol red was replaced every 2 to 3 days.

[0141] The cells were cultured in the above-mentioned medium for induction of corneal endothelial replacement cells for 14 days. After 3, 5 and 7 days of culture, the cell status was confirmed under a microscope ( Figure 2A). After 14 days of culture, for the group in which cell implantation was confirmed under a microscope, cells were collected and the cell number was measured. The cells were collected by removing the culture supernatant, washing twice with DPBS (-) (Thermo Fisher Scientific), stripping with Accutase (Innovative Cell Technologies), and collecting in a centrifuge tube. The cells collected in the centrifuge tube were separated by centrifugation, and after adjusting the cell concentration using culture medium, the cell concentration and viability were measured using an automatic cell counter (ChemoMetec) ( Figure 2 B).

[0142] (result)

[0143] During the passage operation in the differentiation induction step of corneal endothelial replacement cells derived from iPS cells, the number of cells engrafted after cell seeding was studied, and which components in the cell seeding agent had a key influence were explored. It was found that phenol red was a key influencing component.

[0144] After inoculation, the cells were cultured with medium supplemented with or without phenol red, and the cells were observed on days 3, 5, and 7. The results are shown in Figure 2 As shown in (A). When the cells were cultured in a medium supplemented with phenol red after inoculation, the engraftment was significantly better.

[0145] Furthermore, after inoculation, the cells were cultured in a medium supplemented with or without phenol red, and the number of live cells and the survival rate collected on the 14th day were compared. The results showed that the number of live cells and the survival rate collected in the case of culture in a medium supplemented with phenol red were significantly superior.

[0146] Example 3: Effects of phenolphthalein derivatives on engraftment of transplanted cells and corneal edema

[0147] (Materials and methods)

[0148] The cell suspension of corneal endothelial substitute cells derived from iPS cells obtained in Example 1 (8×10 5 Cells / 160 μl) were transplanted into the anterior part of the eye of a cynomolgus macaque model of bullous keratopathy (contracted to Shin Nippon Scientific Co., Ltd.). Two types of substrates for red cell transplantation (containing insulin, insulin-like growth factor, etc.) were prepared, with or without phenol added.

[0149] The corneal endothelial replacement cell suspension was transplanted into the anterior chamber of the eye according to the following sequence.

[0150] (1) Using a 27-gauge syringe, corneal endothelial replacement cells suspended in two transplant base materials with or without phenol red were injected into the anterior chamber.

[0151] (2) The cynomolgus macaques were placed face down for 3 hours.

[0152] (3) After 28 days of observation, the eyeballs were collected and observed with the naked eye and a microscope.

[0153] The formation of corneal endothelial replacement cell layer and corneal thickness were measured in transplanted eyes.

[0154] Corneal thickness was measured using a corneal thickness measurement device (Tomey Corporation; SP-100).

[0155] (result)

[0156] The results are shown in Figure 3 and Figure 4 When transplantation was performed using a transplant base material to which phenol red was added, corneal endothelial replacement cells were confirmed to be implanted and spread evenly on the posterior surface of the cornea. On the other hand, when transplantation was performed using a transplant base material to which phenol red was not added, the implantation of the transplanted cells was uneven ( Figure 4 ). In addition, regarding corneal thickness, when the transplant base material to which phenol red was added was used, a reduction in corneal thickness was observed, and thus, an effect of improving corneal edema was confirmed. When the transplant base material to which phenol red was not added was used, no significant reduction in corneal thickness was confirmed.

[0157] Example 4: Phenolphthalein corneal endothelial replacement cell induction scheme

[0158] 1. iPS cell expansion culture (thawing)

[0159] T12.5 flasks (Corning / 353107) were coated with iMatrix511 (Matrixome / 892005) at a concentration of 6 μg / mL. iPS cells (FF-I01s04 strain) were suspended in AK03N+Y medium and cultured at 8.0×10 3 cells / cm 2 The T12.5 flask was inoculated at a density of 1.5 μg / mL and incubated at 37 °C in CO 2 The cells were cultured in an incubator and the next day, the medium was replaced with AK03N medium without Y-27632. After an interval of 2 days, the medium was replaced for 2 consecutive days and the cells were subcultured on the 6th day of culture.

[0160] T25 flasks (Sumitomo Bakelite / MS-23050) were coated with iMatrix511 (Matrixome / 892005) at a concentration of 6 μg / mL. iPS cells were suspended in AK03N+Y medium and cultured at 2.2×10 3 cells / cm 2The T25 flask was inoculated at a density of 1.54 g / cm2 and incubated at 37°C in CO 2 The cells were cultured in an incubator and the next day, the medium was replaced with AK03N medium without Y-27632. After an interval of 2 days, the medium was replaced for 2 consecutive days and the cells were subcultured on the 6th day of culture.

[0161] 2. Preparation of Phenol Red-free Culture Medium for Induction of Corneal Endothelial Replacement Cells

[0162] A phenol red-free medium for inducing corneal endothelial replacement cells (hereinafter referred to as a phenol red-free medium in this example) was prepared using DMEM / F12 non-phenol red (Thermo Fisher / 11039-021) as a basal medium. As additives, ITS-supplement (Ventria / 777ITS091), IGF1 (Orphan Pacific; Mecasermin Injection; Somazon Injection / 858100075, 1 mg / mL), LIF (Fuji Film Wako Pure Chemical / 125-06661), IL-6 (Miltenyi Biotec / 170-076-161), IL-11 (Peprotech / AF-200-11), TNFα (Miltenyi Biotec / 170-076-178), and water-soluble hydrocortisone sodium phosphate injection (Nippon Ikko / 27126333) described in Table 1 were added to 500 mL of the basal medium.

[0163] Table 1

[0164] Reagent name Addition amount Final concentration LIF 200μL 10 ng / mL IL-11 250μL 5ng / mL IL-6 250μL 10 ng / mL TNFα 100μL 10 ng / mL ITS-Additives 5mL 10mL / L IGF1 5μL 10 ng / mL Hydrocortisone sodium phosphate 20μL 200nM

[0165] 3. Preparation of phenolphthalein solution

[0166] Phenolphthalein derivatives B to D shown in Table 2 below were dissolved in ethanol to a concentration of 6.6×10 -6mol / mL. The culture medium for inducing corneal endothelial replacement cells without phenol red prepared in 2. above was dispensed into 4 50 mL tubes at 30 mL / each, and 40.5 μL of 0.6% A phenolsulfonphthalein injection (AFP) was added to one of the 4 tubes. In addition, 100 μL of the B cresol red solution after adjusting the concentration was added to one of the remaining 3 tubes. Similarly, C thymol blue solution and D bromocresol purple solution were added to the remaining two tubes, respectively. A total of four different culture media containing phenolphthalein derivatives were prepared and used as cell inoculants with the addition of phenolphthalein derivatives. Each inoculant was adjusted to a phenolphthalein derivative concentration of 0.22 mM.

[0167] Table 2

[0168]

[0169] 4. Differentiation induction (seeding density is fixed at 1.04×10 4 cells / cm 2 )

[0170] 90 μL of laminin 511-E8 fragment (iMatrix511MG) was added to 30 mL of DPBS (-), added to a 6-well plate at a concentration of 1.5 mL / well, coated as a scaffold material at a concentration of 3 μg / ml, and allowed to stand at 37°C for more than 1 hour. The supernatant was removed, and the cell inoculum prepared in 3. above with various phenolphthalein derivatives A to D added was added at 2 mL / well and incubated at 37°C. After the iPS cells were expanded and cultured in 1. above, the collected iPS cells were centrifuged at 140 × g for 5 minutes at 25°C, the supernatant was removed, and the cells were suspended in a cell inoculum with various phenolphthalein derivatives A to D added or in a culture medium without phenolphthalein at a seeding density of 1.04 × 10 4 cells / cm 2 The cells were seeded into iMatrix-coated 6-well plates. The cells were kept at 37°C in a CO 2 The cells were incubated in an incubator, with the time of inoculation being designated as day 0. The medium was replaced for the first time at 2 mL / well on day 3, and then every other day. Cells were fixed and stained on day 7 after inoculation.

[0171] 5. Differentiation induction (changing seeding density)

[0172] As in 4. above, after iMatrix coating, the supernatant was removed, and the usual differentiation induction medium (a medium obtained by adding the additives described in the above Table 1 to DMEM / F12 containing phenol red (ThermoFisher / 11330057)) as a positive control, a medium containing no phenol red using DMEM / F12 non-phenol red (Thermo Fisher / 11039-021) instead of DMEM / F12 as a negative control, a medium supplemented with A phenol red cell inoculant, and a medium supplemented with B cresol red cell inoculant were added to 5 wells at 2 mL / well, respectively, and incubated at 37°C. After expansion and culture in the same manner as in 1. above, the collected iPS cells were centrifuged at 140 × g for 5 minutes at 25°C, the supernatant was removed, and the cells were suspended in the positive control, negative control, medium supplemented with A-phenol red cell inoculant, and medium supplemented with B-cresol red cell inoculant at a seeding density of 1.04 × 10 3 cells / cm 2 , 5.0 2 ×10 3 cells / cm 2 , 1.04×10 4 cells / cm 2 , 5.02×10 5 cells / cm 2 , 1.04×10 5 cells / cm 2 The cells were inoculated into the wells of the 6-well plate coated with iMatrix, which contained A-phenol red cell inoculum, B-cresol red cell inoculum, positive control, and negative control. 2 The cells were incubated in an incubator, with the time of inoculation being designated as day 0. The medium was replaced for the first time at 2 mL / well on day 3, and then every other day. Cells were fixed and stained on day 7 after inoculation.

[0173] 6. Results

[0174] In the medium without phenol red, when the inoculation density is specified, 1.04×10 4 cells / cm 2 The following vaccinations will not be implanted.

[0175] In the medium supplemented with phenol red and cresol red, cell engraftment was confirmed at a seeding number lower than the predetermined cell seeding density.

[0176] In the medium containing thymol blue and bromocresol purple, 1.04×104 cells / cm 2 During vaccination, implantation was also confirmed.

[0177] Industrial Applicability

[0178] According to the present invention, subculture operation can be performed without causing cell transformation and cells can be effectively transplanted. It can mass-produce and culture corneal endothelial cells and corneal endothelial replacement cells and improve the implantation rate of transplanted cells, thereby improving the therapeutic effect.

[0179] This application is based on U.S. Provisional Invention Application No. 63 / 219,101 filed in the United States (filing date: July 7, 2021), the contents of which are incorporated herein in their entirety.

Claims

1. An inoculant for promoting implantation of cultured corneal endothelial cells or corneal endothelial substitute cells, comprising a phenolphthalein derivative.

2. An inoculant for promoting implantation of corneal endothelial replacement cells, comprising a phenolphthalein derivative.

3. The inoculant according to claim 2, wherein The corneal endothelial replacement cells are derived from stem cells.

4. The inoculant according to claim 3, wherein The stem cells are pluripotent stem cells.

5. The inoculant according to claim 4, wherein The pluripotent stem cells are derived from iPS cells, and include cells that have been endowed with additional functions through gene editing or gene introduction.

6. The inoculant according to any one of claims 1 to 5, wherein The phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

7. The inoculant according to any one of claims 1 to 6, wherein A phenolphthalein derivative is phenol red.

8. The inoculant according to claim 7, wherein The concentration of phenol red is 1-20 mg / L.

9. The inoculant according to any one of claims 1 to 6, wherein A derivative of phenolphthalein is cresol red.

10. The inoculant according to claim 9, wherein The concentration of cresol red is 1-20 mg / L.

11. The inoculant according to any one of claims 1 to 10, characterized in that Does not contain ROCK inhibitor.

12. A transplantation substrate for culturing corneal endothelial cells or corneal endothelial replacement cells, comprising a phenolphthalein derivative.

13. A base material for transplantation of corneal endothelial replacement cells, comprising a phenolphthalein derivative.

14. The substrate according to claim 12 or 13, wherein The corneal endothelial replacement cells are derived from stem cells.

15. The substrate according to claim 14, wherein The stem cells are pluripotent stem cells.

16. The substrate according to claim 15, wherein The pluripotent stem cells are derived from iPS cells, and include cells that have been endowed with additional functions through gene editing or gene introduction.

17. The substrate according to any one of claims 12 to 16, wherein The phenolphthalein derivative is selected from phenolsulfonphthalein (phenol red), phenolphthalein, bromophenol blue, isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronide, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue and bromocresol purple.

18. The substrate according to any one of claims 12 to 17, wherein A phenolphthalein derivative is phenol red.

19. The substrate according to claim 18, wherein The concentration of phenol red is 1-20 mg / L.

20. The substrate according to any one of claims 12 to 17, wherein A derivative of phenolphthalein is cresol red.

21. The substrate according to claim 20, wherein The concentration of cresol red is 1-20 mg / L.

22. The substrate according to any one of claims 12 to 21, characterized in that Does not contain ROCK inhibitor.

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