Cellular inocula and substrates for cell transplantation

Abstract

The present invention aims to provide a cell inoculum and a cell transplantation substrate which can be subcultured without causing cell transformation and can effectively implant cells. An inoculum and a transplantation substrate containing a phenolphthalein derivative for facilitating implantation of cultured corneal endothelial cells or corneal endothelial replacement cells. The inoculum and the transplantation substrate can facilitate adhesion of cells during subculture and implantation of cells during transplantation.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202280039468.4, filed on July 7, 2022, entitled "Cell Inoculation Agent and Substrate for Cell Transplantation". Technical Field

[0002] This invention relates to a cell inoculation agent and a substrate for cell transplantation, and more particularly to an inoculation agent and a substrate for transplantation of corneal endothelial cells or corneal endothelial substitute cells. Background Technology

[0003] Damage to the corneal endothelium, such as a reduction in corneal endothelial cells, impairs the function of these cells and leads to corneal stromal edema. This results in decreased corneal transparency and decreased vision. This condition is called bullous keratosis. On the other hand, it is known that human corneal endothelial cells have almost no regenerative capacity once damaged. Therefore, when corneal endothelial cells are reduced due to certain injuries, corneal transplantation is the only effective treatment. In fact, bullous keratosis 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 keratoplasty, which transplants all three layers of the cornea: epithelium, stroma, and endothelium. While full-thickness keratoplasty is a mature technique, Japan currently faces a shortage of corneal tissue and issues with rejection. To address these problems, "partial transplantation," which transplants only the damaged tissue, is becoming more common. Known methods include deep lamellar keratoplasty (DLKP), which preserves the corneal endothelium while transplanting only the donor's epithelium and stroma, and corneal endothelial transplantation, which transplants only a portion of the cornea containing the endothelium. However, in corneal endothelial transplantation, for example, the source of the transplant material is still the corneal endothelium itself. Given the limited number of corneal donors, the problem of donor shortage remains unresolved, similar to full-thickness keratoplasty. Furthermore, corneal endothelial cells are difficult to culture, and the time and cost required to prepare a sufficient number of cultured cells for transplantation are substantial.

[0005] To address the shortage of donor cells, researchers are attempting to create cells that function as corneal endothelial cells or as substitutes for them.

[0006] For example, methods for inducing cells from iPS cells or ES cells to become replacement corneal endothelial cells have been reported. Methods for inducing and culturing cells from iPS cells or ES cells into corneal endothelial cells (actually, since specific surface markers for corneal endothelial cells have not yet been established, these are cells with properties similar to corneal endothelial cells, i.e., so-called corneal endothelial-like cells) are reviewed in Non-Patent Literature 1. Furthermore, the inventors of this application previously established methods for manufacturing cells that become replacement corneal endothelial cells with equivalent functions to corneal endothelial cells (Patent Literature 1-3), and named the corneal endothelial-like cells obtained by this method "corneal endothelial replacement cells." Clinical studies of corneal endothelial replacement cells derived from iPS cells for bullous keratosis have also begun.

[0007] Existing technical documents

[0008] Patent documents

[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] The technical problem that the invention aims to solve

[0015] To mass-produce target cells, especially corneal endothelial replacement cells induced from iPS or ES cells, and convert them into the final product, the manufacturing process requires not only cell proliferation but also efficient cell engraftment during passage operations (the process of collecting cells from cell culture containers and reseeding them into new cell culture containers) across various steps. Furthermore, the composition of the culture medium used during passage operations is crucial to ensure that passage operations do not induce cell transformation. In addition, the process of applying a cell suspension to the anterior chamber and uniformly implanting it onto the posterior corneal surface (endothelial surface) is called in vivo passage, and the composition of the transplant substrate is also important, similar to the culture medium used for cell seeding.

[0016] In the passage process of iPS cell expansion culture, a method is used to temporarily add a ROCK inhibitor, such as Y-27632, to the culture medium. However, the inventors of this application have discovered 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, leading to a transition considered to be epithelial-mesenchymal transition (EMT). When EMT occurs, the function of corneal epithelial cells as epithelial cells (e.g., barrier function) is reduced.

[0017] Therefore, the object of the present invention is to provide a cell inoculation agent and a cell transplantation substrate that can be passaged without causing cell transformation and can be effectively implanted into cells.

[0018] means of solving technical problems

[0019] In view of the above problems, the inventors of this application conducted in-depth research and found that during the passage operation of inducing corneal endothelial cells or corneal endothelial replacement cells from iPS cells or ES cells, even without ROCK inhibitors, the inclusion of phenolphthalein derivatives can effectively promote cell implantation. Furthermore, phenolphthalein derivatives in the cell transplantation substrate can also promote uniform implantation on the posterior surface of the cornea, thereby improving the therapeutic effect of transplanted cells, thus completing the present invention.

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

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

[0022] [2] An inoculum for promoting the implantation of corneal endothelial replacement cells, which contains a phenolphthalein derivative.

[0023] [3] According to the inoculum described in [2] above, the corneal endothelial replacement cells are derived from stem cells.

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

[0025] [5] According to the inoculum described in [4] above, wherein the pluripotent stem cells are derived from iPS cells (including cells that have been given additional functions through gene editing or gene introduction).

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

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

[0028] [8] The inoculum described in [7] above, wherein the concentration of phenol red is 1 to 20 mg / L.

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

[0030]

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

[0031]

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

[10] above is characterized in that it does not contain ROCK inhibitors.

[0032]

[12] Use of phenolphthalein derivatives in the manufacture of inoculants that promote the implantation of cultured corneal endothelial cells or corneal endothelial replacement cells.

[0033]

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

[0034]

[14] In the use described above in

[12] or

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

[0035]

[15] According to the use described above

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

[0036]

[16] According to the use described above

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

[0037]

[17] In any one of the above

[12] to

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

[0038]

[18] In any one of the above

[12] to

[17] , the phenolphthalein derivative is phenol red.

[0039]

[19] In any one of the above

[12] to

[17] , the phenolphthalein derivative is cresol red.

[0040]

[20] A phenolphthalein derivative used to promote the implantation of corneal endothelial cells or corneal endothelial replacement cells during inoculation.

[0041]

[21] A phenolphthalein derivative used to promote the implantation of corneal endothelial replacement cells during inoculation.

[0042]

[22] According to the derivatives described in

[20] or

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

[0043]

[23] According to the derivative described in

[22] above, wherein the stem cell is a pluripotent stem cell.

[0044]

[24] According to the derivative described above

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

[0045]

[25] The derivative according to any one of

[20] to

[24] above, wherein the phenolphthalein derivative is selected from phenol sulfonylphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronic acid, 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 substitute cells are inoculated in the presence of phenolphthalein derivatives.

[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] According to the method described in

[28] or

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

[0051]

[31] According to the method described in

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

[0052]

[32] According to the method described in

[31] above, 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.

[0053]

[33] The method according to any one of

[28] to

[32] above, wherein the phenolphthalein derivative is selected from phenol sulfonylphthalein (phenol red), isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronic acid, 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 containing the inoculant described in any one of [2] to

[11] above and corneal endothelial replacement cells.

[0057]

[37] The cell suspension described in

[36] above, wherein the cell concentration is 1.0 to 20 × 10⁻⁶. 4 Cells / mL.

[0058]

[38] The cell suspension according to

[36] or

[37] above is characterized by having a concentration of 1.04 × 10⁻⁶. 3 cells / cm 2 Above and less than 1.04 × 10 4 cells / cm 2 Administer at the required vaccination density.

[0059]

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

[0060]

[40] A substrate for transplanting corneal endothelial replacement cells, which contains a phenolphthalein derivative.

[0061]

[41] The substrate described in

[39] or

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

[0062]

[42] According to the substrate described in

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

[0063]

[43] According to the substrate described in

[42] above, 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.

[0064]

[44] The substrate according to any one of

[39] to

[43] above, wherein the phenolphthalein derivative is selected from phenol sulfonylphthalein (phenol red), phenolphthalein, bromophenol blue, isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronic acid, 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 concentration of phenol red 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 described in

[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 is characterized in that it does not contain ROCK inhibitor.

[0070]

[50] A cell suspension containing the substrate described in any one of

[39] to

[49] above and corneal endothelial replacement cells.

[0071]

[51] The cell suspension described in

[50] above is used for transplantation into the anterior chamber.

[0072]

[52] According to the cell suspension described in

[51] above, the cell concentration is 0.5 to 10 × 10⁻⁶. 6 Cells / mL.

[0073] The effects of the invention

[0074] According to the present invention, passage operations can be performed without inducing cell transformation, and cells can be effectively implanted. Large-scale production and culture of corneal endothelial cells and corneal endothelial replacement cells can be achieved, further increasing the implantation rate of transplanted cells, thus improving therapeutic efficacy. Attached Figure Description

[0075] Figure 1This graph illustrates the results of tight junction formation during passage operations in the differentiation induction step of corneal endothelial replacement cells derived from iPS cells, with and without the use of the ROCK inhibitor (Y-27632). The use of the ROCK inhibitor suppressed tight junction formation.

[0076] Figure 2 This figure illustrates the effects of phenolphthalein derivatives (phenol red) during passage operations in the differentiation induction step of corneal endothelial replacement cells derived from iPS cells. The top section (A) shows microscopic images of cell states on days 3, 5, and 7 of culture with or without phenol red. The bottom section (B) is a graph showing the number and viability of cells collected on day 14 of culture with or without phenol red.

[0077] Figure 3 This graph illustrates the results of corneal thickness measurements using a cynomolgus macaque (Macaca fascicularis) bullous keratopathy model, with and without phenol red, when corneal endothelial cells derived from iPS cells were transplanted as replacement cells. Transplantation in the presence of phenol red reduced corneal thickness and confirmed an improvement in corneal edema.

[0078] Figure 4 This figure illustrates the distribution of corneal endothelial replacement cells in the anterior chamber of the brain when transplanted with iPS cells, in the presence or absence of phenol red, using a cynomolgus macaque bullous keratopathy model. Detailed Implementation

[0079] The present invention will now be described. Unless otherwise stated, the terms used in this specification have their common meanings in the art.

[0080] 1. Cell inoculation agents and substrates for cell transplantation

[0081] This invention provides a novel cell inoculum. In cell culture technology, a cell inoculum refers to a liquid (solution, suspension, etc.) used to mix cells when they are seeded into a new culture vessel during passage operations.

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

[0083] In addition, the present invention provides a novel substrate for transplantation. A substrate for transplantation refers to a liquid (solution, suspension, etc.) containing mixed cells when cells are administered to a human or animal.

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

[0085] The cell inoculation agent of the present invention is suitable for passage operations of culturing corneal endothelial cells or corneal endothelial substitute cells, preferably corneal endothelial substitute cells (the operation of collecting cells from a cell culture container and re-inoculating 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 inoculation agent or cell transplantation substrate of the present invention, the target cell is cultured corneal endothelial cells or corneal endothelial substitute cells, preferably corneal endothelial substitute cells, and more preferably corneal endothelial substitute cells derived from stem cells. Here, the stem cells are preferably pluripotent stem cells, and more preferably iPS cells.

[0088] As a medium for culturing corneal endothelial cells, primary cultured cells or cell lines can be used, but since corneal endothelial cells are difficult to culture in their primary form, cell lines are preferred.

[0089] In this invention, corneal endothelial replacement cells are cells induced from stem cells such as iPS cells, ES cells, or cells endowed with additional functions through gene editing or gene introduction, which can treat corneal endothelial dysfunction and replace corneal endothelial cells. That is, corneal endothelial replacement cells have the same physiological functions as corneal endothelial cells.

[0090] Stem cells are cells that can be cultured in vitro and differentiate into various cell lineages that constitute an organism. Specific examples include embryonic stem cells (ES cells), pluripotent stem cells derived from fetal primordial germ cells (EG cells), pluripotent stem cells derived from the testis (GS cells), induced pluripotent stem cells (iPS cells) derived from somatic cells, and human adult stem cells (tissue stem cells). As iPS cells, cells from any warm-blooded animal (preferably a mammal) can be used. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, monkeys, and humans. Human iPS cells are preferred.

[0091] Specifically, examples of iPS cells include cells that acquire the same pluripotency as ES cells by introducing multiple genes into somatic cells such as skin cells. Examples include iPS cells obtained by introducing Oct3 / 4, Klf4, C-Myc and Sox2 genes, and iPS cells obtained by introducing Oct3 / 4, Klf4 and Sox2 genes (Nat Biotechnol 2008; 26:101-106). In addition, there are ongoing technical improvements to the methods for producing iPS cells, such as methods to further reduce the amount of introduced genes (Nature. 2008 Jul 31; 454(7204): 646-50), methods 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 using transcription factor proteins to replace genes (Cell Stem Cell. 2009 May 8; 4(5): 381-4). However, regardless of the method of production, as long as the basic characteristics of the iPS cells produced (i.e., pluripotency) are the same, they can all be used as a source of corneal endothelial replacement cells in this invention.

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

[0093] The corneal endothelial substitute cells used 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 this application (Patent Documents 1-3), manufactured and prepared by the methods described in Patent Documents 1-3. Preferably, they are corneal endothelial cell substitute cells from iPS cells (including cells that have been endowed with additional functions through gene editing or gene introduction) (CECSi cells), characterized by having corneal endothelial cell-like traits and functions, and enhanced gene expression of NR3C2 (nuclear receptor third subfamily, group C, member 2) (Patent Document 3).

[0094] The corneal endothelial cell-like characteristics and functions of the corneal endothelial replacement cells can be specifically listed as the following features (i) to (iv), having at least one of these features, preferably two, more preferably three, and even more preferably having all four features.

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

[0096] (ii) Tight junctions form between cells.

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

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

[0099] Whether intercellular adhesion is composed of N-cadherin can be confirmed by immunostaining of N-cadherin.

[0100] Whether tight junctions form between cells can be confirmed by observing the presence of the protein ZO-1, which constitutes tight junctions, through immunostaining. Alternatively, it can be confirmed by directly observing the structure using an electron microscope.

[0101] Whether the Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining ZO-1 and the Na,K-ATPase α1 subunit through immunostaining.

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

[0103] In one embodiment of the present invention, the corneal endothelial replacement cells targeted in the cell inoculum 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, as well as non-hematopoietic cells such as nerve cells, ganglion cells, epithelial cells, keratinocytes, muscle cells, pancreatic cells, and epithelial stem cells. CD24 has been reported to have the function of preventing macrophage phagocytosis in recent years. Therefore, the cell inoculum or cell transplantation substrate of the present invention containing corneal endothelial replacement cells with a CD24-positive phenotype is expected to reduce rejection reactions during transplantation. On the other hand, human corneal endothelial cells that are CD24-negative have been reported (Japanese Patent Application Publication No. 2019-510509, Japanese Patent Application Publication No. 2020-073602).

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

[0105] All immunostaining procedures were performed routinely in the field, and the reagents and equipment used were commercially available or could be prepared based on previous reports.

[0106] In the passage operation of the corneal endothelial replacement cell induction differentiation step described above, iPS cells, ES cells, or cells endowed with additional functions through gene editing or gene introduction are added to the cell seeding agent, typically at a concentration of 1.0–20 × 10⁻⁶. 4 Cells / mL, preferably 2.5–10 × 10⁶ cells / mL 4 Cell suspensions were obtained by suspending cells at a concentration of 1.04 × 10⁻⁶ cells / mL. 3 cells / cm 2 Above and less than 1.04 × 10 4 cells / cm 2 The preferred value is 1.04×10 3 cells / cm 2 Above and 5.02×10 3 cells / cm 2 The following inoculation density is used to inoculate culture containers such as petri dishes.

[0107] During transplantation into the eye, especially into the anterior chamber, the aforementioned cultured corneal endothelial cells or corneal endothelial substitute cells, preferably corneal endothelial substitute cells, are added to the cell transplantation substrate, and are typically transplanted at a density of 0.5–10 × 10⁻⁶. 6 Cells / mL, preferably 1.0–5 × 10⁻⁶. 6 The cell suspension is obtained by suspending cells at a concentration of 100 cells / mL. If the cell concentration and / or seeding density is too high or too low, the cell implantation efficiency will be reduced.

[0108] In this invention, the cultured corneal endothelial cells or corneal endothelial substitute cells can be spheroidized cell masses (hereinafter referred to as spheroids) obtained by assembling and spheroidizing dozens to hundreds of cells through suspension culture. Here, "spheroid" includes not only completely spherical shapes, but also oval, rugby ball-shaped, and other approximately spherical 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 even more preferably in the range of 40 to 80 μm. If the spheroid is too large, implantation will be misaligned and cannot be uniformly implanted; if the spheroid is too small, adhesion will be difficult and implantation efficiency will be reduced. When the size of the spheroid is within this range, the cells can be effectively implanted at the transplantation site. "Area equivalent diameter" refers to the diameter of a circle with the same projected area as 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 ​​a sphere that is approximately spherical in shape.

[0109] The cell concentration or seeding density is determined as follows: a portion of the cell suspension is sampled, and the number of cells in the sample is measured; for spheroid suspension, a portion of the spheroid suspension is sampled, and the spheroids in the sample are subjected to enzyme treatment to disperse the cells one by one to measure the cell number. The result is used to determine the cell concentration or seeding density.

[0110] (Phenolphthalein derivatives)

[0111] The cell inoculum or cell transplantation substrate of the present invention is characterized by comprising a phenolphthalein derivative. The phenolphthalein derivative is not particularly limited, as long as it has a basic framework represented by the following formula and can achieve the desired effect.

[0112]

[0113] In the formula, -X- is -SO2- or -CO-, and ring Ar1 and ring Ar2 may be the same or different, and are optional aromatic rings (e.g., benzene, naphthalene) with 1 to 3 substituents (e.g., optional alkyl groups with 1 to 3 carbon atoms such as methyl, ethyl, propyl, isopropyl, etc.; halogen atoms such as bromine, chlorine, etc.; acyl groups such as acetyl and isovaleryl).

[0114] Preferably, -X- is -SO2. The ring Ar1 and ring Ar2 may be the same or different, and may be benzene having 1 to 3, preferably 1 or 2, substituents (e.g., methyl, isopropyl, bromine atoms).

[0115] The phenolphthalein derivative may optionally be its salt or ester. Alternatively, it may optionally be an acid adduct.

[0116] Examples of phenolphthalein derivatives include phenolsulfonphthalein (phenol red), phenolphthalein, bromophenol blue, isovalerylphenolphthalein, acetylphenolphthalein, phenolphthalein dibutyrate, phenolphthalein diphosphate, phenolphthalein disulfate, phenolphthalein glucuronic acid, phenolphthalein mono-β-glucuronic acid, phenolphthalein mono-β-glucuronic acid, phenolphthalein monophosphate, cresol red, thymol blue, and bromocresol purple. Phenolphthalein derivatives can be prepared by methods known per se. It is known that phenolphthalein derivatives can be synthesized using phthalic acid and various corresponding phenols; if phenol is used, phenolphthalein can be synthesized. Furthermore, various phenolphthalein derivatives are commercially available. From the viewpoint of convenience and quality stability, commercially available derivatives are preferred. Phenol red (also known as phenolsulfonphthalein) (left) and cresol red (right) with the following structures are preferred as phenolphthalein derivatives. Phenol red is typically included in culture media as a pH indicator with a color change range (yellow to red) of pH 6.8 to pH 8.4. However, from a cell culture point of view, it is an optional additive, and commercially available culture media are also available without phenol red. Additionally, cresol red is known to be a pH indicator with a color change range (yellow to red) of pH 7.2 to pH 8.8. The concentration of phenol red contained in the cell inoculum or cell transplantation substrate of the present invention is typically 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 inoculum or cell transplantation substrate of the present invention is typically 1 to 20 mg / L during differentiation induction, preferably 1 to 15 mg / L, and more preferably 5 to 10 mg / L. During transplantation, it is 1 to 16 mg / L, preferably 1 to 12 mg / L, and more preferably 4 to 8 mg / L. The concentration of phenolphthalein derivatives 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 "desired effect" of phenolphthalein derivatives refers to their ability to promote cell implantation after passage or transplantation.

[0119] Examples of mammals that can be used as the substrate for cell transplantation according to the present invention include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cattle, horses, sheep, monkeys, and humans. Humans are preferred.

[0120] The dosage of the cell transplantation substrate of the present invention varies depending on the recipient's weight, age, symptoms, etc. However, for individuals weighing 25–300 kg, aged 16–100 years, and exhibiting bullous keratopathy, 50–200 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 its application as a cell suspension to the anterior chamber does not cause side effects such as increased intraocular pressure or significant inflammation, making it safe. Suitable diseases for transplantation include those requiring 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 manufacturing cell inoculum or substrate for cell transplantation

[0122] The cell inoculation agent or cell transplantation substrate of the present invention contains a medium for dissolving and / or diluting phenolphthalein derivatives and maintaining cell viability.

[0123] There are no particular restrictions on the medium used as a cell inoculation agent, as long as it has the ability to promote cell proliferation and implantation after inoculation. Physiological saline, various physiological buffers (e.g., PBS, HBSS, etc.), and various media based on basal media used for cell culture can be used. There are also no particular restrictions on the basal media, as long as it can be used for animal cell culture. Examples include MEM medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium (e.g., F10, F12), RPMI 1640 medium, Fischer's medium, and mixtures thereof. These media are all commercially available. Furthermore, these media can be serum-containing or serum-free. Serum-free media are preferred. When the culture medium used in this invention is a serum-containing culture medium, mammalian serum such as bovine serum or fetal bovine serum can be used, and the concentration of the serum in the culture medium is 0.1-20% (v / v), preferably 1-10% (v / v).

[0124] There are no particular restrictions on the medium used as a transplant substrate, as long as it is suitable for intraocular administration, especially intra-anterior chamber administration. Non-irritating isotonic culture media, physiological saline, buffer solutions, etc., can be used. Preferably, the same culture medium used for culturing corneal endothelial cells or corneal endothelial replacement cells is used. The transplant substrate may contain various components as needed, except for phenolphthalein derivatives. There are no particular restrictions on these components, as long as they are effective in preventing the cultured corneal endothelial cells or corneal endothelial replacement cells administered (transplanted) into the anterior chamber from detaching and differentiating into the corneal endothelial cell layer. Insulin or insulin-like growth factor is preferred. Additionally, retinoic acid, EGF, and bFGF are also preferred components.

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

[0126] Example

[0127] The present invention will now be described in detail with reference to the embodiments, but the invention is not limited to these embodiments. Furthermore, unless otherwise stated, all reagents and materials used are commercially available. Unless otherwise stated, the abbreviations used herein are the same as those commonly used in the art.

[0128] (Materials and methods)

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

[0130] The effect of ROCK inhibitors during passage operations in the differentiation induction of corneal endothelial replacement cells derived from iPS cells was investigated. ZO-1 expression was studied according to Example 1 and Experimental 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 study found that the use of ROCK inhibitors suppressed tight junction formation and led to a transformation known as epithelial-mesenchymal transition. These results indicate a need to develop a cell inoculum that does not contain ROCK inhibitors.

[0131] Example 1: Effects of phenolphthalein derivatives on cell implantation and survival after inoculation

[0132] (Materials and methods)

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

[0134] DMEM / F12 medium (Nacalai Tesque, #08460-95) was used as the basal medium. The following were added to the DMEM / F12 basal medium: N-2MAX medium additive (100X) (R&D Systems, #AR009), ascorbic acid (Fuso Pharmaceutical Co., Ltd.; ascorbic acid injection (50 μg / mL)), IGF1 (Orphan Pacific; Mecasermin Injection; Somazon Injection) (20 ng / mL)), KGF (Fujifilm Wako Pure Chemical, #119-00661 (5 ng / mL)), LIF (Sigma-Aldrich, #L5283 (1 ng / mL)), and IL6 (Fujifilm Wako Pure Chemical). Chemical); #099-04631 (1 ng / mL)), adrenaline (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 added, and culture dishes or flasks coated with laminin 511-E8 fragment (iMatrix511, Nippi) at a concentration of 3 μg / mL were used as scaffold material.

[0135] 2. Induction protocol for corneal endothelial replacement cells

[0136] The method is implemented according to the embodiments described in Patent Document 3. Specifically, it is as follows.

[0137] Y-27632 (Wako / 039-24591) was added to iPS cell culture medium AK03N (Ajinomoto) at a concentration of 10 μM (hereinafter referred to as AK03N+Y medium). Six-well plates (Greiner 657160) were coated with iMatrix511 at a concentration of 6 μg / ml. The culture was then incubated at 1.0 × 10⁻⁶. 4 cells / cm 2 The iPS cells (FF-I01s04 strain) were suspended in AK03N+Y medium at a specific density and seeded into 6-well plates. The cells were then incubated at 37°C using CO2. 2Incubate the cells in an incubator. The following day, replace the medium with AK03N medium that does not contain Y-27632. Thereafter, change the medium every 2–3 days, maintaining the same density as described above, and passage weekly in 6-well plates coated with iMatrix511 using TrypLE-Select (Thermo Fisher Scientific). To induce corneal endothelial replacement cells, use iPS cells from passage 3 to 29 or higher.

[0138] At the start of induction, iPS cells were collected from 6-well plates using TrypLE-Select and suspended in the culture medium described above for corneal endothelial replacement cell induction. 35mm or 100mm culture dishes were pre-coated with iMatrix511 at a concentration of 3 μg / ml and incubated at 2.0 × 10⁻⁶ cells / well. 4 cells / cm 2 Suspended iPS cells were seeded at a density of [insert density here]. Incubated at 37°C with CO2. 2 Start culturing in an incubator, and then change the culture medium every 2 to 3 days.

[0139] Example 2: Effect of phenolphthalein derivatives during passage operations in the differentiation induction step of corneal endothelial replacement cells derived from iPS cells.

[0140] When inducing corneal endothelial replacement cells, iPS cells collected using TrypLE-Select (Thermo Fisher Scientific) were suspended in seeding medium with or 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 inoculated at 1.0 × 10⁻⁶ cells / ml. 4 cells / cm 2 iPS cells were seeded at a density suspended in various cell inoculums. Cultures were initiated in a CO2 incubator at 37°C, and then the medium was changed every 2–3 days between the addition of phenol red to the medium used for corneal endothelial cell replacement induction and the absence of phenol red.

[0141] Under the above conditions, cells were cultured for 14 days in various media used for inducing corneal endothelial replacement cells. The cell status was confirmed under a microscope after 3, 5, and 7 days of culture. Figure 2A). After 14 days of culture, for the group where cell implantation was confirmed under a microscope, cells were collected and cell counts were measured. Cell collection was performed by removing the culture supernatant, washing twice with DPBS (Thermo Fisher Scientific), peeling with Accutase (Innovative Cell Technologies), and then collecting the cells into centrifuge tubes. Cells were separated by centrifugation, and after adjusting cell concentration using culture medium, cell concentration and viability were measured using an automated cell counter (ChemoMetec). Figure 2 B).

[0142] (result)

[0143] During the passage process in the differentiation induction step of corneal endothelial replacement cells derived from iPS cells, the number of cells implanted after seeding was investigated, and the key components of the seeding agent were explored. The results showed that phenol red was a key influencing component.

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

[0145] Furthermore, after inoculation, the number of viable cells and the survival rate collected on day 14 were compared between culture media with and without phenol red. The results showed that the number of viable cells and the survival rate collected from culture media with phenol red were significantly superior.

[0146] Example 3: Effects of phenolphthalein derivatives on cell implantation and corneal edema after transplantation

[0147] (Materials and methods)

[0148] The cell suspension of corneal endothelial replacement cells obtained from iPS cells in Example 1 (8 × 10⁸) 5 One cell / 160 μl was transplanted into the anterior space of a cynomolgus macaque bullous keratopathy model (conducted by Shin Nippon Scientific Co., Ltd.). Two types of substrates were prepared: one with added phenol red cell transplantation material and one without (containing insulin, insulin-like growth factor, etc.).

[0149] The corneal endothelial replacement cell suspension was transplanted into the anterior chamber in the following order.

[0150] (1) Using a 27-gauge syringe, corneal endothelial replacement cells suspended with two types of transplant substrates, one with added and one without phenol red, were injected into the anterior chamber.

[0151] (2) Place the crab-eating macaque face down for 3 hours.

[0152] (3) After 28 days of observation, collect eyeballs and observe them with the naked eye and a microscope.

[0153] Measure the formation of the corneal endothelial replacement cell layer and corneal thickness in the transplanted eye.

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

[0155] (result)

[0156] The results are shown in Figure 3 and Figure 4 When using a grafting substrate with added phenol red, corneal endothelial replacement cells were confirmed to implant and spread evenly on the posterior corneal surface. On the other hand, when using a grafting substrate without added phenol red, the implantation of transplanted cells was uneven. Figure 4 Regarding corneal thickness, a reduction in corneal thickness was observed when using graft substrates with added phenol red, thus confirming an effect in improving corneal edema. No significant reduction in corneal thickness was observed when using graft substrates without added phenol red.

[0157] Example 4: Phenolphthalein-based corneal endothelial cell replacement induction protocol

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

[0159] A T12.5 flask (Corning / 353107) was coated with iMatrix511 (Matrixome / 892005) at a concentration of 6 μg / mL. iPS cells (FF-I01s04 line) were suspended in AK03N+Y medium and incubated at 8.0 × 10⁻⁶. 3 cells / cm 2 The inoculum was planted at a density in the T12.5 flask. The inoculum was then incubated at 37°C using CO2. 2 Incubation began in an incubator. The following day, the culture medium was replaced with AK03N medium that did not contain Y-27632. The culture medium was changed for two consecutive days after a two-day interval, and the culture was passaged on the sixth day.

[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 incubated at 2.2 × 10⁻⁶ ppm. 3 cells / cm 2The inoculum was injected into the T25 flask at a density equal to that of the inoculum. The inoculum was then incubated at 37°C using CO2. 2 Incubation began in an incubator. The following day, the culture medium was replaced with AK03N medium that did not contain Y-27632. The culture medium was changed for two consecutive days after a two-day interval, and the culture was passaged on the sixth day.

[0161] 2. Preparation of a phenol red-free culture medium for inducing corneal endothelial replacement cells.

[0162] A phenol-free medium for inducing corneal endothelial replacement cells was prepared using DMEM / F12 non-phenol red (Thermo Fisher / 11039-021) as the basal medium (hereinafter referred to as phenol-free medium in this example). The following ITS additives listed in Table 1 were added to 500 mL of basal culture medium: Ventria (777ITS091), IGF1 (Orphan Pacific; Mecasermin Injection; Somazon Injection) (858100075, 1 mg / mL), LIF (Fuji FilmWako 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 (Niki Kogyo) (27126333).

[0163] Table 1

[0164] Reagent Name Added 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 Sodium hydrocortisone phosphate 20μL 200nM

[0165] 3. Preparation of phenolphthalein solutions

[0166] The phenolphthalein derivatives B to D shown in Table 2 below were dissolved in ethanol to a concentration of 6.6 × 10⁻⁶. -6mol / mL. The phenol red-free culture medium for inducing corneal endothelial replacement cells prepared as described in step 2 above was dispensed into four 50 mL tubes at a concentration of 30 mL each. 40.5 μL of 0.6% A phenolsulfonphthalein injection (AFP) was added to one of the four tubes. Additionally, 100 μL of B cresol red solution (adjusted to the desired concentration) was added to one of the remaining three 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 added phenolphthalein derivatives. Each inoculant was adjusted to a phenolphthalein derivative concentration of 0.22 mM.

[0167] Table 2

[0168]

[0169] 4. Differentiation induction (inoculation density fixed at 1.04 × 10⁻⁶) 4 cells / cm 2 )

[0170] 90 μL of laminin 511-E8 fragment (iMatrix511MG) was added to 30 mL of DPBS(-), and 1.5 mL was added to each well of a 6-well plate to coat the scaffold material at a concentration of 3 μg / mL. The plate was incubated at 37°C for at least 1 hour. The supernatant was removed, and 2 mL of the cell seeding medium prepared in step 3 above, containing various A-D phenolphthalein derivatives, was added to each well. The plate was incubated at 37°C. After iPS cell expansion culture as described in step 1, the collected iPS cells were centrifuged at 140 × g for 5 minutes at 25°C. The supernatant was removed, and the cells were resuspended in cell seeding medium containing various A-D phenolphthalein derivatives or phenolphthalein-free medium at a seeding density of 1.04 × 10⁴ cells / well. 4 cells / cm 2 Inoculate into iMatrix-coated 6-well plates. Incubate with CO2 at 37°C. 2 The cells were incubated in an incubator, with the inoculation time designated as day 0. The culture medium was changed for the first time on day 3 at a dose of 2 mL / well. The culture medium was then changed every other day. Cells were fixed and stained on day 7 after inoculation.

[0171] 5. Differentiation induction (altering inoculation density)

[0172] Similar to step 4 above, after iMatrix coating, the supernatant was removed, and the following media were added to 5 wells at 2 mL / well: the usual differentiation induction medium (medium containing phenol red in DMEM / F12 (Thermo Fisher / 11330057) with the additives listed in Table 1 above) as a positive control, the medium containing non-phenol red in DMEM / F12 (Thermo Fisher / 11039-021) instead of phenol red-free DMEM / F12 as a negative control, the medium containing phenol red A and the medium containing cresol red B, respectively. After expansion and culture as described in section 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 resuspended in positive control, negative control, culture medium supplemented with phenol red blood cell inoculum A, and culture medium supplemented with cresol red blood cell inoculum B, respectively, at an inoculation density of 1.04×10⁻⁶ cells / year. 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 wells of a 6-well plate coated with iMatrix, containing either A-type erythrocyte inoculum, B-type erythrocyte inoculum, a positive control, or a negative control. The cells were then inoculated using CO2 at 37°C. 2 The cells were incubated in an incubator, with the inoculation time designated as day 0. The culture medium was changed for the first time on day 3 at a dose of 2 mL / well. The culture medium was then changed every other day. Cells were fixed and stained on day 7 after inoculation.

[0173] 6. Results

[0174] In a phenol red-free culture medium, when inoculated at the specified density of 1.04 × 10⁻⁶, 4 cells / cm 2 The following vaccinations will not result in implantation.

[0175] Cell implantation was confirmed in a culture medium supplemented with phenol red and cresol red, at a seeding density lower than the prescribed cell seeding density.

[0176] In media containing thymol blue and bromocresol purple, at a concentration of 1.04 × 10⁻⁶...4 cells / cm 2 During the vaccination, implantation was also confirmed.

[0177] Industrial availability

[0178] According to the present invention, passage operations can be performed and cells can be effectively transplanted without inducing cell transformation. It can mass-produce cultured corneal endothelial cells and corneal endothelial replacement cells, and improve the engraftment rate of transplanted cells, thereby enhancing therapeutic efficacy.

[0179] This application is based on U.S. Provisional Patent Application No. 63 / 219,101 filed in the United States on July 7, 2021, the entire contents of which are incorporated herein by reference.

Claims

1. Use of a phenolphthalein derivative as the sole active ingredient in the manufacture of an accelerant for promoting the implantation of corneal endothelial replacement cells, wherein, The phenolphthalein derivative is selected from the group consisting of phenol red, cresol red, thymolphthalein blue, and bromocresol purple. The corneal endothelial substitute cell is a CECSi cell from an iPS cell, the iPS cell including a cell which is given an additional function by gene editing or gene introduction, characterized in that the corneal endothelial substitute cell has a corneal endothelial cell-like property and function, and a gene expression amount of NR3C2 is enhanced.

2. Use according to claim 1, wherein, The corneal endothelial substitute cell has at least one of the following characteristics (i) to (iv) of a corneal endothelial cell-like property and function, (i) intercellular adhesion is constituted by N-cadherin; (ii) intercellular tight junction is formed; (iii) Na, K-ATPase alpha 1 subunit is expressed on a cell membrane; (iv) expression of a transcription factor PITX2 is observed in a cell nucleus.

3. Use according to claim 1 or 2, wherein, The phenolphthalein derivative is phenol red.

4. Use according to claim 3, wherein, The phenol red concentration is 1 to 20 mg / L.

5. Use according to claim 1 or 2, wherein, The phenolphthalein derivative is cresol red.

6. Use according to claim 5, wherein, The cresol red concentration is 1 to 20 mg / L.

7. Use of a phenolphthalein derivative as the only active ingredient in the preparation of a substrate for transplantation for promoting the implantation of corneal endothelial replacement cells, wherein, The phenolphthalein derivative is phenol red. The corneal endothelial substitute cell is a CECSi cell from an iPS cell, the iPS cell including a cell which is given an additional function by gene editing or gene introduction, characterized in that the corneal endothelial substitute cell has a corneal endothelial cell-like property and function, and a gene expression amount of NR3C2 is enhanced.

8. Use according to claim 7, wherein, The corneal endothelial substitute cell has at least one of the following characteristics (i) to (iv) of a corneal endothelial cell-like property and function, (i) intercellular adhesion is constituted by N-cadherin; (ii) intercellular tight junction is formed; (iii) Na, K-ATPase alpha 1 subunit is expressed on a cell membrane; (iv) expression of a transcription factor PITX2 is observed in a cell nucleus.

9. The use according to claim 7, wherein, The phenol red concentration is 1 to 20 mg / L.

10. Use according to claim 7 or 8, characterized in that, The substrate for the transplantation does not contain a ROCK inhibitor.

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