JP6664755B1 - Method and container for storing corneal endothelial cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preserving corneal endothelial cells with high cell viability. The present invention provides a method for preserving corneal endothelial cells and / or corneal endothelium-like cells, which comprises storing the corneal endothelial cells and / or corneal endothelium-like cells in a container having a bottom area of at least about 0.7 cm 2. A method including the steps of: According to the present invention, corneal endothelial cells can be preserved with high cell viability. The corneal endothelial cells thus preserved have the function of normal corneal endothelial cells and can be used as cells for treating corneal endothelial diseases and the like. [Selection diagram] None

Description

  The present invention relates to a technique for preserving corneal endothelial cells.

  As for the visual information, light taken from the cornea, the transparent tissue in the foreground of the eyeball, reaches the retina and excites nerve cells in the retina, and the generated electrical signal is transmitted to the visual cortex through the optic nerve It is recognized by doing. In order to obtain good vision, the cornea needs to be transparent. The transparency of the cornea is maintained by keeping the water content constant by the pump function and the barrier function of the corneal endothelial cells.

  At birth, human corneal endothelial cells exist at a density of about 3000 cells per square millimeter, but their ability to regenerate is limited and severely impaired, preventing them from maintaining their function. Is lost. BACKGROUND ART In bullous keratopathy caused by corneal endothelial degeneration or corneal endothelial dysfunction due to various causes, the cornea becomes edematous and turbid, resulting in a marked decrease in visual acuity. Currently, for bullous keratopathy, allogeneic corneal transplantation using a donor cornea is performed. However, there are many solutions to current corneal transplants, such as surgical invasion, rejection, and lack of donors.

  In order to overcome these problems, a less invasive cell transplantation treatment has recently been developed as a treatment for corneal endothelial disease (Non-Patent Document 1). Cultivation of corneal endothelial cells is difficult, and there are problems that they do not proliferate by ordinary methods (Non-Patent Documents 2 and 3), become fibroblasts (Non-Patent Document 4), and become senescent (Non-Patent Document 5). Yes, corneal endothelial cell culturing methods have been actively studied, and clinical application has become possible (Non-Patent Document 1).

Kinoshita S, Koizumi N, Ueno M, et al. Injection of cultured cells with a ROCK inhibitor for bullous keratopathy.N Engl J Med 2018; 378: 995-1003. Okumura N, Ueno M, Koizumi N, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.Invest Ophthalmol Vis Sci 2009; 50: 3680-3687. Nakahara M, Okumura N, Kay EP, et al. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.PLoS One 2013; 8: e69009. Okumura N, Kay EP, Nakahara M, Hamuro J, Kinoshita S, Koizumi N. Inhibition of TGF-beta Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine.PLoS One 2013; 8: e58000. Hongo A, Okumura N, Nakahara M, Kay EP, Koizumi N. The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.Invest Ophthalmol Vis Sci 2017; 58: 3325-3334.

  The inventors of the present invention have devised a method for preserving corneal endothelial cells with a high viability as a result. Specifically, the present inventors have found that storing corneal endothelial cells in a container having a specific bottom area maintains high viability. The corneal endothelial cells thus preserved had the function of normal corneal endothelial cells and could be used as therapeutic cells.

Accordingly, the present invention provides the following.
(Item 1)
A method of storing corneal endothelial cells and / or corneal endothelium-like cells, comprising storing the corneal endothelial cells and / or corneal endothelium-like cells in a container having a bottom area of at least about 0.7 cm ² .
(Item 2)
Item 2. The method according to Item 1, wherein a liquid level of the liquid for storing the cells is about 0.75 mm or more.
(Item 3)
Bottom area of the container, about 0.7 to about 4 cm ^2, The method of claim 1 or 2.
(Item 4)
Bottom area of the container is about 1.5 to about 3 cm ^2, The method according to any one of items 1-3.
(Item 5)
Bottom area of the container is about 1.8 to about 2 cm ^2, The method according to any one of items 1-4.
(Item 6)
The method according to any one of items 1 to 5, wherein the container has not been subjected to a surface treatment for adhesion culture.
(Item 7)
7. The method according to any one of items 1 to 6, wherein the container is a low-adhesion surface container or a non-surface treated container.
(Item 8)
Item 8. The method of any of items 1 to 7, wherein the container is made of polystyrene, polypropylene, or glass.
(Item 9)
The method according to any one of items 1 to 8, wherein the container is selected from the group consisting of a 24-well plate, a vial, a syringe, and a dish.
(Item 10)
Item 10. The method according to any one of items 1 to 9, wherein the storage of the cells is performed at a temperature from about 12C to about 42C.
(Item 11)
11. The method of any one of the preceding items, wherein the storage of the cells is performed at a temperature of about 17C to about 39C.
(Item 12)
12. The method according to any of the preceding items, wherein the storage of the cells is performed at a temperature of about 27C to about 37C.
(Item 13)
13. The method according to any of the preceding items, wherein the storage of the cells is performed at a temperature of about 37 <0> C.
(Item 14)
A container for storing corneal endothelial cells and / or corneal endothelial-like cells, wherein the container has a bottom area of at least about 0.7 cm ² .
(Item 15)
Item 15. The container according to Item 14, wherein the container has a structure that allows a liquid level to be about 0.75 mm or more.
(Item 16)
16. A container for storing corneal endothelial cells and / or corneal endothelium-like cells, wherein the container has a bottom area of about 0.7 to about 4 cm ² .
(Item 17)
Bottom area of the container is about 1.5 to about 3 cm ^2, the container according to any one of items 14 to 16.
(Item 18)
Bottom area of the container is about 1.8 to about 2 cm ^2, the container according to any one of items 14 to 17.
(Item 19)
19. The container according to any one of items 14 to 18, wherein the container has not been subjected to adhesion culture surface treatment.
(Item 20)
20. The container according to any one of items 14 to 19, wherein the container is a low-adhesion surface container or a non-surface-treated container.
(Item 21)
21. The container of any one of items 14 to 20, wherein the container is made of polystyrene, polypropylene, or glass.
(Item 22)
The container according to any one of items 14 to 21, wherein the container is selected from the group consisting of a 24-well plate, a vial, a syringe, and a dish.
(Item 23)
14. A corneal endothelial cell or a corneal endothelium-like cell preserved by the method according to any one of items 1 to 13.
(Item 24)
A composition for treating or preventing a corneal endothelial disorder, disease or condition, comprising the corneal endothelial cell and / or corneal endothelium-like cell according to item 23.
(Item 25)
25. The composition according to item 24, further comprising a ROCK inhibitor.
(Item 26)
26. The composition according to item 25, wherein the composition is administered in combination with a ROCK inhibitor.
(Item 27)
27. The composition according to item 25 or 26, wherein said ROCK inhibitor is Y-27632. (Item 28)
A cell-containing container comprising (A) a corneal endothelial cell and / or a corneal endothelium-like cell and (B) a container for storing the corneal endothelial cell and / or a corneal endothelium-like cell, wherein the cell-containing container has at least a bottom area. A cell-containing container that is about 0.7 cm ² .
(Item 29)
29. The cell-containing container according to item 28, further comprising the feature according to any one or more of items 15 to 22.
(Item 30)
30. The cell-containing container according to item 28 or 29, further comprising a ROCK inhibitor.

  In the present invention, it is intended that one or more of the features described above can be provided in addition to the explicitly specified combinations. Still further embodiments and advantages of the invention will be apparent to those skilled in the art upon reading and understanding the following detailed description, as appropriate.

  According to the present invention, corneal endothelial cells can be stored at a high cell viability. The corneal endothelial cells thus preserved have the function of normal corneal endothelial cells and can be used as cells for treating corneal endothelial diseases and the like.

FIG. 1A outlines an exemplary procedure for preservation of corneal endothelial cells. FIG. 1B shows that after storing corneal endothelial cells in 24-well plates (cell culture plates), 24-well plates (ultra-low adhesion), 48-well plates (suspension culture) and 96-well plates (ultra-low adhesion), The phase contrast microscope image of each plate from which cells were collected by pipetting is shown. FIG. 2A shows 24 well plate (ultra low adhesion), 48 well plate (suspension culture), 96 well plate (ultra low adhesion, flat bottom), 96 well plate (ultra low adhesion, round bottom), 15 ml conical tube (ultra low adhesion) (Low adhesion) and the ratio of living cells to dead cells of corneal endothelial cells stored in 2 ml cryotubes. FIG. 2B shows live and dead corneal endothelial cells stored at 37 ° C. in a 24-well plate (ultra-low adhesion), 4 ° C. in a 24-well plate (ultra-low adhesion), and 37 ° C. in a 24-well plate (cell culture plate). Shows the percentage of cells. FIG. 2C shows the percentage of live and dead corneal endothelial cells stored in 24-well plates (ultra-low adhesion), 24-well plates (untreated) and 10 ml glass vial tubes. FIG. 2D shows the ratio of living cells to dead cells of corneal endothelial cells stored in a 24-well plate (ultra-low adhesion) at a storage volume of 300 μl, 1000 μl, 1500 μl and 2500 μl. The white part of the bar indicates dead cells, and the black part indicates live cells. FIG. 2E shows phase contrast microscopy images at 3 hours and 2 weeks after seeding corneal endothelial cells stored in a 24-well plate (ultra-low adhesion) on a culture plate. FIG. 3A shows a slit lamp image in a rabbit model with corneal endothelial cells stored in a 24-well plate (ultra-low adhesion) injected into the anterior chamber. FIG. 3A shows images from day 1, day 7, day 14 after injection from the left, control eye (without cell injection), eye injected with cell stored for 24 hours, eye injected with cell stored for 48 hours, and 72 hours from the top. 3 shows an eye with preserved cells injected. FIG. 3B shows a Scheimpflug image 14 days after injection. FIG. 4A shows a color map of corneal thickness obtained with Pentacam ^™ 14 days after injection in a rabbit model in which corneal endothelial cells stored in a 24-well plate (ultra-low adhesion) were injected into the anterior chamber. FIG. 4B shows a graph of central corneal thickness after injection of preserved corneal endothelial cells. FIG. 4C shows a graph of corneal volume after injection of preserved corneal endothelial cells. FIG. 4D shows a graph of eye cell density 14 days after injection of preserved corneal endothelial cells. FIG. 4E shows a fluorescence microscope image of the immunofluorescently stained rabbit cornea. Blue indicates DAPI staining (cell nuclei). Green indicates Na ⁺ / K ⁺ -ATPase, ZO-1, and N-cadherin from the left, respectively. FIG. 5 shows a state in which 500,000 corneal endothelial cells cultured in a 1 ml syringe are suspended in 300 μl of a cell preservation solution and stored. FIG. 6 shows a phase contrast micrograph after storing the corneal endothelial cells in a 1 ml syringe for 72 hours. Cells have settled on the side of the syringe. FIG. 7 shows a phase contrast micrograph of the corneal endothelial cells stored in a 1 ml syringe for 72 hours and then lightly tapped. Cells are rising from the side of the syringe. FIG. 8 shows a phase contrast micrograph of the corneal endothelial cells stored in a 1 ml syringe for 72 hours, lightly tapped, and the syringe gently washed with a cell preservation solution. No cell adhesion to the side of the syringe. FIG. 9 shows the ratio of living cells to dead cells of corneal endothelial cells stored at 12 ° C., 17 ° C., 22 ° C., 27 ° C., 32 ° C., 35 ° C., 37 ° C., 39 ° C., and 42 ° C. The white part of the bar indicates dead cells, and the black part indicates live cells. FIG. 10 shows (1) vial bottle (Maruem, Osaka, 0501-02), (2) vial bottle (2 ml, diameter 16 mm, height 33 mm, IRAS treatment, Iwata Glass Industry Co., Ltd., Osaka, lot: 181024 And (3) vial bottle (2 ml, diameter 16 mm, height 33 mm, IRAS treatment and silica coating (SiO ₂ coating), Iwata Glass Industry Co., Ltd., Osaka, lot: 181022). FIG. 11 shows (1) vial bottles (Maruem Co., Osaka, 0501-02), (2) vial bottles (2 ml, diameter 16 mm, height 33 mm, IRAS treatment, Iwata Glass Industry Co., Ltd., Osaka), and ( 3) Percentage of live and dead corneal endothelial cells stored in vials (2 ml, diameter 16 mm, height 33 mm, IRAS treatment and silica coating (SiO ₂ coating), Iwata Glass Industry Co., Ltd., Osaka) . The white part of the bar indicates dead cells, and the black part indicates live cells.

  Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. Thus, it is to be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept unless specifically stated otherwise. It should also be understood that the terms used in this specification are used in a meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. As used herein, “about” means ± 10% of the value that follows.

(Definition)
As used herein, “corneal endothelial cell” is used in the ordinary meaning used in the art. The cornea is one of layered tissues constituting the eye, is transparent, and is located at a portion closest to the outside world. The cornea is considered to be composed of five layers in order from the outside (body surface) in humans, and is composed of a corneal epithelium, Bowman's membrane, lamina propria, Descemet's membrane (corneal endothelial basement membrane), and corneal endothelium from the outside. In particular, unless otherwise specified, parts other than epithelium and endothelium are sometimes collectively referred to as "corneal stroma" and are so referred to herein. In the present specification, “HCEC” (human
The term “corneal endothelial cells” is an abbreviation for human corneal endothelial cells.

  As used herein, the term “corneal endothelium-like cell” refers to a cell differentiated from a stem cell, for example, a cell differentiated from an iPS cell or the like, and has substantially the same function as a corneal endothelial cell. A method for differentiating a stem cell, for example, an ES cell, an iPS cell, or the like into a corneal endothelium-like cell is well known in the art (McCabe et al., PLoS One. 2015 Dec 21; 10 (12): e0145266; Ali et. al., Invest Ophthalmol Vis Sci. 2018 May 1; 59 (6): 2437-2444). In a typical example, briefly, iPS cells are seeded at a 1:12 dilution on day 0 into 35 mm Matrigel coated plates (Corning) using cell dissociation buffer (Life Technologies) (80% confluent plate Divide into 12 plates). The iPS cells are grown for 4 days in medium (mTeSR1; STEMCELL Technologies Inc.). On the fourth day, the mTeSR1 medium was added to 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (STEMCELLTechnologies, 0%). In a basal medium of 1 mM β-mercaptoethanol (Millipore Sigma) and 8 ng / mL βFGF (Millipore Sigma), 500 ng / mL human recombinant Noggin (R & D Systems, Minneapolis, MN, USA) and 10 μM SBLipor (Mg. Replace with Smad inhibitor medium. On the sixth day, the Smad inhibitor medium was supplemented with 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), and 1 mM L-glutamine (STEMCELLTechnologies, Inc.). , 0.1 mM β-mercaptoethanol (MilliporeSigma), and 8 ng / mL βFGF (MilliporeSigma) in a basal medium of 0.1 × B27 supplement (Life Technologies), 10 ng / mL recombinant human platelet-derived growth factor-BB ( PDGF-BB; PeproTech, Rocky Hill, NJ, USA) and 10 ng / mL recombinant human Dickkopf-related protein Click protein -2 (DKK-2; R & D Systems) is replaced with corneal medium containing. On day 7, differentiating CECs are transferred to fresh matrigel coated plates (35 mm) and grown in corneal medium for an additional 13 days. The differentiated CEC is collected on day 20. The above examples are typical examples, and those skilled in the art will recognize other methods known in the art (Fukuta et al., PLoS One. 2014 Dec 2; 9 (12): e112291; Hayashi et al., Nature. 2016 Mar 17; 531 (7594): 376-80) may also be used. Moreover, those skilled in the art can produce corneal endothelium-like cells by appropriately adjusting the conditions of a method well known in the art.

  The “corneal endothelial cells” and “corneal endothelial-like cells” may include a magnetic substance (for example, iron). For example, when a corneal endothelial cell containing a magnetic substance is injected into the anterior chamber, it can be attracted to the inside of the cornea (for example, Descemet's membrane) by magnetic force to promote adhesion (Patel et al., Invest Ophthalmol Vis Sci. 2009 May; 50 (5): 2123-31; Mimura et al., Exp Eye Res. 2003 Jun; 76 (6): 745-51; and Mimura et al., Exp Eye Res. 2005 Feb; 80 (2) : 149-57). “Magnetic material” refers to a substance that is magnetized by a magnetic field, and includes, for example, iron, cobalt, nickel, and ferrite.

  As used herein, the term “adhesion culture” refers to culturing cells by attaching them to a container. As the container for the adhesion culture, for example, a container subjected to a cell culture (TC) treatment can be mentioned.

As used herein, "low adhesion" refers to the fact that cells rarely adhere to the container during cell culture or storage. Low adhesion surface containers are, for example, surface coated (eg, by covalent bonding) with a hydrogel and / or silica (SiO ₂ ) coated. Another example is a glass container whose surface has been subjected to an IRAS treatment. Vials that have been IRAS treated are available from Iwata Glass Industry Co., Ltd. (Osaka). By the IRAS treatment, elution of alkali from the borosilicate glass is suppressed. Low-adhesion surface vessels may be commercially available as vessels for suspension (floating) culture. Containers for suspension (floating) culture should be treated as long as they are surface-treated so that cells do not adhere to the vessel. , Is referred to herein as a low adhesion surface container.

  In the present specification, the term "non-treated surface" means that the surface is not treated, and the "untreated surface container" means that the cells are cultured or adhered without the cells adhering to the container similarly to the low-adhesion surface container. Refers to a container that can be stored. Containers with no surface treatment may be commercially available as containers for suspension (floating) culture. Containers for suspension (floating) culture are not surface-treated and cells adhere to the container. A non-surface treated container is considered as long as cells can be cultured or stored without any treatment.

  As used herein, the term "preservation" of cells refers to maintaining cells in a solution while maintaining their functions without growing the cells, and "culturing" for the purpose of growing cells different.

  As used herein, the term "cell-containing container" refers to a container containing cells (typically, corneal endothelial cells or corneal endothelial-like cells), and typically has the features disclosed herein. .

(Preferred embodiment)
A description of the preferred embodiment is set forth below, but it should be understood that this embodiment is exemplary of the invention and that the scope of the invention is not limited to such preferred embodiments. It should be understood that those skilled in the art can also easily make modifications, changes and the like within the scope of the present invention by referring to the following preferred embodiments. Those skilled in the art can appropriately combine any of these embodiments.

(Preservation method)
In one embodiment, the present invention provides a method for storing corneal endothelial cells and / or corneal endothelial-like cells, comprising the step of storing the corneal endothelial cells and / or corneal endothelial-like cells in a container having a specific bottom area. Provide methods, including: Bottom area of the container used in the method of the present invention can be at least about 0.7 cm ^2, usually, about 0.7 to about 4 cm ^2. While not wishing to be bound by theory, in the case of the bottom area than 0.7 cm ² is small (e.g., 12-well plates), not achieved a high cell viability, larger bottom area than 4 cm ² In the case of a container (for example, a 48-well plate), if a cell suspension or a liquid for cell preservation (for example, about 300 μl) that can be used for corneal endothelial cell injection therapy is placed in the container, the liquid level becomes too low. Not suitable for storage. In some embodiments, the liquid level for cell preservation is at least about 0.5 mm, at least about 0.6 mm, at least about 0.7 mm, at least about 0.75 mm, at least about 0.8 mm, at least about 0.9 mm. As described above, it is preferably about 1.0 mm or more, about 1.2 mm or more, about 1.4 mm or more, about 1.6 mm or more, about 1.8 mm or more, and about 2 mm or more. In some embodiments, the level of the liquid for cell preservation is about 1.0 mm or less, about 1.5 mm or less, about 1.8 mm or less, about 2 mm or less, about 3 mm or less, about 4 mm or less, about 5 mm or less, It can be about 6 mm or less, about 7 mm or less, about 8 mm or less, about 9 mm or less, about 10 mm or less, about 15 mm or less, about 20 mm or less, and the like. In some embodiments, the bottom area of the container used in the method of the present invention is from about 0.7 to about 3.5 cm ^2, from about 0.7 to about 3 cm ^2, from about 0.7 to about 2.5cm ^2, about 0.7 to about 2.0 cm ^2, from about 1 to about 4 cm ^2, from about 1 to about 3.5 cm ^2, from about 1 to about 3 cm ^2, from about 1 to about 2.5 cm ^2, from about 1 to about 2cm ^2, about 1.5 to about 3 cm ^2, from about 1.5 to about 4 cm ^2, may be from about 1.6 to about 2.2 m ² or from about 1.8 to about 2m ^2,. When the amount of the preserved corneal endothelial cell suspension is larger than the amount that can be used in corneal endothelial cell infusion therapy, the upper limit of the bottom area of the container is not particularly limited, for example, in the range of the bottom area, The upper limit is about 4.5 cm ² , about 5 cm ² , about 5.5 cm ² , about 6 cm ² , about 7 cm ² , about 8 cm ² , about 9 cm ² , about 10 cm ² , about 15 cm ² , about 20 cm ² , about 25 cm ² , It can be about 30 cm ² , about 40 cm ² , about 50 cm ² , about 60 cm ² , about 70 cm ² , about 80 cm ² , about 90 cm ² , about 100 cm ² . Bottom area of the container is preferably about 1.5 to about 3 cm ^2, more preferably about 2 cm ^2. In certain embodiments, the bottom area of the vessel is about 1.88 cm ^2.

  The method of the present invention allows for preservation of corneal endothelial cells while maintaining a high cell count and high cell viability from the time of storage initiation. For example, in the method of the present invention, assuming that all cells at the start of storage are 100%, at least the total number of cells after storage is about 30% or more and the viable cell ratio (live cells / live cells + dead cells) is about 70%. As described above, preferably, the total cell number after storage is about 50% or more and the viable cell ratio is about 80% or more, and most preferably, the total cell number after storage is 70% or more and the viable cell ratio is about 90% or more. Of corneal endothelial cells. In certain embodiments, the method of the present invention provides that the cell viability (percentage of live cells as compared to total cells at the start of storage) is preferably at least about 30%, more preferably at least about 60%, It is more preferably at least about 80%, most preferably at least about 90%. In a further specific embodiment, in addition to the cell viability, the viable cell ratio (live cells / live cells + dead cells) is preferably about 70% or more, more preferably about 80% or more, and most preferably about 90%. % Or more.

  The bottom area refers to the area of a surface called the bottom surface of the container. The method of the present invention also contemplates storing the syringe in an upright position, in which case the bottom area of the container will be equal to the area of the lower half surface of the cylindrical surface where the cell suspension contacts. I do.

  Storage temperatures can range from about 10 ° C to about 50 ° C. This is because the cell survival rate is reduced if the cell is too high or too low. Those skilled in the art can appropriately determine a suitable temperature for storage. In some embodiments, the storage temperature is about 10 ° C, about 11 ° C, about 12 ° C, about 13 ° C, about 14 ° C, about 15 ° C, about 16 ° C, about 17 ° C, about 18 ° C, About 19 ° C, about 20 ° C, about 21 ° C, about 22 ° C, about 23 ° C, about 24 ° C, about 25 ° C, about 26 ° C, about 27 ° C, about 28 ° C, about 29 ° C, about 30 ° C, about 31 ° C. ° C, about 32 ° C, about 33 ° C, about 34 ° C, about 35 ° C, about 36 ° C, about 37 ° C, about 38 ° C, about 39 ° C, about 40 ° C, about 41 ° C, about 42 ° C, about 43 ° C, It can be about 44 ° C, about 45 ° C, about 46 ° C, about 47 ° C, about 48 ° C, about 49 ° C, or about 50 ° C. A preferred storage temperature range is from about 12C to about 42C, more preferably from about 17C to about 39C, and even more preferably from about 27C to about 37C. In a preferred embodiment, the temperature for storage can be room temperature or about 37 ° C. In a most preferred embodiment, the temperature during storage can be about 37 ° C.

  The corneal endothelial cells can be cells derived from mammals (human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkey, etc.), but are preferably derived from primates, particularly human. preferable.

  In some embodiments, the container has not been surface treated for adherent culture. In some embodiments, the container is a low adhesion surface container or a non-surface treated container. In some embodiments, the container is made of polystyrene, polypropylene, or glass. In specific embodiments, containers include, but are not limited to, for example, 24-well plates, vials, syringes, and dishes.

  The container may or may not be made of an oxygen-permeable material as long as it can be stored without cell adhesion. Examples of the oxygen-permeable material include, for example, polyethylene and any other material that transmits oxygen. Further, a material that does not transmit oxygen can be processed so as to transmit oxygen by a method known in the art.

  As a preservation solution for corneal endothelial cells or corneal endothelium-like cells, a well-known preservation solution used in the art may be used. Examples of the preservative solution that can be used include, for example, OptiMEM-I (registered trademark) (Thermo Fisher Scientific), MEM, DMEM, M199, and corneal endothelial cell preservative solution (the preservative solution used in the present specification, Generation and Feasibility Assessment). . a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction Okumura N, kakutani K, Inoue R, Matsumoto D, Shimada T, Nakahara M, Kiyanagi Y, Itoh T, Koizumi N.PLoS One 2016 Jun 29;. 11 ( 6 :. E0158427 doi:. 10.1371 / journal.pone.0158427 eCollection 2016.PMID: 27355373) but the like, but not limited to.

  In some embodiments, the volume of the solution during storage can be, for example, about 300 μl, which is an appropriate volume for subsequent cell injection, but can be appropriately changed depending on the purpose. For example, the volume of the solution during storage is about 100 μl, about 200 μl, about 300 μl, about 400 μl, about 500 μl, about 600 μl, about 700 μl, about 800 μl, about 900 μl, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 4 ml, It can be 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, or about 10 ml. For the range of the liquid amount, the above numerical values can be appropriately combined.

(container)
In another aspect, the present invention provides a container for storing corneal endothelial cells or corneal endothelial-like cells, the container having a specific bottom area. As the specific characteristics of the container, any embodiment specifically described above, particularly, (storage method) can be adopted.

(container)
In another aspect, the present invention is a cell-containing container comprising a corneal endothelial cell or a corneal endothelial-like cell and a container for storing the corneal endothelial cell or the corneal endothelial-like cell, wherein the container is used. Provides a container having a specific bottom area. As the specific characteristics of the container, any embodiment specifically described above, particularly, (storage method) can be adopted. In some embodiments, a Rho kinase (ROCK) inhibitor may be included in a container of the invention. A ROCK inhibitor as used herein can be any of the embodiments described herein.

(Corneal endothelial cells)
In another aspect, the present invention provides a corneal endothelial cell or a corneal endothelium-like cell preserved by the above method. In a further aspect, the present invention provides a composition for treating or preventing a corneal endothelial disorder, disease or condition, comprising a corneal endothelial cell or a corneal endothelial-like cell preserved by the above method.

  In some embodiments, the corneal endothelial disorder, disease or condition is Fuchs corneal endothelial dystrophy, corneal post-transplant disorder, corneal endotheliitis, trauma, ophthalmic surgery, ophthalmic laser surgery disorder, aging, posterior polymorphism. It is selected from the group consisting of corneal dystrophy (PPD), congenital hereditary corneal endothelial dystrophy (CHED), idiopathic corneal endothelial dysfunction, and cytomegalovirus corneal endotheliitis.

  In some embodiments, a composition of the invention may include a Rho kinase (ROCK) inhibitor or may be administered in combination with a ROCK inhibitor. Examples of the ROCK inhibitor include the following documents: US Pat. No. 4,678,783, Patent No. 3,421,217, WO 95/28387, WO 99/20620, WO 99/61403, WO 02/076976, WO 02/076977, WO 2002/083175, WO 02/100833, WO 03/059913, WO 03/062227, WO 2004/009555, WO 2004/0222541, WO 2004/108724, WO 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 005/035506, WO 2005/080394, WO 2005/103050, WO 2006/057270, compounds disclosed in international publication 2007/026664, and the like. Such compounds can be produced by the methods described in the respective disclosed documents. As a specific example, 1- (5-isoquinoline sulfonyl) homopiperazine or a salt thereof (for example, fasudil (1- (5-isoquinoline sulfonyl) homopiperazine)), (+)-trans-4- (1-aminoethyl)- 1- (4-pyridylcarbamoyl) cyclohexane ((R)-(+)-trans- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide) or a salt thereof (for example, Y-27632 ((R )-(+)-Trans- (4-pyridyl) -4- (1-aminoethyl) -cyclohexanecarboxamide dihydrochloride monohydrate) and the like, and these compounds are commercially available products (Wako Pure Chemical Industries, Ltd.). Pharmaceutical Co., Ltd., Asahi Kasei Pharmaceuticals, etc.) can also be suitably used.

  The present invention has been described above by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

Hereinafter, the present invention will be described more specifically based on examples. It is understood that various reagents used in this example may be those obtained from Sigma-Aldrich, BASF Japan, etc., in addition to those specifically shown. Human tissues were treated according to the ethical rules of the Helsinki Declaration. Human donor corneas were provided by LightLife ^™ (Seattle, WA). For eye donations for research purposes, corneas were collected under the UAGA standards of the state after obtaining written consent from a close relative of the deceased donor. The experiments in rabbits were performed based on the guidelines approved by the Doshisha University Animal Experiment Committee (approval number A18003).

(Example 1: Screening of material and size of container in cell storage)
(Materials and methods)
(Culture of corneal endothelial cells)
Five human donor corneas were obtained from diseased donors over the age of 40. All corneas were stored in Optisol (Chiron Vision, Irvine, CA) at 4 ° C for up to 14 days before use. Human corneal endothelial cells (HCEC) were cultured as follows. Briefly, the Descemet's membrane containing the corneal endothelium was mechanically detached from the donor cornea and digested by incubating for 12 hours at 37 ° C. in 1 mg / mL collagenase A (Roche Applied Science, Penzberg, Germany). After washing HCEC with OptiMEM-I (Life Technologies Corp., Carlsbad, Calif.), It was seeded in one well of a 48-well plate coated with laminin E8 fragment (iMatrix-511; Nippi, Tokyo).

  The medium was prepared according to the following. Briefly, 8% fetal bovine serum (FBS), 5 ng / mL epidermal growth factor (EGF; Thermo Fisher Scientific), 20 μg / mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), 200 mg / L calcium chloride, OptiMEM-I supplemented with 0.08% chondroitin sulfate (Sigma-Aldrich) and 50 μg / mL gentamicin (Thermo Fisher Scientific) was conditioned with NIH-3T3 for 24 hours. The conditioned medium was then collected and filtered through a 0.22 μm filter unit (EMD Millipore Corporation, Billerica, Mass.) And used as HCEC medium.

HCEC was cultured at 37 ° C. in a humidified atmosphere containing 5% CO ₂ , and the medium was changed three times a week. In the subculture of HCEC, cells were trypsinized with TrypLE ^™ Select Enzyme (10X) (Thermo Fisher Scientific) for 5 minutes at 37 ° C and seeded at a ratio of 1: 2. In this test, HCEC cultured for 5 to 9 passages was used.

HCECs were washed with Ca ²⁺ or Mg ²⁺ free phosphate buffered saline (PBS) and trypsinized with TrypLE ^™ Select Enzyme (10X) for 15 minutes at 37 ° C. After the cells were collected from the culture plate, they were washed twice, centrifuged at 280 G for 3 minutes, and suspended in OptiMEM-I. HCECs were cultured at 4 ° C. or 37 ° C. for 72 hours at a cell density of 1.0 × 10 ⁶ cells / 300 μl in the above serum-free culture medium (cell therapy culture medium provided by Institute for Cell Science (Miyagi)). Stored in suspension form. The following cell culture plates, test tubes, and vials were used to screen for size and material for HCEC storage: 24-well plates (ultra-low adhesion) (Corning Inc. Corning, New York), 24-well plates (Cell culture) (Corning Inc. Corning), 24-well plate (untreated) (AGC Technoglass Co., Shizuoka), 48-well plate (suspension culture) (Sumitomo Bakelite Co., Ltd.), 96-well plate (ultra low) Adhesion, round bottom (Corning Inc.), 96-well plate (ultra low adhesion, flat bottom) (Corning Inc.), 15 ml conical tube (ultra low adhesion) (Sumitomo Bakelite Co., Ltd., Tokyo), 2 ml cryovial (Corning)
Inc. Corning), and a 10 ml glass vial (Marem, Inc., Osaka) (Table 1).

After storage for 72 hours, HCECs are gently pipetted from cell culture plates, test tubes, or vials, centrifuged at 280 G for 3 minutes, and used for cell therapy at a cell density of 1.0 × 10 ⁶ cells / 600 μl. Resuspended in culture. The cell viability was calculated by staining dead cells with 0.5% trypan blue staining solution (Nacalai Tesque, Kyoto). As a control, HCEC recovered by trypsinization with TrypLE ^™ Select Enzyme (10X) was centrifuged at 280 G for 3 minutes and resuspended in cell therapy culture. Thereafter, cell viability was immediately assessed without storage in the form of a cell suspension.

(result)
(Effect of cell suspension form storage conditions on cell viability)
HCEC were stored in cell suspension form at 1.0 × 10 ⁶ cells / 300 μl cell density in CTV for 72 hours, cells were harvested by gentle pipetting and cell viability was assessed (FIG. 1A). Phase contrast images showed that 80-90% of the area of the adherent culture plate was covered with HCEC after gentle pipetting for cell recovery. On the other hand, almost no cells were observed in any of the 24-well plate, the 48-well plate, and the 96-well plate for suspension cell culture (FIG. 1B). Since the surface treatment for adherent culture was not applicable to HCEC storage as a form of cell suspension, the effects of the size and shape of commercially available culture plates and test tubes on cell viability were evaluated in this example. The ratio of viable cells to dead cells in the control immediately after being trypsinized and collected from the culture plate is 90.3% and 9.7%, and that in the cell stored in a 24-well plate (ultra-low adhesion) is 82. 6% and 10.1% (relative to the original cell number stored). However, the percentage of viable cells was 48-well plates (suspension culture), 96-well plates (ultra-low adhesion, flat bottom), 96-well plates (ultra-low adhesion, round bottom), 15 ml conical tubes (ultra-low adhesion), and It was significantly reduced compared to the 2 ml cryovial (p <0.01) control (FIG. 2A). After storage in 48 well plates (suspension culture), 15 ml conical tubes (ultra low attachment), and 2 ml cryovials, the number of cells harvested is likely due to cell attachment to the bottom or wall of the culture plate or test tube Was decreasing. The authors further evaluated the effect of temperature on cell viability; 95.1% of cells survived at 37 ° C, but after storage in 4 well plates at 24 ° C (ultra low adherence) due to the death of large numbers of cells. It showed that cells were almost not harvested (FIG. 2B). Other culture plates or tubes were also evaluated without surface treatment for adherent culture. 24 Like the ultra low attachment in well plates, untreated 24-well plates and 10ml glass vial tube (24-well plates (approximately 1.9 cm ²⁾ in the same manner as in the bottom area of about 2.7 cm ²⁾ of the 90% Cell viability was maintained (FIG. 2C). Intermediate volumes did not significantly alter cell viability (FIG. 2D).

  HCECK stored in the form of a cell suspension in a 24-well plate (ultra low adhesion) was stored on a culture plate after being stored at 37 ° C. for 72 hours. Representative phase contrast images showed that the preserved HCEC, like the control, adhered to the culture plate with apparent cell death after 3 hours of seeding. Two weeks later, the stored HCEC had formed a polygonal, single-layer sheet-like structure similar to the control (FIG. 2E).

(Example 2: Injection of RCEC into rabbit corneal endothelial decompensation model)
(Materials and methods)
(Rabbit CEC culture)
In this example, 10 rabbit eyes purchased from Funakoshi Co., Ltd. (Tokyo) were used. Rabbit CEC (RCEC) was cultured as described above. Briefly, stripping Descemet's membrane for 15 minutes at 37 ℃ 0.6U / mLAccutase (Nacalai Tesque, Kyoto) containing RCEC incubated in the recovered RCEC the FNC Coating Mix ^(TM) (Athena Environmental Sciences, Inc. , (Baltimore, MD). Dulbecco's modified Eagle's medium (Life Technologies Corp.) supplemented with RCEC with 10% fetal bovine serum (FBS), 50 U / mL penicillin, 50 μg / mL streptomycin, and 2 ng / mL fibroblast growth factor 2 (Life Technologies Corp.). , Carlsbad, CA). Cultured RCECs that had been passaged from one to three passages were used.

(Injection of RCEC into rabbit corneal endothelial decompensation model)
The right eye of 12 Japanese White Rabbits was used for the study, and the left eye was not used to avoid blindness. The rabbit corneal endothelial decompensation model was prepared as described above. Briefly, the lens was removed one week ago to deepen the anterior chamber, and the corneal endothelium was mechanically removed using a 20G silicon needle (Soft Tapered Needle, Inami, Tokyo). The complete detachment of the corneal endothelium from the Descemet's membrane was confirmed by staining the Descemet's membrane with 0.1% trypan blue staining solution.

The cultured RCEC was washed with PBS, trypsinized with 0.05% Trypsin-EDTA (Life Technologies) at 37 ° C. for 5 minutes, and then neutralized with the medium. The RCEC were washed three times and stored in a 24-well plate (ultra-low adhesion) at a cell density of 1.0 × 10 ⁶ cells / 300 μl CTV in the form of a cell suspension. 24, 48 or 72 hours after storage,, RCEC for injection into a rabbit eye (1.0 × 10 ⁶ cells / 600μl CTV, Y-27632 of 100 [mu] M) in order to prepare, RCEC (1.0 × 10 ⁶ cells / 300 μl CTV) was gently mixed with a ROCK inhibitor (200 μM Y-27632 (Wako Pure Chemical Industries, Ltd.) / 300 μl CTV). The anterior chamber of the corneal endothelial decompensation model was injected with 300 μl of CTV containing 100 μM of Y-27632 together with a total of 5.0 × 10 ⁵ RCEC by using a 26 G syringe. The rabbits were maintained under general anesthesia with the endothelial surface down for 3 hours. As a control, CTV containing Y-27632 (final concentration: 100 μM) was injected into the anterior chamber of the corneal endothelial decompensation model. Three rabbit eyes were used for each group (control, 24 hours, 48 hours, and 72 hours of cell storage).

  The anterior part of the eye was evaluated by the slit lamp test for 14 days. Scheimpflug images were acquired on a Pentacam® (OCULUS Optikgate GmbH, Wetzlar, Germany) and the volume of the cornea at 7 mm diameter was also measured using the Scheimpflug images. The central thickness of the cornea was measured using an ultrasonic pachymeter (SP-2000; Tome, Nagoya). The corneal thickness could not be measured due to severe edema and was considered to be the instrument's maximum reading of 1200 μm.

(Immunofluorescence and actin staining)
Rabbit corneas were harvested and fixed with 4% formaldehyde for 10 minutes at room temperature. Specimens were incubated with 1% bovine serum albumin for 45 minutes at 37 ° C., and antibodies to Na ⁺ / K ⁺ -ATPase (1: 300, Upstate Biotechnology, Lake Placid, NY), ZO-1 (1: 300, Life Technologies Corp.) , Carlsbad, CA), N-cadrelin (1: 300, BD Biosciences, San Jose, CA)) at 4 ° C overnight. After three times washing the sample with PBS, specimen Alexa Fluor ^(R) 488-labeled goat anti-mouse: were incubated for 60 minutes at room temperature (1 1000, Life Technologies). The incubated sample was subjected to actin staining with Alexa Fluor ^{(registered trademark)} 488-labeled phalloidin (1: 400, Life Technologies) for 60 minutes at room temperature. Cell nuclei were stained with DAPI (Dojindo Laboratories, Kumamoto). Specimens were observed with a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Wetzlar Germany).

(Statistical analysis)
Statistical significance (p value) in comparison of multiple sample groups was calculated by Kruskal-Wallis test. Results are expressed as mean ± SD. A p-value less than 0.05 was considered statistically significant.

(result)
Feasibility of Conserved RCEC for Cell Therapy in Rabbit Model
Cultured RCECs were harvested and stored in cell culture form in 24-well plates (ultra low adhesion) at 37 ° C. for 24, 48, or 72 hours. Prior to injection into the rabbit model, Y-27632 was added to the RCEC and injected into the anterior chamber of the rabbit corneal endothelial decompensation model. Slit lamp tests showed that in eyes injected with RCEC after 24, 48, or 72 hours of storage, all corneas exhibited transparency within 7 days (FIG. 3A). In all control eyes, the cornea was opaque due to corneal endothelial decompensation. In the Scheimpflug image, infusion of RCEC stored for 24, 48, and 72 hours shows successful regeneration of anatomically normal corneas, whereas control eyes show severe corneal edema. (FIG. 3B).

The corneal thickness color map obtained with Pentacam ^™ shows that the RCEC stored for 24 and 48 hours regenerated normal corneal thickness from the center to the periphery of the cornea 14 days after the injection. On the other hand, the eyes injected with RCEC that had been stored for 72 hours had clear corneas but were thicker when observed in the slit lamp test (FIGS. 3A and 4A). Ultrasonic pachymetry showed that the central corneal thickness was approximately 400 μm (normal range) after 10 days in eyes injected with RCEC stored for 24 and 48 hours. However, eyes injected with RCEC stored for 72 hours had less decrease in central corneal thickness than eyes injected with RCEC stored for 24 or 48 hours, and were significantly higher throughout the 14 days (p <0.01) (FIG. 4B). ). Similar to the central corneal thickness, the corneal volume identified in the Scheimpflug image was lower in eyes injected with RCEC stored for 24 or 48 hours than in eyes injected with RCEC stored for 72 hours (FIG. 4C). The cell density of regenerated corneal endothelium as assessed by actin and DAPI was 2465 cells / mm ² in eyes injected with RCEC stored for 24 hours and 2368 cells / mm ² in eyes injected with RCEC stored for 48 hours. there were. However, the cell density of eyes injected with RCEC stored for 72 hours was 1548 cells / mm ² , which was significantly lower than that of eyes injected with RCEC stored for 24 or 48 (p <0.05). ) (FIG. 4D).

Immunofluorescence staining revealed that the function-related markers Na ⁺ / K ⁺ -ATPase (a marker of pump function), ZO-1 (a marker of tight junction), and N-cadherin (a marker of tight junction) were 24, 48, and 72. It was demonstrated that time-conserved RCEC was expressed in the lateral membrane of all regenerated CECs in injected eyes. Actin staining showed that the regenerated corneal endothelium formed a polygonal monolayer sheet-like structure. On the other hand, in the control eyes, due to corneal endothelial decompensation, almost no cells expressing a function-related marker associated with fibrosis were observed (FIG. 4E).

(Example 3: Preservation of corneal endothelial cells using a 1 ml syringe)
(Materials and methods)
The HCEC was stored in a 1 ml syringe (disposable syringe, notification number: 08B2X10007000001, Misawa Medical Industry Co., Ibaraki Prefecture) at a cell density of 1.0 × 10 ⁶ cells / 300 μl in cell suspension form on a CTV for 72 hours. The syringe was left standing sideways and stored at 37 ° C. for 72 hours. After storage, the syringe was shaken with a fingertip, the cells were taken out of the syringe with a 26G needle, and the cell survival rate was evaluated. After storage, shake the syringe with your fingertips to remove the cells from the syringe and evaluate the presence or absence of adhesion of the cells to the inner surface of the syringe during storage. Was observed.

(Calculation of bottom area)
In each experiment, the liquid volume was fixed at 300 μl, and the bottom area was adjusted by adjusting the position of the piston. The inner diameter of the used 1 ml syringe is 6 mm. When the tip of the piston (rubber portion) is arranged at a position 10 mm from the tip of the syringe and the syringe is turned sideways, the bottom area is (6 × 3.14 ÷ 2) × according to the definition in this specification. 10 = 94.2 mm ² = 0.942 cm ² . The amount of air at this time is about 0 μl.

When the tip of the piston (rubber portion) is disposed at a position 20 mm from the tip of the syringe and the syringe is turned sideways, the bottom area is (6 × 3.14 ÷ 2) × according to the definition in this specification. 20 = 188.4 mm ² = 1.884 cm ² . At this time, the amount of air is about 300 μl.

When the tip of the piston (rubber portion) is arranged at a position 20 mm from the tip of the syringe and the syringe is lengthened, the bottom area is 6 × 6 × 3.14 ≒ 28 mm ² ≒ about 0.28 cm ² .

(result)
500,000 corneal endothelial cells cultured in a 1 ml syringe were suspended in 300 μl of a cell preservation solution and stored (FIG. 5). When the corneal endothelial cells were stored in the syringe for 72 hours, precipitation of the cells on the side of the syringe was observed (lower left in FIG. 5). When lightly tapped, the cells were lifted from the side of the syringe, indicating that the cells did not adhere to the inner surface of the syringe during storage (bottom in FIG. 5). After storage for 72 hours, light tapping was performed, and the syringe was gently washed with a cell preservation solution. As a result, no adhesion of cells to the side surface of the syringe was observed (lower right in FIG. 5).

The viable cell rate after 72 hours of storage in a syringe is 100% of the total cells at the start of storage (live cell rate: 90.3%, dead cell rate: 9.7%), compared to the viable cell rate. : 70.9%, dead cell rate: 8.2%, and about 90% of the cells were alive, which was equivalent to that at the start of storage (FIG. 6). On the other hand, when cells were stored in the syringe, air was introduced and the syringe was laid down, so that the number of cells recovered was high when the bottom area of the syringe was about 2 cm ² , but it did not contain air. When the bottom area in the syringe was set to about 1 cm ² , the number of cells recovered decreased to about half. Similarly, when the syringe was set up with air in the syringe, the bottom area was about 0.28 cm ² , but the number of cells recovered was significantly reduced (FIG. 7). From these results, it was shown that, even when cells were stored in a syringe, the bottom area of about 2 cm ² increased the cell recovery rate and the cell survival rate.

(Example 4: Detailed examination of optimal temperature in cell storage)
(Materials and methods)
(Culture of corneal endothelial cells)
Five human donor corneas were obtained from diseased donors over the age of 40. All corneas were stored in Optisol (Chiron Vision, Irvine, CA) at 4 ° C for up to 14 days before use. Human corneal endothelial cells (HCEC) were cultured as follows. Briefly, the Descemet's membrane containing the corneal endothelium was mechanically detached from the donor cornea and digested by incubating for 12 hours at 37 ° C. in 1 mg / mL collagenase A (Roche Applied Science, Penzberg, Germany). After washing HCEC with OptiMEM-I (Life Technologies Corp., Carlsbad, Calif.), It was seeded in one well of a 48-well plate coated with laminin E8 fragment (iMatrix-511; Nippi, Tokyo).

  The medium was prepared according to the following. Briefly, 8% fetal bovine serum (FBS), 5 ng / mL epidermal growth factor (EGF; Thermo Fisher Scientific), 20 μg / mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), 200 mg / L calcium chloride, OptiMEM-I supplemented with 0.08% chondroitin sulfate (Sigma-Aldrich) and 50 μg / mL gentamicin (Thermo Fisher Scientific) was conditioned with NIH-3T3 for 24 hours. The conditioned medium was then collected and filtered through a 0.22 μm filter unit (EMD Millipore Corporation, Billerica, Mass.) And used as HCEC medium.

HCEC was cultured at 37 ° C. in a humidified atmosphere containing 5% CO ₂ , and the medium was changed three times a week. In the subculture of HCEC, cells were trypsinized with TrypLE ^™ Select Enzyme (10X) (Thermo Fisher Scientific) for 5 minutes at 37 ° C and seeded at a ratio of 1: 2. In this test, HCEC cultured for 5 to 9 passages was used.

The HCEC, washed with ^{Ca 2+} and phosphate buffered saline without ^{Mg 2+} (PBS), trypsinized for 15 minutes at 37 ° C. with TrypLE ^(TM) Select Enzyme (10X). After the cells were collected from the culture plate, they were washed twice, centrifuged at 280 G for 3 minutes, and suspended in OptiMEM-I. HCEC were cultured in the above serum-free culture medium (cell therapy culture medium provided by Cell Science Research Institute, Inc. (Miyagi)) at a cell density of 1.0 × 10 ⁶ cells / 300 μl in a 24-well plate (untreated) ( (AGC Techno Glass Co., Shizuoka) in the form of a cell suspension for 72 hours. In order to examine the optimal temperature for HCEC storage, it was stored under nine temperature conditions of 12 ° C, 17 ° C, 22 ° C, 27 ° C, 32 ° C, 35 ° C, 37 ° C, 39 ° C, and 42 ° C.

HCECs were stored for 72 hours, then gently pipetted from the cell culture plate, centrifuged at 280 G for 3 minutes, and resuspended in a cell therapy culture at a cell density of 1.0 × 10 ⁶ cells / 600 μl. did. The cell viability was calculated by staining dead cells with 0.5% trypan blue staining solution (Nacalai Tesque, Kyoto). As a control, HCEC recovered by trypsinization with TrypLE ^™ Select Enzyme (10X) was centrifuged at 280 G for 3 minutes and resuspended in cell therapy culture. Thereafter, cell viability was immediately assessed without storage in the form of a cell suspension.

(result)
(Effect of temperature conditions on cell viability in cell suspension storage)
HCECs were stored in cell suspension form at 1.0 × 10 ⁶ cells / 300 μl cell density in CTV for 72 hours, cells were harvested by gentle pipetting and cell viability was assessed (FIG. 9).

  The storage rate at 37 ° C. under the nine temperature conditions was highest for the viable cell rate, and the viable cell rate after 72 hours storage was 92.4% for the viable cell rate and 100% for all cells at the start of storage. The cell ratio was 7.6%, whereas the live cell ratio was 95.4% and the dead cell ratio was 4.9%, indicating a cell viability equivalent to that at the start of storage. In addition, under a temperature condition lower than 37 ° C., the viable cell rate decreases in proportion to the decrease in temperature, and the storage rate at 12 ° C., 17 ° C., and 22 ° C. is significantly lower than the viable cell rate at the start of storage. It showed a viable cell rate, but maintained a viability of 30% or more. Under the conditions of 39 ° C. and 42 ° C., the viable cell rate decreased in proportion to the rise in temperature, showing a significantly lower viable cell rate than the viable cell rate at the start of storage, but a viability of 30% or more. Was maintained. As described above, it is preferable to store at a temperature in the range of 12 ° C. to 42 ° C. that can maintain a viability of 30% or more, and to store at 17 ° C. to 39 ° C. that can maintain a viability of 60% or more. More preferably, it is more preferably stored at 27 ° C. to 37 ° C. that can maintain a viability of 80% or more, and it can be stored at 37 ° C. that can maintain a viability of 90% or more equivalent to a control. Is most preferred.

(Example 5: Examination of cell preservation using a glass vial)
(Materials and methods)
HCECs were stored in cell suspension form at 1.0 × 10 ⁶ cells / 300 μl cell density in CTV in glass vials for 72 hours. The following three glass vials were used: a vial having a normal glass surface (Maruem Co., Ltd., Osaka, 0501-02), and a vial having a low adsorption treatment (IRAS treatment) (Iwata Glass Industrial Co., Ltd.). Company, Osaka, lot: 181024), vial bottles with surface treatment with silica (IRAS treatment and SiO ₂ coating) (Iwata Glass Industry Co., Ltd., Osaka, lot: 181022) (FIG. 10). The bottom area of these vials is about 2 cm ² .

After storage for 72 hours, the cells were collected from the vial by gentle pipetting, centrifuged at 280 G for 3 minutes, and resuspended in a cell therapy culture at a cell density of 1.0 × 10 ⁶ cells / 600 μl. The cell viability was calculated by staining dead cells with 0.5% trypan blue staining solution (Nacalai Tesque, Kyoto). As a control, HCEC recovered by trypsinization with TrypLE ^™ Select Enzyme (10X) was centrifuged at 280 G for 3 minutes and resuspended in cell therapy culture. Thereafter, cell viability was immediately assessed without storage in the form of a cell suspension.

(result)
HCECs were stored in cell suspension form at 1.0 × 10 ⁶ cells / 300 μl cell density in CTV for 72 hours, cells were harvested by gentle pipetting and cell viability was assessed (FIG. 11).

  In the vial (Maruem Co., Osaka, 0501-02), adhesion of cells to the vial was observed. On the other hand, in the vial bottle (Iwata Glass Industry Co., Ltd., Osaka, 181024) and the vial bottle (Iwata Glass Industry Co., Ltd., Osaka, 181102), there was no adhesion of the cells to the vial bottle, and the collection was performed by gentle pipetting. It was possible. Assuming that all cells at the start of storage are 100%, the control is stored in a vial (Maruem Co., Osaka, 0501-02) against a live cell ratio of 92.4% and a dead cell ratio of 7.6%. The cells thus obtained had a viable cell ratio of 38.9% and a dead cell ratio of 7.4%, showing a significantly lower viable cell count than the viable cell count at the start of storage. Cells stored in a vial (Iwata Glass Industry Co., Ltd., Osaka, 181024) have a live cell ratio of 90.7% and a dead cell ratio of 7.8%, and a vial bottle (Iwata Glass Industry Co., Osaka, 181102). The cells stored in the above-mentioned cells had a viable cell ratio of 84.6% and a dead cell ratio of 8.4%, and exhibited the same cell viability as that at the time of starting the storage. Thus, all glass vials showed a cell viability of 30%, but those treated with a low-adhesion surface treatment showed higher cell viability.

(Example 6: Production example and transport example of cell-containing product)
Corneal endothelial cells cultured from the donor cornea, or corneal endothelial cells differentiated from iPS cells, ES cells, neural crest cells, or the like, or cells having the same function as corneal endothelial cells are collected from the culture dish by enzyme treatment. The collected cells are suspended in, for example, about 500,000 to about 1,000,000 cells in 300 μl of the cell injection solution, and about 500 μl to about 800 μl of the cell suspension is placed in a vial (Iwata Glass Industrial Co., Ltd.). Company, Osaka, lot: 181024) or vial (Iwata Glass Industry Co., Ltd., Osaka, lot: 181022). The vial is sealed with a rubber stopper and aluminum (primary container). The vial is further housed in a secondary container that is sealed and leakproof. The secondary container is stored in an incubator maintained at 37 ° C. The secondary container is housed in an external container that absorbs external shock, and transported to a medical institution while being maintained at 37 ° C.

  As described above, the present invention has been exemplified by the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the appended claims. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as if the content itself were specifically described herein. Understood.

  A method for storing corneal endothelial cells is provided. According to the present invention, corneal endothelial cells can be stored at a high cell viability. The thus preserved corneal endothelial cells have the function of normal corneal endothelial cells, and can be used as therapeutic cells for corneal endothelial diseases and the like, and thus can be used in the field of pharmaceuticals and the like. is there.

Claims (12)

A method of storing corneal endothelial cells and / or corneal endothelium-like cells in a cell suspension that can be used for cell infusion therapy, wherein the corneal endothelial cells and / or corneas are stored in a container having a bottom area of at least about 0.7 cm ^2. A step of preserving the endothelial-like cells, wherein the level of the liquid for preserving the cells is about 0.75 mm or more, and the corneal endothelial cells and / or the corneal endothelial-like cells are about 24-72. Time saved, method. The method of claim 1, wherein the bottom area of the container is between about 0.7 and about 4 cm ² . Bottom area of the container is about 1.5 to about 3 cm ^2, The method of claim 1 or 2. Bottom area of the container is about 1.8 to about 2 cm ^2, The method according to any one of claims 1 to 3.   The method according to any one of claims 1 to 4, wherein the container has not been subjected to a surface treatment for adhesion culture.   The method according to claim 1, wherein the container is a low-adhesion surface container or a non-surface treated container.   The method according to any one of claims 1 to 6, wherein the container is made of polystyrene, polypropylene, or glass.   The method according to any one of claims 1 to 7, wherein the container is selected from the group consisting of a 24-well plate, a vial, a syringe, and a dish.   9. The method according to any one of the preceding claims, wherein the storage of the cells is performed at a temperature from about 12C to about 42C.   10. The method according to any one of the preceding claims, wherein the storage of the cells is performed at a temperature from about 17C to about 39C.   The method of any one of claims 1 to 10, wherein the storage of the cells is performed at a temperature of about 27C to about 37C.   12. The method according to any one of the preceding claims, wherein the storage of the cells is performed at a temperature of about 37 <0> C.