Method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells

Abstract

To provide a preservation method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells while maintaining a high survival rate and for minimizing the generation rate of contaminant cells in culture after preservation.SOLUTION: Provided is a method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells, characterized by collecting at a time when specific conditions are met human corneal endothelial cells and / or human corneal endothelial progenitor cells proliferated using a medium containing a ROCK inhibitor and containing epidermal growth factor (EGF) below the concentration that causes transformation, and preserving them in a suspended state.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to a method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells that can differentiate and mature after storage and re-culture, thereby expressing the functional characteristics of human corneal endothelial cells. [Background technology]

[0002] As a method for culturing human corneal endothelial cells in vitro, as shown in Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2, a method is known in which epidermal growth factor (EGF) is administered, and another factor is further administered to suppress the adverse effects of EGF on the maturation and differentiation of human corneal endothelial cells.

[0003] In response to this, the present inventors have developed a method for culturing human corneal endothelial cells with as little epidermal growth factor (EGF) as possible, as shown in Patent Documents 2 and 3. According to the culture method described in Patent Document 2 or Patent Document 3, compared to the culture methods described in Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2, it is possible to obtain a cell population with an improved content of functional human corneal endothelial cells (hereinafter also referred to as effector cells) that have corneal endothelial functional characteristics equivalent to those of corneal endothelial cells present in the human body, while minimizing the generation of contaminating cells that do not possess human corneal endothelial functional characteristics.

[0004] However, a method for storing a cell population cultured by the method described in Patent Document 2 or Patent Document 3, or functional human corneal endothelial cells contained in this cell population, in a suspension state has not yet been established. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2017 / 110094 [Patent Document 2] International Publication No. 2009 / 028631 [Patent Document 3] Japanese Patent Application No. 2020-032139 [Non-patent literature]

[0006] [Non-Patent Document 1] Vianna LMM, et al. (2016) Arg Bras Oftalmol.;79(1):37 [Non-Patent Document 2] Okumura N., et al. (2019) PLos ONE 14(6):e0218431 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In order to supply a cell population cultured by the method described in Patent Document 2 or Patent Document 3, or functional human corneal endothelial cells contained in this cell population, internationally as a regenerative medicine product, it is desirable that the cell population or the functional human corneal endothelial cells be stored in a suspension state for at least several days, rather than in a monolayer culture state. Therefore, the present inventors investigated a method for storing a cell population or functional human corneal endothelial cells cultured by the method described in Patent Document 2 or Patent Document 3 in a suspension state for at least several days. In addition, cryopreservation is beneficial for long-term storage of cells over several years and for creating cell banks, and we also investigated the establishment of a method for cryopreserving functional human corneal endothelial cells.

[0008] In order to proliferate human corneal endothelial cells using the method described in Patent Document 2 or Patent Document 3 and mature and differentiate them until the cell population has a functional human corneal endothelial cell content (hereinafter also referred to as E-ratio) of more than 90%, it is necessary to culture them for 4 to 5 weeks or more. Therefore, we first attempted to collect a cell population that had been cultured for more than four weeks using the method described in Patent Document 2 or Patent Document 3, and matured and differentiated until it contained more than 90% functional human corneal endothelial cells, then suspended and stored it. As a result, it was found that when cell populations that had been matured and differentiated to a level with a functional human corneal endothelial cell content of more than 90% were stored in a suspension state, the survival rate after storage was low, and the proportion of contaminating cells increased during subsequent culture.

[0009] Based on these results, the inventors developed a method for storing human functional corneal endothelial cells (cultured using the method described in Patent Document 2 or Patent Document 3) in a suspension state while maintaining a favorably high viability rate, and for suppressing the generation of contaminating cells. As a result, the inventors discovered that by collecting a cell population immediately after its fate to differentiate into functional human corneal endothelial cells has been determined and at a time of high proliferative capacity, and then suspending it, it is possible to preserve it while maintaining a high viability rate, and to suppress the rate of contamination in subsequent culture. This led to the establishment of a novel and improved method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells (and related compositions used therein). Furthermore, the inventors of this invention believe that if a cell population can be collected and preserved immediately after its fate as a functional human corneal endothelial cell has been determined and while its proliferative capacity is high, then by collecting cells at the same time and repeatedly subculturing them, it will be possible to expand the culture of functional human corneal endothelial cells in a much shorter period than conventional methods. Based on this, they have also established a method for expanding the culture of functional human corneal endothelial cells. Thus, the present invention was only completed when the inventors discovered that if cells are destined to become functional human corneal endothelial cells when cultured under appropriate culture conditions, then by collecting and preserving or subculturing these cells in an immature state, functional human corneal endothelial cells with the desired properties of improved viability and reduced contamination can be obtained. [Means for solving the problem]

[0010] In other words, the method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells according to the present invention is as follows. [Item 1] A method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells, characterized by harvesting human corneal endothelial cells and / or human corneal endothelial progenitor cells grown in a medium containing a ROCK inhibitor and free of epidermal growth factor (EGF) or containing EGF at a concentration below the level that causes transformation, at a time when any one or more of the following conditions (a) to (d) are met, and storing them in a suspension state. (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells changes from a spindle-shaped fibroblast-like morphology with irregular elongated projections to a cobblestone-like morphology (e.g., polygonal or circular) in which the ratio of the diameter including the projections is close to 1 (e.g., about 2:1 to about 1:2), until immediately before the boundaries between cells become indistinct. (b) The period from when the expression level of CD44 falls to less than half of the maximum value observed in the most recent passage until it reaches a plateau. (c) The cell density of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells is approximately 900 cells / mm 2 More than about 2500cells / mm 2 The following applies: (d) The number of days of cultivation after sowing is approximately 4 days or more and approximately 14 days or less. [Item 2] The storage method described in [Item 1], wherein the human corneal endothelial cells and / or the human corneal endothelial progenitor cells are stored at approximately 10 degrees Celsius or below. [Item 3] The storage method described in [Item 1] or [Item 2], wherein the human corneal endothelial cells and / or the human corneal endothelial progenitor cells are stored at approximately -30 degrees Celsius or below. [Item 4] A storage method according to any one of [Item 1] to [Item 3], wherein the human corneal endothelial cells and / or the human corneal endothelial progenitor cells are suspended and stored for approximately 24 hours or more. [Item 5] The method according to any one of [Items 1] to [Item 4], wherein the transformation includes endodermal-mesenchymal transformation. [Item 6] The preservation method according to any one of [Items 1] to [Item 5], wherein the human corneal endothelial cells and / or the human corneal endothelial progenitor cells are prepared from cells selected from the group consisting of corneal endothelial cell-derived cells, pluripotent stem cells, mesenchymal stem cells, corneal endothelial progenitor cells collected from corneal endothelium, cells collected from corneal endothelium, and corneal endothelial progenitor cells and corneal endothelial-like cells prepared by the direct programming method. [Item 7] The preservation method according to any one of [Items 1] to [Item 6], wherein the cell population of human corneal endothelial cells obtained by growing and / or differentiating / maturing the human corneal endothelial cells and / or the human corneal endothelial progenitor cells after preservation using a medium containing a ROCK inhibitor and having an epithelial growth factor (EGF) content less than the concentration causing transformation satisfies one or more items of the following (1) to (8). (1) In the appearance inspection by phase-contrast imaging, there are no fibroblasts, foreign substances, discoloration, or other abnormalities. (2) The cell viability is 70% or more by trypan blue staining. (3) In the purity test by ELISA of the cell supernatant, PDGF-BB is 100 pg / mL or more. (4) The purity test result by FACS of the cells satisfies all of the following ranges. CD166 + >99% CD24 + <5% CD44 high <5% CD44 neg~low >90% CD90 + <5% (5) The content ratio (E-ratio) of effector cells having human corneal endothelial function is higher than 90%. (6) The pump function (Na + / K + ATPase) is positive. (7) The barrier function (ZO-1) is positive. (8) The human corneal endothelial cell density (ECD) is 1500 cells / mm 2 or more. [Item 8] The preservation method described in [Item 7], wherein the effector cells have not undergone endothelial-mesenchymal transition. [Item 9] The preservation method according to item 7 or item 8, wherein the effector cells express functional proteins necessary for exhibiting the corneal endothelial functional characteristics, or inhibitory proteins that inhibit the corneal endothelial functional characteristics are not expressed or the expression of inhibitory proteins is reduced. [Item 10] The functional protein is Na + / K + The preservation method described in [Item 9], comprising one or more selected from the group consisting of ATPase, ZO-1, sodium / hydrogen exchanger 1 (NHE1), aquaporin 1 (AQP-1), and carbonic anhydrase 5B (CA5B). [Item 11] The preservation method according to [Item 9] or [Item 10], wherein the functional protein further comprises one or more metabolism-related enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malate enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain keto acid dehydrogenase 2 (BCKDH2). [Item 12] The preservation method according to any one of [Items 9] to [Item 11], wherein the inhibitory protein comprises at least one metabolism-related enzyme selected from the group consisting of ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malate enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and lactate dehydrogenase (LDH).

[0011] Furthermore, the present invention also provides the following inventions. Human corneal intracellular cells and / or human corneal endothelial progenitor cells preserved using the preservation method described in any one of items 1 through 12. [Item 14] A suspension of human corneal intracellular cells and / or human corneal endothelial progenitor cells preserved by the preservation method described in any one of [Item 1] to [Item 12]. A method for preparing human corneal endothelial cells, characterized by growing and / or differentiating and maturing human corneal endothelial cells and / or human corneal endothelial progenitor cells, which have been preserved by the preservation method described in any one of items [1] to

[12] , using a culture medium containing a ROCK inhibitor and in which the epidermal growth factor (EGF) content is below the concentration that causes transformation. [Item 16] A method for preparing a suspension of human corneal endothelial cells and / or human corneal endothelial progenitor cells, characterized by harvesting human corneal endothelial cells and / or human corneal endothelial progenitor cells, which have been grown using a medium containing a ROCK inhibitor and in which the epidermal growth factor (EGF) content is below the concentration that causes transformation, at a time when any one or more of the following conditions (a) to (d) are met, and then suspending them. (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells changes from a spindle-shaped fibroblast-like morphology with irregular elongated projections to a cobblestone-like morphology in which the ratio of the major axis to the minor axis, including the projections, is close to 1, until immediately before the boundaries between cells become indistinct. (b) The period from when the expression level of CD44 falls to less than half of the maximum value observed in the most recent passage until it reaches a plateau. (c) The cell density of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells is 900 cells / mm 2 More than 2500cells / mm 2 The following applies: (d) The culture period since the most recent passage is between 4 and 14 days. [Item 17] A method for culturing human corneal endothelial cells and / or human corneal endothelial progenitor cells, characterized by harvesting and subculturing human corneal endothelial cells and / or human corneal endothelial progenitor cells grown in a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below the concentration that causes transformation, at a time when any one or more of the following conditions (a) to (d) are met. (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells changes from a spindle-shaped fibroblast-like morphology with irregular elongated projections to a cobblestone-like morphology with a major-minus-minus ratio close to 1, until immediately before the boundaries between cells become indistinct. (b) The period from when the expression level of CD44 falls to less than half of the maximum value observed in the most recent passage until it reaches a plateau. (c) The cell density of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells is 900 cells / mm 2 More than 2500cells / mm 2 The following applies: (d) The culture period since the most recent passage is between 4 and 14 days. [Effects of the Invention]

[0012] According to the present invention, human corneal endothelial cells and / or human corneal endothelial progenitor cells, which can be obtained by culturing again after storage to obtain a cell population with a functional human corneal endothelial cell content of approximately 90% or higher, can be stored for at least several days while maintaining a high viability rate. As a result, by culturing the cells again, it becomes possible to supply a cell population with a functional human corneal endothelial cell content of approximately 90% or higher internationally as a regenerative medicine product. Furthermore, cryopreservation enables the creation of a master cell bank, allowing for more efficient and planned manufacturing of regenerative medicine products. [Brief explanation of the drawing]

[0013] [Figure 1] This is a phase-contrast microscope image showing differences in cell morphology of cultured human corneal endothelial cells under different culture conditions (Passage 3). [Figure 2] This is a phase-contrast micrograph of cultured human corneal endothelial cells that were subcultured up to Passage 7 under culture condition 2. [Figure 3]This graph shows the results of cell surface antigen analysis of Passage 3 cells cultured under culture condition 1 and Passage 3 or Passage 7 cells cultured under culture condition 2. [Figure 4] This photograph shows the results of immunohistochemical staining of Passage 7 cells cultured under culture condition 2, examining the expression of Na+ / K+ ATPase, ZO-1, and N-Cadherin. [Figure 5] This is a phase-contrast micrograph of cells cultured under culture condition 2 from Passage 0, taken in Passage 7. [Figure 6] Figure 5 shows photographs and graphs illustrating the expression of cell surface antigens and other factors in the cells shown. [Figure 7] This is a photograph showing the results of trypan blue staining of cells after cryopreservation according to one embodiment of the present invention. [Figure 8] This is a phase-contrast microscope image showing the time course of cell morphology in cultured human corneal endothelial cells cultured under culture condition 2 after cryopreservation. [Figure 9] This is a phase-contrast microscope image comparing the morphology of cells that were cryopreserved with cells that were cultured without cryopreservation. [Figure 10] This graph shows the results of cell surface antigen analysis between cells that were cryopreserved and cells that were continuously cultured without cryopreservation. [Figure 11] This is a fluorescence micrograph showing the expression of Na+ / K+ ATPase, ZO-1, and N-Cadherin in cells after cryopreservation. [Figure 12] This diagram illustrates the procedure for comparing and analyzing the optimal timing for cell collection and preservation. [Figure 13] Figure 12 shows phase-contrast microscope images illustrating cell morphology at different sampling times. [Figure 14] These are graphs and photographs showing the cell survival rate after cryopreservation at each collection time. [Figure 15] Figure 14 is a phase-contrast microscope image comparing the morphology of cells that were thawed after cryopreservation and cultured for 35 days under culture condition 2 with cells that were subcultured without cryopreservation. [Figure 16] Figure 14 is a graph comparing the surface antigens of cells that were thawed after cryopreservation and cultured for 35 days under culture condition 2, with those of cells that were subcultured without cryopreservation. [Figure 17] Figure 14 is a photograph comparing the immunostaining results of cells that were thawed after cryopreservation and cultured for 35 days under culture condition 2, with cells that were subcultured without cryopreservation. [Figure 18] This diagram illustrates the procedure for comparing and analyzing the optimal timing for cell collection and preservation. [Figure 19] Figure 18 shows a graph illustrating the survival rate of cells collected and cryopreserved at each collection time. [Figure 20] Figure 18 is a graph showing the expression of cell surface antigens and other factors in cells collected and cryopreserved at each collection time. [Figure 21] Figure 18 is a graph showing the expression of cell surface antigens and other factors in cells collected and cryopreserved at each collection time. [Figure 22] Figure 18 is a graph showing the expression of cell surface antigens and other factors in cells collected and cryopreserved at each collection time. [Figure 23] Figure 18 shows fluorescence micrographs confirming the expression of Na+ / K+ ATPase, ZO-1, and N-Cadherin in cells collected and cryopreserved at each collection time. [Figure 24] Figure 18 shows fluorescence micrographs confirming the expression of Na+ / K+ ATPase, ZO-1, and N-Cadherin in cells collected and cryopreserved at each collection time. [Figure 25] Figure 18 shows fluorescence micrographs confirming the expression of Na+ / K+ ATPase, ZO-1, and N-Cadherin in cells collected and cryopreserved at each collection time. [Figure 26] This is a phase-contrast microscope image showing the changes in cell morphology over time when cultured under culture condition 2. [Figure 27] This is a phase-contrast microscope image showing the changes in cell morphology over time when cultured under culture condition 2. [Figure 28]This graph shows the daily change in CD44 expression levels when cultured under culture condition 2. [Figure 29] This graph shows the daily change in ECD when cultured under culture condition 2. [Figure 30] These images show a phase-contrast microscope photograph of cells cultured under culture condition 2 after cryopreservation, a graph showing the results of surface antigen analysis, and a photograph showing the results of immunohistochemical staining. [Figure 31] This photograph shows the results of trypan blue staining of cultured human corneal endothelial cells after storage at ice temperature. [Figure 32] This graph shows the survival rate calculated from the results in Figure 31. [Figure 33] These are photographs showing the results of trypan blue staining of cells after storage at ice temperature using each preservation solution. [Figure 34] This graph shows the survival rate calculated from the results in Figure 33. [Figure 35] These are phase-contrast microscope images of cells that were recovered and cultured after being stored at ice-cold temperatures in a conditioned medium. [Figure 36] This photograph shows the immunostaining results of cells that were recovered and cultured after being stored at ice temperature in a conditioned medium. [Figure 37] These are the proliferation rates and phase-contrast micrographs of cells that were subcultured up to Passage 8 at 8-10 day intervals after thawing. [Figure 38] Figure 29 shows a phase-contrast microscope image of cells cultured for 34 days under culture condition 2 in Passage 9, which was obtained by passage of the cells shown in Figure 29, and a graph showing the results of surface antigen analysis. [Figure 39] This diagram illustrates the cell culture conditions in Example 13. [Figure 40] Figure 39 is a graph showing the changes in cell yield and culture scale under each culture condition. [Figure 41] Figure 39 shows the cell yield under each culture condition and phase-contrast micrographs of the cultured cells. [Figure 42] Figure 39 shows the cell yield under each culture condition and phase-contrast micrographs of the cultured cells. [Figure 43]Figure 39 shows the cell yield under each culture condition and phase-contrast micrographs of the cultured cells. [Figure 44] Figure 39 shows the cell yield under each culture condition and phase-contrast micrographs of the cultured cells. [Modes for carrying out the invention]

[0014] The following describes one embodiment of the present invention. The preservation method according to this embodiment is a method for preserving human corneal endothelial cells and / or human corneal endothelial progenitor cells. These human corneal endothelial cells and / or human corneal endothelial progenitor cells were cultured in a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below the concentration that causes transformation, for example, according to the method described in Patent Document 2 or Patent Document 3. In this specification, the descriptions in Japanese Patent Application No. 2020-032139, cited as Patent Document 2, and the descriptions in International Publication No. 2017 / 110094, cited as Patent Document 3, will be appropriately referenced and incorporated.

[0015] The aforementioned human corneal endothelial cells or human corneal endothelial progenitor cells are preferably prepared from cells selected from the group consisting of human corneal endothelial cell-derived cells, pluripotent stem cells, mesenchymal stem cells, corneal endothelial progenitor cells collected from corneal endothelium, cells collected from corneal endothelium, and corneal endothelial progenitor cells and corneal endothelial-like cells produced by direct programming.

[0016] In this specification, the term "human corneal endothelial progenitor cell" refers to a cell destined to become a human corneal endothelial cell through differentiation and maturation, and includes cells in the intermediate stages of determining their differentiation fate.

[0017] The culture medium can be a basic medium used for culturing human corneal endothelial cells, such as Opti-MEM-I, Essential 6, or DMEM / F12, to which appropriate additives have been added.

[0018] ROCK inhibitors (also known as Rho kinase inhibitors) are described in the following documents: U.S. Patent No. 4678783, Patent No. 3421217, International Publications 95 / 28387, 99 / 20620, 99 / 61403, 02 / 076976, 02 / 076977, 2002 / 083175, 02 / 100833, 03 / 059913, 03 / 062227, 2004 / 009555, 2004 / 022541, and 20 Examples of compounds disclosed in 04 / 108724, International Publication 2005 / 003101, International Publication 2005 / 039564, International Publication 2005 / 034866, International Publication 2005 / 037197, International Publication 2005 / 037198, International Publication 2005 / 035501, International Publication 2005 / 035503, International Publication 2005 / 035506, International Publication 2005 / 080394, International Publication 2005 / 103050, International Publication 2006 / 057270, International Publication 2007 / 026664, etc. These compounds can be produced by the methods described in the respective documents. Specific examples include 1-(5-isoquinoline sulfonyl)homopiperazine or its salts (e.g., Fasudil(1-(5-isoquinoline sulfonyl)homopiperazine)), (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) or its salts (e.g., Y-27632((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate). These compounds can also be purchased from companies such as Wako Pure Chemical Industries, Ltd. and Asahi Kasei Pharma. The concentration of the ROCK inhibitor in the culture medium is preferably, for example, 1 to 100 μM, more preferably 5 to 20 μM, and particularly preferably 10 μM.

[0019] A concentration below the level that induces transformation means that the concentration of epidermal growth factor (EGF) is lower than the level at which transformation (e.g., endothelial-mesenchymal transition) occurs in the target corneal endothelial cells, or that no EGF is present at all. In some embodiments, EGF concentrations lower than the level that induces transformation are, for example, about 0 ng / ml to about 5 ng / ml, about 1 ng / ml to about 4 ng / ml, about 2 ng / ml to about 3 ng / ml, about 0 ng / ml to about 2 ng / ml, about 0 ng / ml to about 1 ng / ml, and any amount in between. More specifically, EGF concentrations below the level that induces transformation are, for example, less than 2 ng / mL, preferably less than 1 ng / mL, more preferably less than 0.5 ng / mL, and particularly preferably 0 ng / mL (no EGF added) from the viewpoint of completely eliminating the adverse effects of EGF addition on the differentiation and maturation process of human corneal endothelial cells. In some embodiments, the culture medium does not contain other growth factors that mimic the EGF effect by acting on, for example, upstream or downstream signaling molecules.

[0020] Transformation (also called phase transition) refers to a change in the characteristics of a cell that is different from its previous state (abnormal), and includes carcinogenesis, in which cells divide uncontrollably, or dynamic metaplasia, in which cells change beyond the boundaries of the basic tissue structure. Specific examples of transformation include cell state phase transitions (CSTs) such as epithelial-mesenchymal transition (EMT), fibrosis, aging, and endothelial-mesenchymal transition.

[0021] However, the method for preserving human corneal endothelial cells according to this embodiment is characterized by collecting a cell population including human corneal endothelial cells or human corneal endothelial progenitor cells, which have been grown using a medium containing a ROCK inhibitor and in which the EGF content is below the concentration that causes transformation, at a time determined by any one or more of the following indicators (a) to (d) (for example, before a differentiation event occurs), and preserving it in a suspension state. The timing of cell collection is preferably such that all of the following indicators (a) to (d) are met, but it is not necessary to meet all of them; it is acceptable if one or more of them (for example, only (a)) are met.

[0022] (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells changes from a spindle-shaped fibroblast-like morphology with irregular elongated projections to a cobblestone-like morphology in which the ratio of the major axis to the minor axis, including the projections, is close to 1, until immediately before the boundaries between cells become indistinct. (b) The period from when the expression level of CD44 falls to less than half of the maximum value observed in the most recent passage until it reaches a plateau. (c) The cell density of the human corneal endothelial cells and / or the human corneal endothelial progenitor cells is 900 cells / mm 2 More than 2500cells / mm 2 The following applies: (d) The culture period since the most recent passage is between 4 and 14 days.

[0023] The following explains each of the indicators (also called evaluation criteria) listed in (a) to (d). First, let's explain (a). When human corneal endothelial cells are grown in a culture medium containing the aforementioned ROCK inhibitor and with an EGF content below the concentration that induces transformation, the following changes in cell morphology occur during the maturation and differentiation process. Immediately after seeding on a culture plate containing the aforementioned medium, human corneal endothelial cells and / or corneal endothelial progenitor cells have a spindle-shaped morphology with irregular, elongated projections similar to fibroblasts. As the culture period progresses, the elongated projections become smaller, and the ratio of the long axis to the short axis (including the projections) approaches 1 (e.g., approximately 1.5:1, 1.25:1, 1.1:1, 1:1.1, 1:1.25, 1:1.5, and any ratio in between), resulting in a cobblestone-like morphology (e.g., polygonal shapes such as hexagons or circular shapes) similar to corneal endothelial cells in vivo. Subsequently, as the cell size becomes more uniform and the cells become fully differentiated and mature functional corneal endothelial cells, the cells adhere to each other, and the boundaries between cells become indistinct. Thus, one of the distinctive features of the preservation method according to this embodiment is that the cells are collected at the stage described in (a) above, when the morphology of the cells changes depending on the number of days of culture from the passage, that is, when they have a morphology similar to corneal endothelial cells in vivo, but before they have fully differentiated and matured. If cells are collected during this period and stored in a suspension state as described herein, a cell population with a functional human corneal endothelial cell (hereinafter also referred to as effector cells) content (hereinafter also referred to as E-ratio) of approximately 90% or higher can be obtained by culturing it again after storage. Human corneal endothelial cells and / or human corneal endothelial progenitor cells can be stored for at least several days while maintaining a high viability.

[0024] Next, we will explain (b). When human corneal endothelial cells are grown in a culture medium containing the aforementioned ROCK inhibitor and with an EGF content below the concentration that induces transformation, the expression level of the surface antigen CD44 changes during the maturation and differentiation process as follows. CD44 expression levels have been observed to increase significantly immediately after seeding human corneal endothelial cells into culture plates containing the aforementioned medium (days 1-2), then decrease logarithmically as the culture period progresses, and finally stabilize at a certain level (reaching a plateau) once the cells have differentiated and matured to a certain extent. One of the distinctive features of this embodiment is that cells are collected at a stage where the CD44 expression level is as shown in (b), that is, when the CD44 expression level is lower than immediately after passage, but higher than in cells that have fully differentiated and matured and whose CD44 expression level has plateaued. The CD44 expression level can be quantified, for example, by flow cytometry analysis using an antibody against CD44. For example, after staining with PE-Cy7-conjugated CD44 antibody at the antibody-cell ratio of Example 3, FACSCanto TMIn the case of analysis in II, the above-mentioned period can also be described as the period when the relative value of the average fluorescence intensity indicating the expression level of CD44 in the human corneal endothelial cells and / or human corneal endothelial progenitor cells, when cultured for 35 days using the medium, is greater than 1 and 60 or less, more preferably 50 or less. If cells are collected at this period and stored in a suspension state, a cell population with a functional human corneal endothelial cell content of approximately 90% or more can be obtained by culturing again after storage, and human corneal endothelial cells and / or human corneal endothelial progenitor cells can be stored for at least several days while maintaining a high viability. This CD44 expression level can also be measured by methods other than flow cytometry analysis, such as quantitative PCR. However, it should be noted that absolute quantification is not necessary in all embodiments, and rather the expression pattern can be qualitatively evaluated.

[0025] (c) will be explained. It is known that human corneal endothelial cells, after being seeded on a culture plate containing the aforementioned medium, increase almost linearly, and when they have sufficiently differentiated and matured, the corneal endothelial cell density (hereinafter also referred to as ECD) settles at a constant value. One of the distinctive features of this embodiment is that cells are collected at a stage when the ECD value is as shown in (c), that is, during the proliferative phase when cell density is increasing, but at a stage when the fate of differentiating into functional human corneal endothelial cells has already been determined. By collecting cells at this stage and suspending them, it is possible to obtain a cell population with a functional human corneal endothelial cell content of more than 90% when cultured again after storage, and to preserve human corneal endothelial cells and / or human corneal endothelial progenitor cells for at least several days while maintaining a high viability rate. The ECD value is approximately 900 cells / mm² at the time of (c) mentioned above. 2 More than about 2500cells / mm 2 It is preferable to collect the cells in the following locations, but approximately 1000 cells / mm². 2 More than about 2400cells / mm 2 Below, about 1200cells / mm 2More than about 2000cells / mm 2 Below, about 1200cells / mm 2 More than about 1800cells / mm 2 It may also be taken at a time such as less than [a certain age]. In some embodiments, ECD values ​​that fall between the above range combinations can also be used. The corneal endothelial cell density at the start of culture is not particularly limited, but for example, 300 cells / mm³ is a reasonable value. 2 Above 500 cells / mm 2 The following range is preferable. Corneal endothelial cell density (ECD) can be determined, for example, by detaching cells from a culture vessel using a cell detachment solution such as TrypLE to prepare a cell suspension, and then using a portion of this suspension to measure the number of cells in a hemocytometer, or by using analysis software (e.g., KSSE-400EB) based on images taken with a phase-contrast microscope.

[0026] (d) will be explained. It is preferable to collect human corneal endothelial cells and / or human corneal endothelial progenitor cells at the above-described stages (a) to (c) by harvesting approximately 4 to 14 days, more preferably approximately 6 to 11 days, after seeding on a culture plate containing the aforementioned culture medium.

[0027] Human corneal endothelial cells and / or human corneal endothelial progenitor cells collected during the aforementioned period should be stored at an appropriate temperature, but it is preferable to store them at low temperatures such as below approximately 10°C, at freezing temperatures such as below approximately 4°C or below approximately 0°C, or frozen at temperatures such as below approximately -10°C, below approximately -30°C, below approximately -80°C, or in a liquid nitrogen tank. While the temperature during storage may fluctuate somewhat, it is preferable to maintain it within the aforementioned temperature range, for example, from the time of freezing until thawing for use.

[0028] The period for storing human corneal endothelial cells in suspension can be set as appropriate depending on factors such as the transport time from the production site to the treatment facility. However, regardless of whether it is low-temperature storage, ice-temperature storage, or cryopreservation, it is preferable to store them for 24 hours or more, and for longer periods such as 2 days or more, 3 days or more, 4 days or more, or about a week. In particular, with cryopreservation, it is thought that storage for several months to several decades is possible.

[0029] When suspending and preserving human corneal endothelial cells, the suspension or preservation solution should preferably be the acclimatized culture medium used for cultivation in the case of ice-temperature preservation, but it is not limited to this. For example, a preservation solution commonly used for preserving cultured cells can be used. For cryopreservation, a suspension containing cryoprotective agents commonly used for the cryopreservation of animal cells can be used. Examples of such cryoprotective agents include the following cell membrane-permeable cryoprotective agents: Cryoprotective agents that are one or more selected from the group consisting of dimethyl sulfoxide, ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG), and glycerin.

[0030] The preservation methods for human corneal endothelial cells and / or human corneal endothelial progenitor cells described above allow for long-term preservation while maintaining a surprisingly high survival rate. One possible reason for this long-term preservation is that the cells are collected and preserved during a period when ERK (p44 / 42 MAPK) activation (phosphorylation) is enhanced compared to after maturation and differentiation. Furthermore, while overcoming the tendency for phase transitions is necessary to obtain functional corneal endothelial cells, human corneal endothelial cells or human corneal endothelial progenitor cells collected at the above-mentioned timeframe, as well as at the timeframes specified by each of the indicators (a) to (d) above, are collected after their fate to differentiate into functional human corneal cells has already been determined. Therefore, even if culture is resumed after storage in a suspension state, phase transitions are suppressed, and it is considered advantageous to obtain a cell population with a functional human corneal endothelial cell content of more than 90%. Moreover, since the cells collected have already been determined to differentiate into functional human corneal endothelial cells, it is possible to administer the stored cells directly to patients, for example. The stored cells or cells recultured after storage can be made suitable for delivery to the eyes of a subject (patient) for the treatment of eye diseases or injuries, for example, by administration or transplantation. When administering the cells as described above, they may be administered as they are, or they may be used in the preparation of pharmaceuticals containing these cells. Preferably, the disease or injury in question relates to the cornea.

[0031] Whether a cell population with a functional human corneal endothelial cell content of more than 90% can be obtained after the human corneal endothelial cells and / or human corneal endothelial progenitor cells stored using the aforementioned storage method have been returned from a low temperature storage state (e.g., below approximately -20°C) to room temperature such as 25°C to about 37°C, seeded again in culture medium, and cultured for a predetermined period (hereinafter also referred to as "post-recovery") can be confirmed by whether one or more of the following conditions (also referred to as evaluation criteria) (1) to (8) are met. The aforementioned predetermined period may be any period during which human corneal endothelial cells and / or human corneal endothelial progenitor cells are sufficiently differentiated and matured, but it is preferably, for example, from seeding and for 4 weeks (e.g., 28 days) or longer, and more preferably from seeding and for 5 weeks (e.g., 35 days) or longer.

[0032] (1) Visual inspection using phase-contrast imaging reveals no fibroblasts, foreign bodies, discoloration, or other abnormalities. (2) Cell viability is 70% or higher as measured by trypan blue staining. (3) Purity testing of the cell supernatant by ELISA shows that PDGF-BB is at least approximately 100 pg / mL or higher. (4) The results of the FACS purity test of the cells must meet all of the following criteria. CD166 + >99% CD24 + <5% CD26 + <5% CD44 high <5% CD44 neg~low >90% CD90 + <5% (5) Effector cells (E-ratio) > approximately 90%. (6) Pump function (Na + / K + The ATPase test is positive. (7) The barrier function (ZO-1) test is positive. (8) ECD is 1500 cells / mm 2 That's all.

[0033] In this specification, functional human corneal endothelial cells (effector cells) refer to human corneal endothelial cells that have corneal endothelial characteristics similar to those of human corneal endothelial cells present in the human body, or human corneal endothelial cells that can induce human corneal function when injected into the anterior chamber of a human eye. More specifically, effector cells refer to those that meet condition (4) of the aforementioned conditions (1) to (8), particularly CD166 + CD24 - CD44 neg~low This refers to cells that satisfy at least one of the following criteria. Note that among these indicators, CD26 + Regarding <5%, in Patent Document 2, CD44 high It has already been confirmed that this is always achieved in cell populations where the percentage is <5%. The concept of being able to induce human corneal function includes the ability to induce corneal endothelial properties (for example, improving corneal opacity and hydration edema, thereby maintaining corneal endothelial cell density over a long period and leading to improved visual acuity).

[0034] As described in Patent Document 3, it has been found that functional human corneal endothelial cells possessing corneal endothelial characteristics express functional proteins that contribute to corneal endothelial characteristics. The aforementioned functional protein is Na + / K + Examples include ATPase, ZO-1, sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1), carbonic anhydrase 5B (CA5B), and the like.

[0035] Furthermore, as verified in Patent Document 3, human corneal endothelial cells that have been proliferated and / or differentiated and matured in the presence of a ROCK inhibitor and EGF at a concentration below that which causes transformation are thought to have suppressed phase transitions in cultured cells because the activation of epigenetic polygenes, particularly histone acetylation by acetylcoenzyme A (AcCoA), which is produced by metabolites of enzymes involved in the TCA metabolic pathway that function in the cytoplasm and nucleus, is avoided. As evidence, these functional human corneal endothelial cells have been shown to have increased expression of one or more metabolism-related enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malate enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain keto acid dehydrogenase 2 (BCKDH2) in their mitochondria, and these are also included in functional proteins. This suggests that inhibitory proteins, which are at least one metabolism-related enzyme selected from the group consisting of ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malate enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and lactate dehydrogenase (LDH), are not expressed or are hardly expressed.

[0036] The present invention is not limited to what has been described above. For example, the culture medium used does not necessarily have to have the composition described in the above embodiment; it is sufficient as long as it can produce human corneal endothelial cells and / or human corneal endothelial progenitor cells that meet the aforementioned evaluation criteria during cell collection and re-culturing. Furthermore, various modifications and combinations of embodiments are permitted, as long as they do not contradict the spirit of the present invention. [Examples]

[0037] The present invention will be described in more detail below with reference to various embodiments, but the present invention is not limited to these. The following examples were conducted in compliance with medical ethics codes such as the Declaration of Helsinki, regulations such as those of GCH, and regulations established by Kyoto Prefectural University of Medicine and other organizations, and with the approval of the ethics committee related to the organization to which the inventor belongs. The following experiments were conducted after obtaining the necessary informed consent.

[0038] (Example 1: Confirmation of the effect of culture conditions on the appearance rate of phase-transition cells) In this Example 1, it was confirmed that a cell population containing a very high proportion of functional human corneal endothelial cells (effector cells) can be cultured by growing and / or differentiating and maturing cells in a medium containing a ROCK inhibitor and an EGF content below the concentration that causes transformation. Corneal endothelial cells collected from corneas of the same donor were cultured in two types of culture media with different additive compositions, and the effects on cell quality, such as the content of functional human corneal endothelial cells (effector cells), were investigated.

[0039] (Materials used in the experiment) The donor cornea used belonged to an 18-year-old male. The culture medium used was Opti-MEM-I + 8% FBS + 200 mg / ml CaCl2 + 0.08% chondroitin sulfate + 20 μg / ml ascorbic acid + 50 μg / ml gentamicin. In the culture conditions 1 of the following experiment, the culture medium used was prepared by adding 0.5 ng / ml EGF, 10 μM SB203580, and 10 μM Y-27632 as additives to the aforementioned medium. On the other hand, under culture condition 2, a culture medium without EGF and SB203580 was used, in which only 10 μM Y-27632 was added as an additive to the aforementioned medium.

[0040] (Cell culture) Corneal endothelial cells, along with Descemet's membrane, were detached from donor corneas obtained from the Seattle Eye Bank. These cells were treated with collagenase overnight at 37°C, then suspended in culture medium under condition 1. The cells were seeded at a rate of one well per eye in a 6-well plate coated with type I collagen. These wells were then placed in a CO2 incubator and cultured for 34 days. The culture medium was changed twice a week. 10×TrypLE TM After detaching the cells from the culture dish using Select (Thermo Fisher Scientific), the number of recovered cells was measured using a hemocytometer. The cell suspension was divided into two portions; one portion was placed in a new culture vessel containing culture medium under culture condition 1, and the other in a new culture vessel containing culture medium under culture condition 2, each containing 400 cells / mm³. 2 The cells were seeded at a certain density and cultured again in a CO2 incubator for 42 days. 10×TrypLE TM After detaching the cells from the culture dish using Select (Thermo Fisher Scientific), the number of recovered cells was measured using a hemocytometer, with a value of 400 cells / mm³. 2 The cells were seeded at a given cell density and cultured again in a CO2 incubator for 29 to 42 days. This process was repeated under different culture conditions, and subsequent subculturing was performed.

[0041] (Confirmation of cell morphology using phase-contrast photography) Figure 1 shows a phase-contrast image of cells from Passage 3 (the third generation after separating into culture conditions 1 and 2) cultured under culture condition 1 or culture condition 2. Figure 1 shows that under culture condition 1, a large number of significantly larger phase transition cells appear. On the other hand, under culture condition 2, the cell size remains uniform even after multiple passages, and the appearance and increase of phase transition cells are suppressed. Figure 2 shows a phase-contrast image of cells that have been subcultured up to Passage 7 under culture condition 2. Even at this stage, there is almost no difference from the case in Figure 1, indicating that the cell size is uniformly maintained, the appearance and increase of phase-transition cells are suppressed, and there are no foreign bodies, discoloration, or other abnormalities.

[0042] (Analysis of surface antigens) Figure 3 shows the results of flow cytometry analysis of cell surface antigens of Passage 3 cells cultured under culture condition 1 or Passage 3 or Passage 7 cells cultured under culture condition 2. The cell surface antigen analysis was performed in the same manner as in Example 4, which will be described later. As shown in Figure 3, even after repeated subculturing up to Passage 7 under culture condition 2, the surface antigen CD166 in human corneal endothelial cells remained unchanged. + CD24 - CD44 neg~low or CD44 neg~low CD90 neg~low It was found that a cell population exhibiting the desired characteristics was cultured, with a high proportion of effector cells and a high content of the target functional human corneal endothelial cells. Note: CD166 + CD24 - CD44 neg~low Regarding the indicator that this is the case, the upper graph of Figure 3 shows CD166 + CD24 - The cell population contained in gate A is extracted, and the expression level of CD44 is examined in the graph in the middle. neg~low CD90 neg~low The indicator that this is the case is examined in the lower graph of Figure 3, independently of the upper and middle graphs of Figure 3. This is also the case in Figures 6, 10, 16, 20, 21, 22, 30, 38, etc. The expression levels of these surface antigens are defined as follows: "-" or "neg" means that the expression is not substantially observed, i.e., negative. "+" indicates a result that is not negative, meaning that significant expression is observed, or that the result is positive. "Low" refers to a weakly positive result when the expression is classified into three stages: weakly positive, moderately positive, and strongly positive. Results with an expression level below this weakly positive level are considered negative. "High" refers to a strongly positive result when the expression is classified into three stages: weakly positive, moderately positive, and strongly positive. The expression intensity of surface antigens such as the aforementioned CD markers is difficult to define numerically because the fluorescence intensity varies depending on the type of fluorescent label and instrument settings. However, for example, weakly positive, moderately positive, and strongly positive under the following conditions can be defined within the following fluorescence intensity ranges. Conditions: Using PE-Cy7-labeled anti-human CD44 antibody (BD Biosciences), FACSCanto TM When the Area Scaling Factor setting for II is set to Blue laser=0.75 and the voltage setting to PE-Cy7=495. Weakly positive: Fluorescence intensity is approximately less than 3800. Intermediate positive: Fluorescence intensity is approximately 3800 or higher but less than 27500. Strongly positive: Fluorescence intensity is approximately 27,500 or higher. For example, in Figure 3, for the marker used to determine whether a result is negative or positive, a solid line is drawn on the graph to separate the negative and positive regions (also called gates). Furthermore, in Figure 3, for markers that distinguish between weakly positive, moderately positive, and strongly positive levels, three distinct areas (also called gates) are indicated. The percentages in the diagram represent the proportion of cells belonging to each area mentioned above.

[0043] (Confirmation of functional protein expression by immunohistochemistry) Furthermore, for Passage 7 cells cultured under culture condition 2, Na + / K + ATPase and ZO-1 expression were examined by immunohistochemistry. After subculturing to Passage 7, the cells were cultured for 40 days in the same medium, changing the medium twice a week. After removing the culture supernatant, the cells were fixed with ice-cold methanol or 4% paraformaldehyde / phosphate buffer. After washing with PBS(-), the cells were permeabilized with PBS(-) + 0.02% by mass Triton X-100 for 10 minutes. Subsequently, the cells were blocked with PBS(-) + 1% by mass bovine serum albumin at room temperature for more than one hour. After blocking, mouse anti-Na was tested. + / K +The cells were reacted overnight at 4°C with either an ATPase antibody, a mouse anti-ZO-1 antibody, or a mouse anti-N-Cadherin antibody. After washing four times with PBS(-), the cells were reacted with AlexaFluor555-labeled anti-mouse IgG antibody at room temperature for one hour. After washing with PBS(-), the nuclei were stained with 5 μg / ml DAPI for 10 minutes, and after further washing three times with PBS(-), the cells were observed under a fluorescence microscope. The results are shown in Figure 4. As shown in Figure 4, in almost all cells of Passage 7 cultured under culture condition 2, Na, an important functional protein in functional human corneal endothelial cells, was present. + / K + It was confirmed that the organism expresses ATPase, ZO-1, and N-Cadherin.

[0044] (Confirmation of PDGF-BB production) The culture supernatant of Passage 7 cultured under culture condition 2 was collected 34 days after subculturing, and the concentration of PDGF-BB secreted into the culture supernatant was examined using the Human PDGF-BB ELISA Kit (Abcam, #ab184860). As a result, the PDGF-BB concentration in the culture supernatant was 328.4 ± 19.6 pg / mL, confirming that it met the specification value of 100 pg / mL or higher.

[0045] (Consideration) From the above results, it was confirmed that, according to some embodiments disclosed herein, by growing and / or differentiating and maturing cells in a medium containing a ROCK inhibitor and an EGF content below the concentration that causes transformation, the appearance of phase-transition cells can be suppressed, and a cell population containing a very high proportion of functional human corneal endothelial cells can be cultured. Furthermore, from the inventors' experience, it is understood that compounds other than Y-27632 that exhibit ROCK inhibitory properties are similarly or even more effective. In some embodiments, the compounds exhibiting ROCK inhibitory properties may include one or more combinations selected from the group consisting of AT-13148, BA-210, β-elemen, chroman 1, DJ4, fasudil, GSK-576371, GSK429286A, H-1152, hydroxyfasudil, ibuprofen, LX-7101

[24] , netaludil, RKI-1447, ripasudil, TCS-7001, thiazovibin, berosudil (AR-12286), Y-27632, Y-30141, Y-33075, and Y-39983.

[0046] (Example 2: Confirmation of culture conditions that suppress phase transition) In Example 2, it was confirmed that the cell population cultured under the aforementioned culture conditions 2 from primary cultures (Passage 0) of donor cornea contained a high proportion of functional human corneal endothelial cells.

[0047] (Materials used in the experiment) The donor cornea used belonged to a 27-year-old male. The culture medium used was the same as that used in culture condition 2 of Example 1.

[0048] (Cell culture) Corneal endothelial cells, along with Descemet's membrane, were detached from donor corneas obtained from the Seattle Eye Bank. After being treated with collagenase overnight at 37°C, the cells were suspended in the culture medium described in culture condition 2 and seeded at a rate of one well per eye in a 6-well plate coated with type I collagen. These wells were placed in a CO2 incubator and cultured for 40 days. The culture medium was changed once a week. 10×TrypLE TM After detaching the cells from the culture dish using Select (Thermo Fisher Scientific), the number of recovered cells was measured using a hemocytometer. This cell suspension was then transferred to a new culture vessel containing culture medium under culture condition 2, at a rate of 400 cells / mm³ each. 2 The cells were seeded at a certain density and cultured again in a CO2 incubator for 32 days. 10×TrypLE TM After detaching the cells from the culture dish using Select (Thermo Fisher Scientific), the number of recovered cells was measured using a hemocytometer, with a value of 400 cells / mm³. 2 The cells were seeded at a given cell density and cultured again in a CO2 incubator for 38-44 days. This process was repeated under different culture conditions, and subsequent subculturing was performed.

[0049] (Confirmation of cell morphology using phase-contrast photography) Figure 5 shows a phase-contrast image of the Passage 7 cells cultured as described above. From the phase-contrast image in Figure 5, it can be seen that the Passage 7 cells cultured in this example maintain a uniform cell size, and the appearance and increase of phase-transition cells are suppressed.

[0050] (Analysis of surface antigens) The results of analyzing the surface antigens of cells from Passage 7 of this example, using the same procedure as in Example 1, are shown in the graph in Figure 6B. From these results, the surface antigen characteristics of functional human corneal endothelial cells were demonstrated in most of the cells from Passage 7 of this example. These results confirm that, in this example as well, under these culture conditions, it is possible to maintain a high level of cell population with a high content of the target functional human corneal endothelial cells even after long-term subculturing up to Passage 7.

[0051] (Confirmation of functional protein expression by cell immunostaining) Furthermore, regarding the Passage 7 cells cultured in this example, Na + / K +The expression levels of ATPase, ZO-1, and N-Cadherin were examined by immunohistochemical staining in the same manner as in Example 1, and the results are shown in Figure 6A. This result also indicates that in almost all of the Passage 7 cells cultured in this example, Na, an important functional protein in human corneal endothelial cells, was present. + / K + It was confirmed that the organism expresses ATPase, ZO-1, and N-Cadherin.

[0052] (Confirmation of PDGF-BB production) Figure 6C shows the results of examining the concentration of PDGF-BB secreted into the culture supernatant of Passages 0-7 cultured in this example using the Human PDGF-BB ELISA Kit (Abcam, #ab184860). It was confirmed that all Passages met the specification value of 100 pg / mL or higher.

[0053] (Consideration) The results of this Example 2 confirm that even from the primary culture (Passage 0), by using the culture medium under the aforementioned culture conditions 2 (a medium containing a ROCK inhibitor and with an EGF content below the concentration that causes transformation), phase transitions can be suppressed even during long-term culture up to Passage 7, and a cell population containing the target functional human corneal endothelial cells in a very high proportion can be obtained. Phase transitions in human corneal endothelial cells are considered irreversible, and in fact, it has not been possible to revert cells that have undergone a phase transition back to functional human corneal endothelial cells even when cultured in culture medium under condition 2. Therefore, in order to obtain a cell population with a high proportion of functional human corneal endothelial cells, it is preferable to culture the cells in a medium that contains a ROCK inhibitor, such as the one used in culture condition 2, from as early as possible, such as Passage 0 or Passage 1, and in which the EGF content is below the concentration that causes transformation.

[0054] (Example 3: Cell cryopreservation and confirmation of viability) In Example 3, it was confirmed that functional human corneal endothelial cells could be cryopreserved with a high viability rate by harvesting a cell population cultured under the aforementioned culture conditions 2 at a specific culture stage after subculturing, and that a cell population with a high content of functional human corneal endothelial cells could be obtained by culturing them under culture conditions 2 after thawing.

[0055] (Materials used in the experiment) The donor cornea used belonged to a 29-year-old woman. The culture medium used was the same as that used in culture condition 2 of Example 1.

[0056] (Culturing and cryopreservation) Cells cultured up to Passage 3 under the same conditions as in Example 2 (136 days after passage) were subjected to 10×TrypLE TM The cells were separated from the culture dish using Select (Thermo Fisher Scientific) and divided into two. One was suspended in the culture medium under the conditions of Example 1, and then cultured on a culture plate coated with type I collagen at a rate of 400 cells / mm². 2 The cells were seeded at the specified cell density. The remaining cells were suspended in CELLBANKER I (Nippon Zenyaku Kogyo Co., Ltd.) at a concentration of 1,000,000 cells per ml, and this cell suspension was dispensed into cell cryopreservation tubes (Corning Inc.) at a rate of 400 μl per tube. After the cell cryopreservation tubes containing this cell suspension were left to stand on ice for 1 hour, some of the tubes were frozen and stored in the gas phase of a liquid nitrogen tank, while the remaining tubes were placed in an animal cell freezing container BICELL (Nippon Freezer Co., Ltd.) and frozen and stored at -80°C using an ultra-low temperature freezer (PHC Holdings Corporation) (control example). The cells seeded in the former group underwent a medium change on the 4th day after seeding, and on the 5th day, they underwent 10×TrypLE. TM Cells were collected (separated) from the culture plate using Select (Thermo Fisher Scientific). The collected cells were frozen and stored in the same manner as described above (Example 3).

[0057] (Checking the survival rate) Each of the cells that had been cryopreserved in this manner was thawed in a water bath maintained at 37°C, still in its tube, and washed once with 4 ml of culture medium under the culture conditions described in 2. The washed cells were then suspended in 400 μl of culture medium under the culture conditions described in 2. Ten μl of this suspension was mixed with ten μl of 0.4% by mass trypan blue solution (Sigma Aldrich Corp.), and live and dead cells were counted on a hemocytometer. The results are shown in Figure 7 and Table 1. In Figure 7, white cells represent live cells, and blue-stained cells represent dead cells.

[0058] [Table 1]

[0059] (Consideration) From the results of this Example 3, 400 cells / mm 2 When cells were seeded in the culture medium under the aforementioned culture conditions 2 at the cell density and harvested on the 5th day for cryopreservation, it was confirmed that a high survival rate was maintained even after cryopreservation. On the other hand, in a control case where cells were collected and preserved after maturation and differentiation (after 136 days of culture) in the same manner, it was found that the survival rate was significantly reduced. Furthermore, when mature and differentiated cells collected 35 days after subseeding (Passage 5) using corneas from a 14-year-old male donor were cryopreserved using the same method, the survival rate after thawing was 49.5% for those stored in liquid nitrogen and 63% for those stored at -80°C, which was at the same level as the control case described in Table 1.

[0060] (Example 4: Evaluation of cells recovered after cryopreservation) In Example 4, we investigated whether the cell population obtained by thawing and restoring the cells cryopreserved in Example 3 maintained a high content of functional human corneal endothelial cells.

[0061] (Confirmation of cell morphology using phase-contrast photography) 380 μl of cell suspension, obtained by cryopreserving cells from the same donor as in Example 3 (Passage 3) using the same method, was thawed using the same method as in Example 3, and then diluted to 2 ml with the culture medium described in Culture Condition 2. The cells were then seeded into one well of a 6-well cell culture plate coated with type I collagen. The cells were cultured at 37°C under humidified air containing 5% v / v CO2. The culture medium was changed twice a week with the same culture medium described in Culture Condition 2, and observations were made periodically using a phase-contrast microscope. The observed changes in cell morphology over time are shown in Figure 8. The results in Figure 8 show that the cells, after cryopreservation, proliferated and developed into a cobblestone-like (polygonal or circular, etc.) morphology with uniform size. Furthermore, Figure 9 shows the results of comparing cells cultured up to day 35 without cryopreservation in Example 3 with cells cultured up to day 35 by continuing to change the culture medium without being harvested on day 4 in Example 3, using a phase-contrast microscope. The results in Figure 9 show no morphological differences between cells that were cryopreserved and cells that were cultured without cryopreservation.

[0062] (Confirmation of surface antigens: FACS analysis) Cells cultured for 35 days without cryopreservation in Example 3 and cells cultured for 35 days in this example were subjected to 10×TrypLE spectroscopy. TM The cells were collected from the culture plate using Select (Thermo Fisher Scientific) and placed in FACS buffer (PBS + 0.5% BSA + 0.05% NaN3) in 4 × 10⁶ solutions. 6 The cells were suspended to a concentration of cells / ml. 20 μL of this cell suspension was mixed with 20 μL of antibody solution 1 or antibody solution 2, and incubated at 4°C for 1.5 to 2 hours under light protection. The antibody solutions used are as follows. All antibodies without a manufacturer listed were obtained from BD Biosciences. (Antibody solution 1) A solution prepared by diluting FITC-conjugated anti-human CD90 mAb (4 μL), PE-conjugated anti-human CD166 mAb (4 μL), PerCP-Cy5.5-conjugated anti-human CD24 mAb (1 μL), PE-Cy7-conjugated anti-human CD44 mAb (0.25 μL), and APC-conjugated anti-human CD105 (eBioscience, 1 μL) to 20 μL with FACS buffer. After washing with FACS buffer, the cells were analyzed using FACSCanto TM II (BD Biosciences, S / N = V33896101710). The results are shown in Fig. 10. The voltage settings for each fluorescence of FACSCanto TM II were as follows: FSC = 270, SSC = 380, FITC = 290, PE = 290, PerCP-Cy5.5 = 410, PE-Cy7 = 495, APC = 430. The setting values of the Area Scaling Factor were as follows: FSC = 0.5, Blue Lasor = 0.75, Red Lasor = 0.8. From the analysis results of the surface antigens in Fig. 10, there was no difference in the content rate of effector cells showing the characteristics of CD166 + CD24 - CD44 neg~low (Gate 1) or CD44 neg~low CD90 neg~low (the gate at the lower left of the lower panel).

[0063] (Evaluation by immunostaining: Immunocyte staining) The cells cultured for 35 days in this example were collected using 10×TrypLE TM Select (Thermo Fisher Scientific), suspended in the medium of Culture Condition 2, and seeded onto a culture plate coated with type I collagen at a cell density of 400 cells / mm 2 . After culturing for 5 weeks while changing the medium twice a week with the same medium, immunostaining was performed in the same manner as in Example 1. However, the antibody used as the primary antibody was mouse anti-Na + / K +ATPase antibody and rabbit anti-ZO-1 antibody were used, along with AlexaFluor488-labeled anti-rabbit IgG antibody and AlexaFluor555-labeled anti-mouse IgG antibody as secondary antibodies. The results were observed using a fluorescence microscope and are shown in Figure 11. These results suggest that even in cryopreserved cells, recovery culture after thawing can restore the functional protein Na of human corneal endothelial cells. + / K + We were able to confirm that ATPase and ZO-1 were expressed normally.

[0064] (Consideration) From these results, it was confirmed that the cell population that was cryopreserved in Example 3 and then thawed and restored had a high content of functional human corneal endothelial cells, similar to the cells that were cultured without cryopreservation.

[0065] (Example 5: Confirmation of cryopreservation and survival rate when using other culture media) In Example 5, the effect of the type of culture medium on the survival rate after cryopreservation was investigated.

[0066] (Materials used in the experiment) The donor corneas used were from an 18-year-old male and a 10-year-old male. The culture medium used was Essential 6 + 8% FBS by mass + 50 μg / ml gentamicin + 10 μM Y-27632. Essential 6 medium is based on DMEM / F12 medium, which is widely used in cell culture, with the addition of ascorbic acid, insulin, transferrin, selenium, etc., and its composition is publicly available.

[0067] (Confirmation of survival rate after cryopreservation) The survival rate was examined using trypan blue staining in the same procedure as in Example 3 described above. The results are shown in Table 2 below.

[0068] [Table 2]

[0069] (Examination) Since almost the same survival rate was observed in Example 3, it was confirmed that the effects of the present invention can be achieved even when different basal media are used.

[0070] (Example 6: Optimization of Cell Collection Timing) In Example 6, an investigation was conducted to optimize the timing of cell collection during cryopreservation.

[0071] (Example 6-a: Examination of Cell Collection Timing) (Materials Used in the Experiment) The donor cornea used was from a 41-year-old female. The medium used was the same as that in Culture Condition 2 of Example 1.

[0072] (Culture and Cryopreservation) The donor cornea obtained from the Seattle Eye Bank was cultured up to Passage 2 under the same conditions as in Example 2. At the time of passage to Passage 3, the cell suspension was divided into five flasks. One flask was cultured continuously, and for the remaining four flasks, cells were collected on the 3rd, 7th, 10th, and 14th days after passage, respectively. The collected cells were suspended in CELLBANKER I to a concentration of 1,000,000 cells per milliliter. This cell suspension was placed in the gas phase part of a liquid nitrogen tank and frozen for cryopreservation in the liquid nitrogen tank. The cells in the first flask that had been continuously cultured were passaged to a type I collagen-coated 24-well plate at a cell density of 400 cells / mm 2 on the 35th day after passage. For the cells cryopreserved in the liquid nitrogen tank, thawing operations were also performed on the same day as the cells that had been continuously cultured. A ThowSTAR frozen cell thawing station (Model CFTS, Astero Bio) was used for thawing. After thawing, the cells washed with the medium of Culture Condition 2 were seeded onto a type I collagen-coated 2-four-well plate at a cell density of 400 cells / mm 2 to achieve the desired cell density. These cells were cultured at 37°C under humidified air containing 5% v / v CO2. The culture medium was changed twice a week. Protein expression was compared and analyzed by FACS and immunocytochemistry 35 days after seeding in 24-well plates. The procedure is outlined in Figure 12. Phase-contrast microscope images of the cell morphology on the day each cell lot was collected for cryopreservation are shown in Figure 13. Furthermore, the viability of the cryopreserved cells after thawing and the results of trypan blue staining are shown in Figure 14, and phase-contrast microscope images, FACS analysis, and immunocytochemistry results 37 days after recovery culture are shown in Figures 15 to 17. The FACS analysis and immunocytochemistry were performed using the same procedure as in Example 4.

[0073] (Consideration) The results in Figure 13 show that significant changes in both cell size and morphology were observed by 14 days after subsowing. Since the cells at 14 days resembled functional human corneal endothelial cells at 35 days, it can be inferred that the cells were in the differentiation stage up to this point. The results in Figure 14 show that cells collected and cryopreserved 3 days after subculturing had a survival rate of less than 50% after thawing and recovery. However, cells collected and cryopreserved on day 14 had a higher survival rate of approximately 70%, and cells collected and cryopreserved on day 7 or 10 showed even higher survival rates. This confirms that the survival rate differs significantly depending on the timing of collection after subculturing. Furthermore, the results shown in Figures 15-17 indicate that cells collected and cryopreserved on day 7, day 10, or day 14, after thawing and recovery culture, become a cell population containing functional human corneal endothelial cells at a rate equivalent to that of cells cultured without cryopreservation. These results indicate that, in order to obtain a cell population containing functional human corneal endothelial cells at a rate equivalent to that of cells cultured without cryopreservation, with a high survival rate even after cryopreservation, it is preferable to collect and store cells within a period of 4 to 14 days from the most recent passage.

[0074] (Example 6-b: Optimization of cell harvesting timing) This example was conducted to further optimize the timing of cell collection, which was determined in Example 6-a.

[0075] (Materials used in the experiment) The donor cornea used belonged to an 8-year-old male. The culture medium used was the same as that used in culture condition 2 of Example 1.

[0076] (Culturing and cryopreservation) The donor corneas obtained from the Seattle Eye Bank were cultured up to Passage 4 under the same conditions as in Example 2. When subculturing to Passage 5, the cell suspension was divided into five flasks, and cells were harvested on days 6, 7, 8, 9, and 10 after subculturing. The harvested cells were suspended in STEMCELL-BANKER, 10% DMSO / 90% FBS, or BAMBANKER hRM (day 8 only) at a concentration of 1,000,000 cells per ml. This cell suspension was placed in the gas layer of a liquid nitrogen tank, frozen, and then stored cryopreserved within the liquid nitrogen tank. Thawing was performed 200 days after recovery and freezing on day 10 (204 days after recovery and freezing on day 6). A 37°C water bath was used for thawing. After thawing, the cells washed with the culture medium described in culture condition 2 were placed in a type I collagen-coated 24-well plate at a rate of 400 cells / mm². 2 Seeds were seeded to achieve the following cell density. These cells were cultured at 37°C under humidified air containing 5% v / v CO2. The culture medium was changed twice a week. Protein expression was compared and analyzed by FACS and immunocytochemistry 35 days after seeding in 24-well plates. The procedure is outlined in Figure 18. The viability of the cryopreserved cells after thawing is shown in Figure 19, and the results of FACS analysis and immunocytochemistry 35 days after recovery culture are shown in Figures 20 to 25. Viability was confirmed by trypan blue staining, as in the other examples. The FACS analysis and immunocytochemistry were performed using the same procedure as in Example 4. The bar graph on the right side of Figure 19 shows the viability of cells collected on day 8 when STEMCELL-BANKER, 10% DMSO / 90% FBS, or BAMBANKER hRM were used as the cryopreservation solution.

[0077] (Consideration) The results in Figure 19 show that cells collected and cryopreserved 6 to 10 days after the most recent passage showed a high survival rate of over 80% after thawing and recovery, indicating that collection during this period is preferable. Furthermore, the graph on the right side of Figure 19 shows that cells collected and preserved within the aforementioned period maintain a sufficiently high survival rate even when the type of cryopreservation solution is changed. Furthermore, the results shown in Figures 20-25 indicate that cells recovered and cryopreserved during this period become a cell population that maintains high levels of functional human corneal endothelial cells after recovery culture following thawing. It was also confirmed that there were no significant changes in the survival rate after storage or the proportion of functional human corneal endothelial cells after recovery culture, even when the type of preservation solution was changed.

[0078] (Example 7: Regarding other indicators showing the timing of cell collection) In Example 6, the number of days after seeding (passaging) on ​​a plate was examined as an indicator of the timing of cell collection. In Example 7, other indicators for determining the timing of human corneal endothelial cells and / or human corneal endothelial progenitor cells were examined by investigating the characteristics of cells at the time identified in Example 6.

[0079] (Example 7-a: Regarding indicators based on cell morphology) (Materials used in the experiment) The donor cornea used belonged to a 29-year-old male. The culture medium used was the same as that used in culture condition 2 of Example 1.

[0080] (Cell culture and observation of cell morphology) Corneal endothelial cells, along with Descemet's membrane, were detached from the donor corneas obtained from the Seattle Eye Bank. After being treated with collagenase overnight at 37°C, the cells were suspended in the culture medium and seeded at a rate of one well per eye in a 6-well plate coated with type I collagen. The cells were cultured up to the fourth passage using the same procedure as in Example 2 described above. The culture medium was changed twice a week. After passing through five passages, the cells were cultured under the same conditions, and the cell morphology was photographed using a phase-contrast microscope each day. The results are shown in Figure 26, and a magnified view of a portion of Figure 26 is shown in Figure 27 for greater clarity.

[0081] (Consideration) The results shown in Figures 26 and 27 indicate that on the first and second days after subsowing, the cells have a spindle-shaped, fibroblast-like morphology with irregular, elongated projections. However, from the third day onward, the ratio of the major and minor axes becomes closer to approximation, and by the fifth day, they transform into a shape similar to in vivo corneal endothelial cells, with a major-minor-long ratio close to 1, resembling cobblestone-like (polygonal or circular) structures. Such significant morphological changes were observed up to the fifth day after sowing. Two weeks after sowing, the cells become uniform in size, and from the third week onward, the boundaries between individual cells become less distinct, and cell-to-cell adhesion is formed, leading to a maturation stage where the cells become somewhat denser. Based on the above observations, it is presumed that under these culture conditions, redifferentiation occurs within two weeks (14 days) after sowing, and then the plants proceed to the maturation process. In comparison with the results of Example 6-a, the period during which target human corneal endothelial cells and / or human corneal endothelial progenitor cells can be harvested can be defined as "the period from immediately after the cell morphology changes from a spindle-shaped fibroblast-like form with irregular, elongated projections to a shape similar to in vivo corneal endothelial cells with only minute projections and a length-to-short axis ratio close to 1, to just before cell-to-cell adhesion is formed and the boundaries between cells become indistinct." Here, the number of days in Example 6-a is used as a guideline, but it is thought that the number of culture days required for cells to mature may vary depending on the cell density during subculturing and the culture conditions. Even in such cases, it is thought that cells can be collected at the appropriate time by using the cell morphology obtained in this example as an indicator.

[0082] (Example 7-b: Indicators for harvesting timing based on CD44 expression level) The timing of cell collection can also be determined by the expression level of CD44. For cells cultured under the same conditions after the 5th passage of Example 7-a, the CD44 expression level was measured daily using a flow cytometer.

[0083] (Materials used in the experiment) • Same cells as in Example 7-a The same FACS buffer and antibody solution used in Example 4 were employed.

[0084] (Measurement of CD44 expression levels) After subculturing to the 5th passage in Example 7-a and culturing the cells under the same conditions for a predetermined number of days, wash with PBS(-) and then perform 10×TrypLE TM By processing with Select (Thermo Fisher Scientific), the cells are recovered from the culture plates and placed in FACS buffer in a 4 × 10⁶ solution. 6 The cells were suspended to a concentration of cells / ml. 20 μL of this cell suspension was then stained with antibody using the same method as in Example 4, followed by FACSCanto. TM The analysis was performed using II (BD Biosciences). The results are shown in Figure 28.

[0085] (Results / Discussion) The average fluorescence intensity of CD44 rose to over 15,000 immediately after sowing (subculturing), then decreased logarithmically by day 11. By day 17, it had almost reached a plateau, suggesting that differentiation is completed approximately two weeks after subculturing and sowing, and the organisms then move into the maturation process. Based on the results of Example 6-a and Figure 28, it is considered that the period in which target human corneal endothelial cells and / or human corneal endothelial progenitor cells can be collected is from when the CD44 expression level falls to less than half of the maximum value observed on day 1-2 after passage until it reaches a plateau. Here, the number of days in Example 6-a is used as a guideline, but it is thought that the number of culture days required for cells to mature may vary depending on the cell density during subculturing and the culture conditions. Even in such cases, it is thought that cells can be harvested at the appropriate time by using the CD44 expression level obtained in this example as an indicator.

[0086] (Example 7-c: Indicators for timing of harvesting based on cell density) The ECD (corneal endothelial cell density) of the cells from Example 7-a was measured over time using KSSE-400EB after passage.

[0087] (Materials used in the experiment) • Same cells as in Example 7-a • Analysis software: KSSE-400EB (KONAN Medical)

[0088] (Measurement of cell density) 400 cells / mm 2 Cells from Example 7-a, which were subcultured to the 5th passage at a certain density, were washed with PBS(-) at 2, 4, 6, 9, 12, 17, and 35 days after subculturing, and photographed with a phase-contrast microscope. The obtained images were analyzed using KSSE-400EB software. The results are shown in Figure 29.

[0089] (Results / Discussion) The ECD (corneal endothelial cell density) increased linearly from immediately after subculturing to the 5th passage until day 17, reaching a plateau after day 17. This suggests that the proliferation phase after subculturing continues until around day 17, when the cells transition to the maturation process. To collect cells during the limited period from when the fate of differentiation into mature human cells is determined until before the transition to the maturation process, the ECD value should be 900 cells / mm³, in comparison with the results of Example 6-a. 2 That's all, 2500 cells / mm 2 It is considered best to collect them during the following times. Here, the number of days in Example 6-a is used as a guideline, but it is thought that the number of culture days required for cells to mature may vary depending on the cell density during subculturing and the culture conditions. Even in such cases, it is thought that cells can be harvested at the appropriate time by using the cell density obtained in this example as an indicator.

[0090] (Example 8: Confirmation of the viability of cells cryopreserved for a long period and their quality after recovery culture) In Example 8, we investigated the survival rate and quality after recovery culture of cells that were cryopreserved under the conditions optimized in Examples 6 and 7 when stored for a long period of time. (Materials used in the experiment) The donor cornea used belonged to an 8-year-old male. The culture medium used was the same as that used in culture condition 2 of Example 1.

[0091] (Cell culture and cryopreservation) Corneal endothelial cells, along with Descemet's membrane, were detached from the donor corneas obtained from the Seattle Eye Bank. After being treated with collagenase overnight at 37°C, the cells were suspended in the culture medium and seeded at a rate of one well per eye in a 6-well plate coated with type I collagen. The cells were cultured up to the second passage using the same procedure as in Example 2 described above. The culture medium was changed twice a week. On the 7th day after seeding, the cells were harvested, frozen using the same method as in Example 6, and stored in a liquid nitrogen cell storage container. Cells were thawed in a water bath on days 99, 160, and 171 of storage, and their viability was examined by trypan blue staining using the same procedure as in Example 3. In addition, to dispel the suspicion that the apparent viability rate might be inflated due to the destruction of dead cells, the recovery rate (number of cells after freezing / number of cells used for freezing), which is the ratio of the number of cells subjected to cryopreservation to the number of cells when viability was examined after cryopreservation, was also examined. The results are shown in Table 3.

[0092] [Table 3]

[0093] (Quality assessment of cells recovered and cultured after cryopreservation) Of these, cells stored in liquid nitrogen for 99 days were cultured for 33 days under culture condition 2, and the results of evaluating cell morphology, FACS analysis, and functional protein expression by immunocytochemistry are shown in Figures 30A-C. FACS analysis and immunocytochemistry were performed using the same procedure as in Example 4. The cell density analyzed with KSS-400EB software was 3344 ± 150.8 cells / mm². 2 On day 32, the concentration of PDGF-BB in the culture supernatant was 217.5 ± 1.53 pg / mL. As described above, it was confirmed that the present invention allows for the cryopreservation of a cell population with a high content of functional human corneal endothelial cells after recovery culture, while maintaining a viability of 90% or more for a period of 5 months or more.

[0094] (Example 9: Confirmation of cell survival rate through cryogenic storage) In Example 9, the effect of the timing of cell harvesting after subculturing on the viability of cells stored in suspension at ice temperature was investigated. (Materials used in the experiment) The donor cornea used belonged to an 8-year-old male. The culture medium used was the same as that used in culture condition 2 of Example 1. Each sample is as follows: Sample 1: Cells cultured using the same procedure as in Example 2, and cultured in Passage 3 for 94 days without cryopreservation. Sample 2: Cells cultured using the same procedure as in Example 2, harvested on day 7 of Passage 2, cryopreserved using the same procedure as in Example 3, and then cultured in Passage 3 for 33 days after thawing. Sample 3: Cells cultured using the same procedure as in Example 2, harvested on day 7 of Passage 2, cryopreserved using the same procedure as in Example 3, thawed, and then subcultured up to Passage 5, and cultured for 9 days in Passage 6.

[0095] (Subterranean storage and survival rate after storage) Samples 1-3, as described above, were detached from the culture vessels using 10×TrypLE™ Select (Thermo Fisher Scientific) to prepare cell suspensions. After staining a 10 μl sample of each cell suspension with trypan blue, the number of viable and dead cells was measured using a hemocytometer. Based on these results, 3.33 × 10⁻⁶ 6 The cells were suspended in their respective acclimatization media to achieve a cell density of cells / ml, placed in 1.5 mL commercially available microcentrifuge tubes, and stored on ice.

[0096] (Confirmation of survival rate after storage at sub-zero temperatures) On days 1, 2, 3, and 4 after the start of storage, a portion (10 μl) of each cell suspension was collected, stained with trypan blue, and the number of viable and dead cells was measured using a hemocytometer to calculate the cell viability. Figure 31 shows the results of trypan blue staining, and Figure 32 shows the viability calculated from the results in Figure 31. The results in Figure 32 show that while the survival rate for Sample 1 fell below 70% on day 1 and for Sample 2 on day 2, Sample 3 maintained a survival rate of over 80% even on day 4.

[0097] (Consideration) From these results, it was confirmed that, similar to cryopreservation, if the plants are collected and stored between 4 and 14 days after the most recent subculturing, the survival rate can be maintained at a higher level even under sub-zero storage conditions compared to collection after maturation.

[0098] (Example 10: Effect of preservation solution on cell storage at ice temperature) In Example 10, the effect of the type of preservation solution used during ice-temperature storage on the survival rate was investigated. (Materials used in the experiment) The cells used in the experiment were Sample 3 from Example 9. The preservation solutions used in the experiment are as follows: Preservation solution A: Conditioned medium Preservation solution B: Culture medium prepared on the same day under the culture conditions 2 of Example 1. Preservation solution C: Opti-MEM + 100 μM Y-27632 prepared on the same day

[0099] (Preservation at sub-zero temperatures and confirmation of survival rate) The cells from Sample 3 of Example 9 were placed in the aforementioned preservation solutions A, B, or C in a 3.33 × 10⁴ solution. 6 The cells were suspended to a cell density of cells / ml and stored on ice. On days 1, 2, 3, and 4 after the start of storage, a portion (10 μl) of each cell suspension was collected, stained with trypan blue, and the number of viable and dead cells was measured using a hemocytometer to calculate the cell viability. Figure 33 shows the results of trypan blue staining, and Figure 34 shows the viability calculated from the results in Figure 33. The results in Figure 34 show that regardless of which preservation solution was used, a survival rate of 70% or more could be maintained for at least two days. Furthermore, it was found that a high survival rate of over 80% could be maintained even after the fourth day if the culture medium was acclimatized.

[0100] (Consideration) From these results, it can be inferred that a wide variety of preservation solutions can be used for ice-temperature storage. Furthermore, it was found that by using a more suitable preservation solution, the survival rate of human corneal endothelial cells can be maintained at a high level of over 80% for a long period exceeding 4 days, even when stored at freezing temperatures.

[0101] (Example 11: Evaluation of the differentiation and maturation period of cells stored at freezing temperatures into functional human corneal endothelial cells) In Example 11, we confirmed whether cells stored at ice temperature, when seeded at the same cell density as those transplanted into humans, differentiate and mature into functional human corneal endothelial cells at an early stage, similar to when transplanted into humans.

[0102] (Evaluation of cell morphology) In human transplantation, 3.33 × 10 6 300 μl of a cell suspension with a cell density of cells / ml was injected into the anterior chamber. In Example 9, a cell suspension (with a cell density equivalent to that at the time of transplantation into humans) that had been stored at ice temperature for 4 days using preservation solution A was placed in a 24-well plate (diameter approximately 7.78 mm, base area 190 mm²) with an area close to that of a human endothelial surface. 2 300 μl of seeds were seeded onto type I collagen-coated substrate. After seeding, the culture was incubated for 7 days under the culture conditions 2 of Example 1. A phase-contrast microscope image of the culture during this period is shown in Figure 35. Figure 35 shows that even cells stored at ice temperature for four days, when seeded at the same cell density as those used for human transplantation, develop the morphology of functional human corneal endothelial cells within one week of seeding.

[0103] (Evaluation by immunohistochemical staining) The cells in Figure 35 were fixed with ice-cold methanol, and the functional protein of human corneal endothelial cells, Na, was found. + / K + The expression of ATPase and ZO-1 was confirmed by immunohistochemical staining. The results are shown in Figure 36. From the results in Figure 36, in almost all cells, Na + / K + Expression of ATPase and ZO-1 was confirmed.

[0104] (Consideration) From these results, it was confirmed that even cells stored at ice temperature for 4 days, when seeded at a cell density equivalent to that used for human transplantation, differentiated and matured within one week, just as in human transplantation, and expressed human corneal endothelial cell functional proteins. In this Example 11, one reason why a population of functional human corneal endothelial cells could be obtained in a short period of one week after seeding is thought to be that the cells were seeded at a high density, eliminating the need for a proliferation period, and thus the cells immediately proceeded to differentiation and maturation after seeding.

[0105] (Example 12: Short-term expansion culture of human corneal endothelial cells after cryopreservation) In Examples 1 to 11 above, it was confirmed that no phase transition occurred during culture after storage, even when cells were collected within 14 days of the most recent passage. Therefore, in Example 12, we attempted to proliferate human corneal endothelial cells in a shorter period than conventional methods by collecting cells within 14 days and repeating the passage process.

[0106] (Culture conditions) The cells were cultured until day 7 of Passage 2 using the same procedure as in Example 2. These Passage 2 cells were then harvested on day 7 and cryopreserved using the same procedure as in Example 3. The cell suspension, after cryopreservation, was thawed, and the cell density at the start of culture was 400 cells / mm³. 2 The cells were propagated by seeding to achieve a certain cell density and then subculturing every 8 to 10 days, repeating this process up to Passage 8.

[0107] (Results and Discussion) These results are shown in Figure 37 and Table 4. Note that the phase-contrast microscope image in Figure 37 is from day 10 of Passage 8.

[0108] [Table 4] From the results in Figure 37 and Table 4, it was found that while conventional methods took more than six months to increase the cell count by 1000 times, this embodiment can shorten that time to just about 50 days.

[0109] Furthermore, the cells from Passage 8 on day 10 were passaged to Passage 9, and a phase-contrast microscope image of the cells cultured for 34 days under culture condition 2 is shown in Figure 38A, and the results of surface antigen analysis by FACS are shown in Figure 38B. The FACS analysis was performed using the same procedure as in Example 4. It was confirmed that even cells that were repeatedly passaged at intervals of 8 to 10 days after seeding, before differentiation and maturation, could be further cultured in a medium containing a ROCK inhibitor and an EGF content below the concentration that causes transformation, thereby obtaining a cell population with a sufficiently high content of functional human corneal endothelial cells.

[0110] (Example 13: Short-term expansion culture of human corneal endothelial cells) In Example 12 described above, we described an example of thawing a cell suspension after cryopreservation and performing short-term expansion culture. However, in this example, we attempted short-term expansion culture of cells that had not been cryopreserved.

[0111] (Materials used in the experiment) The donor corneas used were from a 29-year-old male and an 8-year-old female.

[0112] (Culture conditions) One cornea from the same donor was cultured by subculturing at 35-day intervals under culture conditions 1 of Example 1 (Control Example 1). The other cornea was cultured under culture conditions 2 of Example 1, and divided into two at the time of subculturing to Passage 1. One was cultured by subculturing at 35-day intervals under culture conditions 2 of Example 1 (Control Example 2), and the other was cultured by repeatedly subculturing at intervals of approximately 8 days (5 to 11 days) as in Example 12 (Example 13). An overview of the procedure is shown in Figure 39.

[0113] (Results and Discussion) Figure 40 shows the yield and culture scale calculated from the growth rate. This short-term expansion culture method, in which subculturing was repeated at intervals of approximately 8 days, was shown to yield 2 billion cells within 80 days, equivalent to 500 T-25 flasks. Furthermore, Figures 41 and 42 show photographs and yield comparisons of cells cultured up to Passage 4 using the short-term expansion culture method described above and then cultured for approximately 5 weeks in Passage 5, as well as cells from control example 2 cultured for approximately the same number of days and cells from control example 2 cultured up to Passage 5. Similarly, Figures 43 and 44 show photographs and yield comparisons of cells cultured using the short-term expansion culture method up to Passage 6 and then cultured for approximately 5 weeks in Passage 7, as well as cells from control example 2 cultured for approximately the same number of days and cells from control example 2 cultured up to Passage 7.

[0114] (Consideration) The results shown in Figures 41-44 demonstrate that even with the short-term expansion culture method used in this example, a cell population with a sufficiently high content of functional human corneal endothelial cells can be obtained in a short period of time and with high yield.

[0115] (Discussion regarding Examples 1-13) From the results of each of the above examples, it was found that if cells are destined to become functional human corneal endothelial cells when cultured under appropriate culture conditions, then the desired functional human corneal endothelial cells can be obtained even if those cells are collected in an immature state and stored or subcultured.

Claims

1. Human corneal endothelial cells and / or human corneal endothelial precursor cells are grown using a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, and the human corneal endothelial cells and / or human corneal endothelial precursor cells are grown and / or differentiated and matured using a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, and the purity test results by FACS for a cell population obtained are within the following ranges: CD166 ＋ >99% CD24 ＋ <5% CD44 ｈｉｇｈ <5% CD44 ｎｅｇ～ｌｏｗ >90% CD90 ＋ <5% A method for preserving human corneal endothelial cells and / or human corneal endothelial precursor cells, comprising collecting human corneal endothelial cells and / or human corneal endothelial precursor cells which satisfy all of the above requirements at a time when any one or more of the following conditions (a) to (d) are satisfied, and preserving them in a suspended state. (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial precursor cells changes from a spindle shape to a polygonal or circular shape with a ratio of major axis to minor axis close to 1, to immediately before the boundaries between the cells become unclear. (b) The period from when the CD44 expression level becomes half or less of the maximum level observed after the most recent passage to when it reaches a plateau. (c) the cell density of the human corneal endothelial cells and / or the human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500cells / mm ２ The following is the result. (d) When the cells are seeded at a cell density of 300 cells / mm 2 or more and 500 cells / mm 2 or less, the number of days since the most recent passage is 4 days or more and 14 days or less.

2. A method for preserving human corneal endothelial cells and / or human corneal endothelial precursor cells, characterized in that human corneal endothelial cells and / or human corneal endothelial precursor cells grown in a medium containing a ROCK inhibitor and not containing epidermal growth factor (EGF) are harvested at a time when any one or more of the following conditions (a) to (d) are satisfied, and preserved in a suspended state. (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial precursor cells changes from a spindle shape to a polygonal or circular shape with a ratio of major axis to minor axis close to 1, to immediately before the boundaries between the cells become unclear. (b) The period from when the CD44 expression level becomes half or less of the maximum level observed after the most recent passage to when it reaches a plateau. (c) the cell density of the human corneal endothelial cells and / or the human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500cells / mm ２ The following is the result. (d) When the cells are seeded at a cell density of 300 cells / mm 2 or more and 500 cells / mm 2 or less, the number of days since the most recent passage is 4 days or more and 14 days or less.

3. Human corneal endothelial cells and / or human corneal endothelial precursor cells are grown using a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, and the human corneal endothelial cells and / or human corneal endothelial precursor cells are grown and / or differentiated and matured using a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, and the purity test results by FACS for a cell population obtained are within the following ranges: CD166 ＋ >99% CD24 ＋ <5% CD44 ｈｉｇｈ <5% CD44 ｎｅｇ～ｌｏｗ >90% CD90 ＋ <5% A method for preparing a suspension of human corneal endothelial cells and / or human corneal endothelial precursor cells, comprising collecting human corneal endothelial cells and / or human corneal endothelial precursor cells which satisfy all of the above at a time when any one or more of the following conditions (a) to (d) are satisfied, and putting them into a suspended state. (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial precursor cells changes from a spindle-shaped fibroblast-like form having irregular, elongated processes to a cobblestone-like form in which the ratio of the long axis to the short axis, including the processes, is close to 1, to immediately before the boundaries between the cells become unclear. (b) The period from when the CD44 expression level becomes half or less of the maximum level observed after the most recent passage to when it reaches a plateau. (c) the cell density of the human corneal endothelial cells and / or the human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500cells / mm ２ The following is the result. (d) When the cells are seeded at a cell density of 300 cells / mm 2 or more and 500 cells / mm 2 or less, the number of days since the most recent passage is 4 days or more and 14 days or less.

4. A method for preparing a suspension of human corneal endothelial cells and / or human corneal endothelial precursor cells, comprising harvesting human corneal endothelial cells and / or human corneal endothelial precursor cells grown in a medium containing a ROCK inhibitor and not containing epidermal growth factor (EGF) at a time when any one or more of the following conditions (a) to (d) are satisfied, and suspending the cells: (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial precursor cells changes from a spindle-shaped fibroblast-like form having irregular, elongated processes to a cobblestone-like form in which the ratio of the long axis to the short axis, including the processes, is close to 1, to immediately before the boundaries between the cells become unclear. (b) The period from when the CD44 expression level becomes half or less of the maximum level observed after the most recent passage to when it reaches a plateau. (c) the cell density of the human corneal endothelial cells and / or the human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500cells / mm ２ The following is the result. (d) When the cells are seeded at a cell density of 300 cells / mm 2 or more and 500 cells / mm 2 or less, the number of days since the most recent passage is 4 days or more and 14 days or less.

5. Human corneal endothelial cells and / or human corneal endothelial precursor cells are grown using a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, and the human corneal endothelial cells and / or human corneal endothelial precursor cells are grown and / or differentiated and matured using a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, and the purity test results by FACS for a cell population obtained are within the following ranges: CD166 ＋ >99% CD24 ＋ <5% CD44 ｈｉｇｈ <5% CD44 ｎｅｇ～ｌｏｗ >90% CD90 ＋ <5% A method for culturing human corneal endothelial cells and / or human corneal endothelial precursor cells, comprising harvesting and subculturing human corneal endothelial cells and / or human corneal endothelial precursor cells which satisfy all of the above at a time when any one or more of the following conditions (a) to (d) are satisfied: (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial precursor cells changes from a spindle-shaped fibroblast-like morphology having irregular elongated processes to a cobblestone-like morphology in which the ratio of the major axis to the minor axis is close to 1, to immediately before the boundaries between the cells become unclear. (b) The period from when the CD44 expression level becomes half or less of the maximum level observed after the most recent passage to when it reaches a plateau. (c) the cell density of the human corneal endothelial cells and / or the human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500cells / mm ２ The following is the result. (d) When the cells are seeded at a cell density of 300 cells / mm 2 or more and 500 cells / mm 2 or less, the number of days since the most recent passage is 4 days or more and 14 days or less.

6. A method for culturing human corneal endothelial cells and / or human corneal endothelial precursor cells, characterized in that human corneal endothelial cells and / or human corneal endothelial precursor cells grown in a medium containing a ROCK inhibitor and not containing epidermal growth factor (EGF) are harvested and passaged at a time when any one or more of the following conditions (a) to (d) are satisfied: (a) The period from immediately after the morphology of the human corneal endothelial cells and / or the human corneal endothelial precursor cells changes from a spindle-shaped fibroblast-like morphology having irregular elongated processes to a cobblestone-like morphology in which the ratio of the major axis to the minor axis is close to 1, to immediately before the boundaries between the cells become unclear. (b) The period from when the CD44 expression level becomes half or less of the maximum level observed after the most recent passage to when it reaches a plateau. (c) the cell density of the human corneal endothelial cells and / or the human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500cells / mm ２ The following is the result. (d) When the cells are seeded at a cell density of 300 cells / mm 2 or more and 500 cells / mm 2 or less, the number of days since the most recent passage is 4 days or more and 14 days or less.

7. A cell population of cultured human corneal endothelial cells and / or human corneal endothelial precursor cells, which is obtained by proliferating and / or differentiating and maturing the human corneal endothelial cells and / or human corneal endothelial precursor cells in a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, has a purity test result by FACS within the following range: CD166 ＋ >99% CD24 ＋ <5% CD44 ｈｉｇｈ <5% CD44 ｎｅｇ～ｌｏｗ >90% CD90 ＋ <5% A method for preserving human corneal endothelial cells and / or human corneal endothelial precursor cells that satisfy all of the above requirements, comprising: Obtaining a plurality of human corneal endothelial cells and / or human corneal endothelial precursor cells; separating at least a portion of the plurality of cells to generate a cell population for evaluation and a cell population for storage; assessing at least a portion of the cell population against one or more of the following assessment criteria: (a), (b), (c), and (d); (a) identifying cells in which the morphology of human corneal endothelial cells and / or human corneal endothelial precursor cells has changed from a spindle shape to either (i) a polygonal shape or (ii) an elliptical shape with a ratio of the major axis to the minor axis close to 1, and in which the boundaries between cells are clearly defined; (b) identifying cells expressing a constant amount of CD44 after the expression of CD44 reaches levels less than half the maximum observed after the previous passage; (c) The cell density of the human corneal endothelial cells and / or human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500 pieces / mm ２ Identifies that: (d) When the cells are seeded at a cell density of 300 cells / mm 2 to 500 cells / mm 2 , it is specified whether the period from the evaluation to the immediately preceding passage is 4 days or more and 14 days or less. and if the cell population for evaluation satisfies one or more of the evaluation criteria (a), (b), (c), or (d), placing at least a portion of the cells from the cell population in a storage medium for preservation.

8. A method for preserving human corneal endothelial cells and / or human corneal endothelial precursor cells cultured in a medium not containing EGF, comprising: Obtaining a plurality of human corneal endothelial cells and / or human corneal endothelial precursor cells; separating at least a portion of the plurality of cells to generate a cell population for evaluation and a cell population for storage; assessing at least a portion of the cell population against one or more of the following assessment criteria: (a), (b), (c), and (d); (a) identifying cells in which the morphology of human corneal endothelial cells and / or human corneal endothelial precursor cells has changed from a spindle shape to either (i) a polygonal shape or (ii) an elliptical shape with a ratio of the major axis to the minor axis close to 1, and in which the boundaries between cells are clearly defined; (b) identifying cells expressing a constant amount of CD44 after the expression of CD44 reaches levels less than half the maximum observed after the previous passage; (c) The cell density of the human corneal endothelial cells and / or human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500 pieces / mm ２ Identifies that: (d) When the cells are seeded at a cell density of 300 cells / mm 2 to 500 cells / mm 2 , it is specified whether the period from the evaluation to the immediately preceding passage is 4 days or more and 14 days or less. and if the cell population for evaluation satisfies one or more of the evaluation criteria (a), (b), (c), or (d), placing at least a portion of the cells from the cell population in a storage medium for preservation.

9. A cell population of cultured human corneal endothelial cells and / or human corneal endothelial precursor cells, which is obtained by proliferating and / or differentiating and maturing the human corneal endothelial cells and / or human corneal endothelial precursor cells in a medium containing a ROCK inhibitor and having an epidermal growth factor (EGF) content below a concentration that causes transformation, has a purity test result by FACS within the following range: CD166 ＋ >99% CD24 ＋ <5% CD44 ｈｉｇｈ <5% CD44 ｎｅｇ～ｌｏｗ >90% CD90 ＋ <5% A method for treating human corneal endothelial cells and / or human corneal endothelial precursor cells that satisfy all of the above, Culturing a plurality of human corneal endothelial cells and / or human corneal endothelial precursor cells in a culture vessel containing a culture medium; Separating at least a portion of the plurality of human corneal endothelial cells and / or human corneal endothelial progenitor cells to generate a cell population for evaluation and a remaining cell population; assessing at least a portion of the cell population for one or more of the following assessment criteria: (a), (b), (c) and (d); (a) identifying cells in which the morphology of human corneal endothelial cells and / or human corneal endothelial precursor cells has changed from a spindle shape to either (i) a polygonal shape or (ii) an elliptical shape with a ratio of the major axis to the minor axis close to 1, and in which the boundaries between cells are clearly defined; (b) identifying cells expressing a constant amount of CD44 after the expression of CD44 reaches levels less than half the maximum observed after the previous passage; (c) The cell density of the human corneal endothelial cells and / or human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500 pieces / mm ２ Identifies that: (d) When the cells are seeded at a cell density of 300 cells / mm 2 to 500 cells / mm 2 , it is specified whether the period from the evaluation to the immediately preceding passage is 4 days or more and 14 days or less. and if the cell population for evaluation satisfies one or more of the evaluation criteria (a), (b), (c), and (d), placing at least a portion of the cells from the cell population in a storage medium to preserve them; or if the cell population for evaluation does not satisfy each of the evaluation criteria (a), (b), (c), and (d), placing at least a portion of the cells from the remaining cell population in a culture vessel containing a culture medium, and further culturing for a certain period of time.

10. A method for treating human corneal endothelial cells and / or human corneal endothelial precursor cells cultured in a medium not supplemented with EGF, comprising: Culturing a plurality of human corneal endothelial cells and / or human corneal endothelial precursor cells in a culture vessel containing a culture medium to which EGF is not added; Separating at least a portion of the plurality of human corneal endothelial cells and / or human corneal endothelial progenitor cells to generate a cell population for evaluation and a remaining cell population; assessing at least a portion of the cell population for one or more of the following assessment criteria: (a), (b), (c) and (d); (a) identifying cells in which the morphology of human corneal endothelial cells and / or human corneal endothelial precursor cells has changed from a spindle shape to either (i) a polygonal shape or (ii) an elliptical shape with a ratio of the major axis to the minor axis close to 1, and in which the boundaries between cells are clearly defined; (b) identifying cells expressing a constant amount of CD44 after the expression of CD44 reaches levels less than half the maximum observed after the previous passage; (c) The cell density of the human corneal endothelial cells and / or human corneal endothelial precursor cells is 900 cells / mm ２ More than 2500 pieces / mm ２ Identifies that: (d) When the cells are seeded at a cell density of 300 cells / mm 2 to 500 cells / mm 2 , it is specified whether the period from the evaluation to the immediately preceding passage is 4 days or more and 14 days or less. and if the cell population for evaluation satisfies one or more of the evaluation criteria (a), (b), (c), and (d), placing at least a portion of the cells from the cell population in a storage medium to preserve them; or if the cell population for evaluation does not satisfy each of the evaluation criteria (a), (b), (c), and (d), placing at least a portion of the cells from the remaining cell population in a culture vessel containing a culture medium, and further culturing for a certain period of time.

Images