Patent Application
20210207088
The present disclosure relates to a technology for preserving ocular cells, or culturing the cells after preservation.
Visual information is recognized when light transmitted into the cornea, which is a transparent tissue at the front-most part of an eye ball, reaches the retina and excites nerve cells of the retina, and a generated electric signal is transmitted through the optic nerve to the visual cortex of the cerebrum. To attain good vision, it is necessary that the cornea is transparent. The transparency of the cornea is maintained by maintaining constant water content by the pump and barrier functions of corneal endothelial cells.
While human corneal endothelial cells are present at a density of about 3000 cell per 1 mm2 at birth, once severely damaged, corneal endothelial cells cannot maintain their function such that transparency of the cornea is lost due to the limited ability of the cells to regenerate. Corneal endothelial dystrophy or bullous keratopathy induced by corneal endothelial dysfunction due to various causes results in edema or turbidity of the cornea, leading to significant deterioration in vision. Currently, heterologous corneal transplant using a donor cornea is performed for bullous keratopathy. However, many problems remain to be solved for corneal transplantation, such as invasiveness of surgery, rejection, and shortage in donors.
To overcome such problems, cell transplant therapy with low invasiveness have been developed in recent years as therapy for corneal endothelial diseases (Non Patent Literature 1). Culture of corneal endothelial cells is challenging, and involves problems such as inability to grow with conventional methods (Non Patent Literatures 2 and 3), fibroblast formation (Non Patent Literature 4), and cellular senescence (Non Patent Literature 5). Culturing methods of corneal endothelial cells are being actively studied, and in fact have become clinically applicable (Non Patent Literature 1).
The inventors have come to discover a method of stably preserving ocular cells (e.g., corneal cells, typically corneal endothelial cells) that can withstand long-term preservation (e.g., cryopreservation) and maintain a cell survival rate and/or cell density after preservation as a result of diligent study. The inventors have also come to apply such a preservation method to provide a cell formulation that can be used directly (ready-to-use) in ocular cell transplant and a therapeutic or prophylactic method using the same. The present disclosure also provides a method of preserving cells that can provide a cell formulation, which can be directly used substantially without additional manipulation, and a composition and method for preservation that can be used in said method.
Specifically, the inventors found that the cell survival rate and/or cell density is maintained and the cell count in culture after preservation is significantly increased by preserving ocular cells (e.g., corneal cells, typically corneal endothelial cells) by using a specific component, especially a component that can be used in clinical applications after preservation. Surprisingly, the cell survival rate and cell density were higher with preservation using a specific medium used herein as compared to using a preservation medium based on Opti-MEM®-I, which is conventionally considered to be the most suitable for culture of corneal endothelial cells, with an addition of DMSO and FBS. The inventors also surprisingly found that ocular cells preserved by such a preservation method can be used in clinical settings even when the cells are not subsequently cultured.
Therefore, the present disclosure provides the following.
A composition for use in preserving ocular cells, or culturing the cells after preservation, comprising albumin and dimethyl sulfoxide.
The composition of item 1, wherein the ocular cells comprise corneal cells.
The composition of any one of the preceding items, wherein the ocular cells comprise corneal endothelial cells.
The composition of any one of the preceding items, wherein the albumin is derived from a cow.
The composition of any one of the preceding items, further comprising a saccharide.
The composition of any one of the preceding items, wherein the saccharide is a monosaccharide.
The composition of any one of the preceding items, wherein the saccharide is a glucose.
The composition of any one of the preceding items, further comprising a phosphate ion.
The composition of any one of the preceding items, further comprising a metal ion.
The composition of any one of the preceding items, wherein the metal ion is a divalent metal ion.
The composition of any one of the preceding items, wherein the metal ion is selected from the group consisting of a calcium ion, a magnesium ion, an iron ion, a zinc ion, a copper ion, an aluminum ion, and any combination thereof.
The composition of any one of the preceding items, wherein the composition comprises (i) a calcium ion and magnesium or (ii) calcium, magnesium, and copper.
The composition of any one of the preceding items, wherein a concentration of the purified albumin is within the range of 0.01 to 25% (w/w).
The composition of any one of the preceding items, wherein a concentration of the purified albumin is within the range of 0.1 to 5% (w/w).
The composition of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is within the range of 0.1 to 50% (w/w).
The composition of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is within the range of 1 to 20% (w/w).
The composition of any one of the preceding items, wherein a concentration of the metal ion is within the range of 0.0000001 to 5% (w/w).
The composition of any one of the preceding items, wherein a concentration of the metal ion is within the range of 0.1 to 5% (w/w).
The composition of any one of the preceding items, wherein the composition comprises (i) 0.1% calcium and magnesium or (ii) 0.1 to 5% calcium, magnesium, and copper.
The composition of any one of the preceding items, wherein a concentration of the saccharide is within the range of 0.1 to 10% (w/w).
The composition of any one of the preceding items, wherein a concentration of the saccharide is within the range of 1 to 5% (w/w).
The composition of any one of the preceding items, wherein the preservation is cryopreservation.
The composition of any one of the preceding items, wherein the composition is Bambanker®.
A method for preserving corneal endothelial cells, comprising:
suspending ocular cells in the composition of any one of the preceding items to provide a suspension; and
freezing the suspension.
The method of any one of the preceding items, wherein the corneal endothelial cells are characterized by being suspended in the composition at 1×103 to 1×108 cells/ml.
The method of any one of the preceding items, wherein freezing the suspension comprises freezing without pre-freezing.
The method of any one of the preceding items, wherein the freezing without pre-freezing comprises freezing rapidly at −80° C.
A frozen cell product comprising corneal endothelial cells that have been cryopreserved in accordance with the method of any one of the preceding items.
A composition for use in preserving ocular cells, or culturing the cells after preservation, comprising albumin and dimethyl sulfoxide.
The composition of the preceding item, wherein the ocular cells comprise corneal cells.
The composition of any one of the preceding items, wherein the ocular cells comprise corneal endothelial cells.
The composition of any one of the preceding items, wherein the ocular cells are isolated cells or cells derived from progenitor cells.
The composition of any one of the preceding items, wherein the albumin is derived from a cow.
The composition of any one of the preceding items, further comprising a saccharide.
The composition of any one of the preceding items, wherein the saccharide is a monosaccharide.
The composition of any one of the preceding items, wherein the saccharide is a glucose.
The composition of any one of the preceding items, further comprising a phosphate ion.
The composition of any one of the preceding items, further comprising a metal ion.
The composition of any one of the preceding items, wherein the metal ion is a divalent metal ion.
The composition of any one of the preceding items, wherein the metal ion is selected from the group consisting of a calcium ion, a magnesium ion, an iron ion, a zinc ion, a copper ion, an aluminum ion, and any combination thereof.
The composition of item 10A, wherein the composition comprises (i) a calcium ion and magnesium or (ii) calcium, magnesium, and copper.
The composition of any one of the preceding items, wherein a concentration of the albumin is within the range of about 0.01 to about 25% (w/w).
The composition of any one of the preceding items, wherein a concentration of the albumin is within the range of about 0.1 to about 5% (w/w).
The composition of any one of the preceding items, wherein a concentration of the albumin is about 1% (w/w).
The composition of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is within the range of about 0.1 to about 50% (w/w).
The composition of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is within the range of about 1 to about 20% (w/w).
The composition of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is about 10% (w/w).
The composition of any one of the preceding items, wherein a concentration of the metal ion is within the range of about 0.0000001 to 5% (w/w).
The composition of any one of items 10A to 19A, wherein a concentration of the metal ion is within the range of about 0.1 to about 5% (w/w).
The composition of any one of the preceding items, wherein the composition comprises (i) about 0.1% calcium and magnesium or (ii) about 0.1 to about 5% calcium, magnesium, and copper.
The composition of any one of the preceding items, wherein a concentration of the saccharide is within the range of about 0.1 to about 10% (w/w).
The composition of any one of the preceding items, wherein a concentration of the saccharide is within the range of about 1 to about 5% (w/w).
The composition of any one of the preceding items, wherein the preservation is cryopreservation.
The composition of any one of the preceding items, wherein the composition is Bambanker®.
The composition of any one of the preceding items, wherein the composition is for administering corneal endothelial cells after preservation to a subject.
The composition of any one of the preceding items, wherein the corneal endothelial cells after preservation are characterized by being administered to a subject without culturing.
The composition of any one of the preceding items, wherein the corneal endothelial cells are characterized by being co-administered with a ROCK inhibitor.
The composition of any one of the preceding items, wherein the ROCK inhibitor is selected from Y-27632 ((+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexa ne), ripasudil (4-fluoro-5-{[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl}isoquinoline), fasudil (1-(5-isoquinolinesulfonyl)homopiperadine), or a pharmaceutically acceptable salt thereof.
A method for preserving ocular cells, comprising:
suspending ocular cells in the composition of any one of the preceding items to provide a suspension; and
freezing the suspension.
The method of any one of the preceding items, wherein the ocular cells are characterized by being suspended in the composition at about 1×103 to about 1×108 cells/ml.
The method of any one of the preceding items, wherein the freezing the suspension comprises freezing without pre-freezing.
The method of any one of the preceding items, wherein the freezing without pre-freezing comprises freezing rapidly at about −80° C.
A frozen cell product comprising corneal endothelial cells that have been cryopreserved in accordance with the method of any one of the preceding items.
A cell formulation comprising corneal endothelial cells, albumin, and dimethyl sulfoxide.
The cell formulation of any one of the preceding items, provided in a frozen state.
The cell formulation of any one of the preceding items for treating or preventing a corneal endothelial disease, disorder, or symptom.
The cell formulation of any one of the preceding items, wherein the albumin is derived from a cow.
The cell formulation of any one of the preceding items, further comprising a saccharide.
The cell formulation of any one of the preceding items, wherein the saccharide is a monosaccharide.
The cell formulation of any one of the preceding items, wherein the saccharide is a glucose.
The cell formulation of any one of the preceding items, further comprising a phosphate ion.
The cell formulation of any one of the preceding items, further comprising a metal ion.
The cell formulation of any one of the preceding items, wherein the metal ion is a divalent metal ion.
The cell formulation of any one of the preceding items, wherein the metal ion is selected from the group consisting of a calcium ion, a magnesium ion, an iron ion, a zinc ion, a copper ion, an aluminum ion, and any combination thereof.
The cell formulation of any one of the preceding items, wherein the cell formulation comprises (i) a calcium ion and a magnesium ion or (ii) a calcium ion, a magnesium ion, and a copper ion.
The cell formulation of any one of the preceding items, wherein a concentration of the albumin is within the range of about 0.01 to about 25% (w/w).
The cell formulation of any one of the preceding items, wherein a concentration of the albumin is within the range of about 0.1 to about 5% (w/w).
The cell formulation of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is within the range of about 0.1 to about 50% (w/w).
The cell formulation of any one of the preceding items, wherein a concentration of the dimethyl sulfoxide is within the range of about 1 to about 20% (w/w).
The cell formulation of any one of the preceding items, wherein a concentration of the metal ion is within the range of about 0.0000001 to about 5% (w/w).
The cell formulation of any one of the preceding items, wherein a concentration of the metal ion is within the range of about 0.1 to about 5% (w/w).
The cell formulation of any one of the preceding items, wherein the cell formulation comprises (i) about 0.1% calcium ion and magnesium ion or (ii) about 0.1 to about 5% calcium ion, magnesium ion, and copper ion.
The cell formulation of any one of the preceding items, wherein a concentration of the saccharide is within the range of about 0.1 to about 10% (w/w).
The cell formulation of any one of the preceding items, wherein a concentration of the saccharide is within the range of about 1 to about 5% (w/w).
The cell formulation of any one of the preceding items, wherein the corneal endothelial cells are at a concentration of about 1×103 to about 1×108 cells/ml.
The cell formulation of any one of the preceding items, further comprising a ROCK inhibitor.
The cell formulation of any one of the preceding items, wherein the ROCK inhibitor is selected from Y-27632, ripasudil, fasudil, or a pharmaceutically acceptable salt thereof.
A method for treating or preventing a corneal endothelial disease, disorder, or symptom in a subject, comprising administering to the subject a therapeutically effective amount of corneal endothelial cells preserved in accordance with the method of any one of items 31A to 34A.
The method of any one of the preceding items, wherein the corneal endothelial cells are administered in conjunction with a ROCK inhibitor.
The method of any one of the preceding items, wherein the ROCK inhibitor is selected from Y-27632, ripasudil, fasudil, or a pharmaceutically acceptable salt thereof.
The method of any one of the preceding items without a step of culturing corneal endothelial cells.
Corneal endothelial cells for treating or preventing a corneal endothelial disease, disorder, or symptom in a subject, wherein the corneal endothelial cells are corneal endothelial cells preserved in accordance with the method of any one of the preceding items.
The corneal endothelial cells of anyone of the preceding items, wherein the corneal endothelial cells are characterized by being administered in conjunction with a ROCK inhibitor.
The corneal endothelial cells of any one of the preceding items, wherein the ROCK inhibitor is selected from Y-27632, ripasudil, fasudil, or a pharmaceutically acceptable salt thereof.
The method of any one of the preceding items, wherein corneal endothelial cells after preservation are characterized by being administered without further culturing.
Use of corneal endothelial cells in the manufacture of a drug for treating or preventing corneal endothelial disease, disorder, or symptom in a subject, wherein the corneal endothelial cells are corneal endothelial cells preserved in accordance with the method of any one of items 31A to 34A.
The use of any one of the preceding items, wherein the corneal endothelial cells are characterized by being administered in conjunction with a ROCK inhibitor.
The use of any one of the preceding items, wherein the ROCK inhibitor is selected from Y-27632, ripasudil, fasudil, or a pharmaceutically acceptable salt thereof.
The use of anyone of the preceding items, wherein corneal endothelial cells after preservation are characterized by being administered without further culturing.
The present disclosure is intended so that one or more of the aforementioned features can be provided not only as the explicitly disclosed combinations, but also as other combinations thereof. Additional embodiments and advantages of the present disclosure are recognized by those skilled in the art by reading and understanding the following detailed description as needed.
According to the present disclosure, ocular cells (e.g., corneal cells, typically corneal endothelial cells) can be preserved at a high cell survival rate and cell density. The cell count also significantly increases by culturing after preservation. Preserved cells can be used in clinical settings even when the cells are not subsequently cultured.
In this present disclosure, a formulation comprising ocular cells (e.g., corneal cells, typically corneal endothelial cells) can be clinically used directly after preservation. Thus, a so-called ready-to-use cell formulation is provided. In regenerative medicine, cumbersome operations are preferably omitted as much as possible in clinical settings. Thus, the present disclosure drastically improves the work efficiency in transplant surgery settings.
The present disclosure is described hereinafter. Throughout the entire specification, a singular expression should be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Thus, singular articles (e.g., “a”, “an”, “the”, and the like in the case of English) should also be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Further, the terms used herein should be understood as being used in the meaning that is commonly used in the art, unless specifically noted otherwise. Therefore, unless defined otherwise, all terminologies and scientific technical terms that are used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of a contradiction, the present specification (including the definitions) takes precedence. As used herein, “about” refers to a range of ±10% of the subsequent value.
The preferred embodiments are described hereinafter. It is understood that the embodiments are exemplification of the present disclosure, so that the scope of the present disclosure is not limited to such preferred embodiments. It is understood that those skilled in the art can refer to the following preferred embodiments to readily make modifications or changes within the scope of the present disclosure. Any of these embodiments can be appropriately combined by those skilled in the art.
(Composition)
In one aspect, the present disclosure provides a composition for preserving (e.g., cryopreserving) ocular cells or culturing the cells after preservation (e.g., cryopreservation), comprising albumin and dimethyl sulfoxide. The inventors found that the cell survival rate and/or cell density is maintained, and the cell count significantly increases in culture after preservation when ocular cells (e.g., corneal cells, typically corneal endothelial cells) are preserved using a composition comprising albumin and dimethyl sulfoxide, compared to using a conventional preservation medium. Examples of ocular cells that can be preserved with the composition of the present disclosure include corneal epithelial cells, corneal stromal cells, corneal endothelial cells, trabecular cells, retinal pigment epithelial cells, bipolar cells, ganglion cells, scleral cells, and lens epithelial cells. Such ocular cells are preferably corneal cells such as corneal epithelial cells, corneal stromal cells, and corneal endothelial cells, and most preferably corneal endothelial cells. Ocular cells can be cells derived from mammals (human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkey, or the like), but are preferably derived from primates and particularly preferably derived from humans. It is more difficult to maintain culture of ocular cells compared to other body parts. For preservation, ocular cells often exhibit behavior that is different from other cells. Therefore, it was unknown to those skilled in the art that ocular cells, especially corneal cells, and more specifically corneal endothelial cells can be efficiently preserved. In particular, it was unknown whether ocular cells, especially corneal cells, and more specifically corneal endothelial cells can be isolated and/or preserved in a cultured state, or preserved in a transplantable state thereafter.
In some embodiments, ocular cells that can be preserved can be isolated cells or cells derived from progenitor cells (e.g., ES cells or iPS cells). It is difficult to maintain ocular cells (particularly corneal endothelial cells) such as isolated cells or cells derived from progenitor cells compared to cells assembled as a tissue. Such cells can be preserved with a high cell survival rate and/or cell density with the composition for preservation of the present disclosure.
In some embodiments, the composition of the present disclosure is substantially free of nutritional factors for growing and/or differentiating cells during preservation.
In some embodiments, the composition of the present disclosure can be for administrating ocular cells after preservation to a subject. Ocular cells preserved using the composition of the present disclosure can be immediately administered without additional culturing, with only a simple operation such as exchanging media as needed. More specifically, the ocular cells can be administered to a subject without cell culture or incubation for 24 hours or more, 18 hours or more, 12 hours or more, preferably 6 hours or more (or any other unit of time) for the purpose of growth and/or re-differentiation of cells after exchanging at least some of the components other than cells in the composition of the present disclosure with other components such as injected medium by means such as centrifugation. Therefore, ocular cells preserved using the composition of the present disclosure can be administered to a subject without culturing. Ocular cells can also be co-administered with a ROCK inhibitor. A ROCK inhibitor can be included in a composition in advance, mixed in immediately before administration, or administered individually upon administration of cells. Examples of ROCK inhibitors include compounds disclosed in the following documents: U.S. Pat. No. 4,678,783, Japanese Patent No. 3421217, International Publication No. WO 95/28387, International Publication No. WO 99/20620, International Publication No. WO 99/61403, International Publication No. WO 02/076976, International Publication No. WO 02/076977, International Publication No. WO 2002/083175, International Publication No. WO 02/100833, International Publication No. WO 03/059913, International Publication No. WO 03/062227, International Publication No. WO 2004/009555, International Publication No. WO 2004/022541, International Publication No. WO 2004/108724, International Publication No. WO 2005/003101, International Publication No. WO 2005/039564, International Publication No. WO 2005/034866, International Publication No. WO 2005/037197, International Publication No. WO 2005/037198, International Publication No. WO 2005/035501, International Publication No. WO 2005/035503, International Publication No. WO 2005/035506, International Publication No. WO 2005/080394, International Publication No. WO 2005/103050, International Publication No. WO 2006/057270, and International Publication No. WO 2007/026664. Specific examples thereof include, but are not limited to, Y-27632 ((+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexa ne), ripasudil (4-fluoro-5-{[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl}isoquinoline), fasudil (1-(5-isoquinolinesulfonyl)homopiperadine), and pharmaceutically acceptable salts thereof.
Albumin that can be used in the present disclosure can be derived from any species, such as cells derived from mammals (human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkey, or the like), but those derived from humans are preferably used when preserving human cells. In an embodiment of preserving human cells, albumin derived from a human can be used or albumin derived from a cow can be used. The albumin used in the present disclosure is generally not used as a component of serum, but in a separated state from other components. In one example, albumin that is used is preferably purified albumin. Purification can be performed using any method in the art. The albumin concentration in a composition below about 0.001% (w/w) results in no effect, and a concentration above about 25% (w/w) results in no change in the effect. Therefore, in some embodiment, the concentration of albumin can be in the range of about 0.001% to about 25% (w/w), such as about 0.01 to about 25% (w/w), about 0.01 to about 20% (w/w), about 0.01 to about 15% (w/w), about 0.01 to about 10% (w/w), about 0.01 to % (w/w), about 5% (w/w), about 0.1 to about 25% (w/w), about 0.1 to % (w/w), about 20% (w/w), about 0.1 to about 15% (w/w), about 0.1 to about 10% (w/w), and more preferably about 0.1 to about 5% (w/w). In a preferred embodiment, the albumin concentration can be about 1% (w/w).
The dimethyl sulfoxide used in the present disclosure can be commonly known dimethyl sulfoxide that is commercially available. The dimethyl sulfoxide concentration in a composition below about 0.1% (w/w) results in a reduced ability to preserve cells, and a concentration above about 50% (w/w) results in no change in the effect. Thus, the concentration is preferably within the range of about 0.1% (w/w) to about 50% (w/w) such as about 0.1 to about 40% (w/w), about 0.1 to about 30% (w/w), about 0.1 to about 20% (w/w), about 1 to about 50% (w/w), about 1 to about 40% (w/w), about 1 to 30% (w/w), and more preferably in the range of about 1 to about 20% (w/w). In a preferred embodiment, the dimethyl sulfoxide concentration can be about 10% (w/w).
In some embodiments, the composition of the present disclosure can further comprise a saccharide. Although not wishing to be bound by any theory, the post-preservation viable cell rate (effective cell rate) and culture efficiency improve by further including a saccharide. Examples of saccharides include, but are not limited to, monosaccharides, disaccharides, and trisaccharides, such as glucose, galactose, fructose, ribose, trehalose, and glucose. In a preferred embodiment, a saccharide that is added is preferably a monosaccharide. In particular, glucose is preferred due to the ease of handling, but other saccharides described above can be used or any combination can be used. The saccharide concentration in a composition of less than about 0.1% (w/w) results in no effect, and a concentration above about 10% (w/w) results in no change in the effect. Thus, the concentration is in the range of about 0.1 to about 10% (w/w), such as about 0.1 to 9% (w/w), about 0.1 to 8% (w/w), about 0.1 to about 7% (w/w), about 0.1 to about 6% (w/w), about 0.1 to about 5% (w/w), about 1 to 10% (w/w), about 1 to about 9% (w/w), about 1 to about 8% (w/w), about 1 to about 7% (w/w), about 1 to about 6% (w/w), and more preferably about 1 to about 5% (w/w).
In some embodiments, the composition of the present disclosure can further comprise a phosphate ion. A phosphate ion can be used as a phosphate buffer. Dulbecco's phosphate buffer, phosphate buffer, or the like can be used.
In some embodiments, the composition of the present disclosure can comprise a metal ion. Although not wishing to be bound by any theory, the post-preservation viable cell rate (effective cell rate) after preservation and culture efficiency improve by further including a metal ion. Examples of metal ions include, but are not limited to, divalent and trivalent metal ions, such as calcium ion, magnesium ion, iron ion, zinc ion, copper ion, and aluminum ion. In a preferred embodiment, a metal ion can be a divalent metal ion. The metal ion concentration in a composition of less than about 0.0000001% (w/w) results in no effect, and a concentration above about 1% (w/w) results in no change in effect. Thus, in a specific embodiment, the metal ion concentration is in the range of about 0.0000001% to about 5% (w/w) such as about 0.1 to about 4% (w/w), about 0.1 to about 3% (w/w), about 0.1 to about 2% (w/w), about 0.1 to about 1% (w/w), about 0.01 to about 5% (w/w), about 0.01 to about 4% (w/w), about 0.01 to about 3% (w/w), about 0.01 to about 3% (w/w), about 0.01 to about 2% (w/w), about 0.01 to about 1% (w/w), and more preferably about 0.1 to about 5% (w/w). In a specific embodiment, the composition of the present disclosure can comprise any combination of the metal ions described above. For example, the composition of the present disclosure can comprise (i) a calcium ion and magnesium or (ii) calcium, magnesium, and copper. In a more specific embodiment, the composition of the present disclosure can comprise (i) about 0.1% calcium and magnesium or (ii) 0.1 to 5% calcium, magnesium, and copper.
In a specific embodiment, the composition of the present disclosure can comprise about 0.1% (w/w) to 5% (w/w) of albumin and about 5% (w/w) to 15% (w/w) of dimethyl sulfoxide.
In a specific embodiment, the composition of the present disclosure can comprise about 1% (w/w) of albumin and about 10% (w/w) of dimethyl sulfoxide.
In a specific embodiment, the composition of the present disclosure is, for example, Bambanker®, Bambanker® hRM, or STEM-CELLBANKER®, or comprises the same. Bambanker® is preferable due to the high ability to grow cells in culture after preservation. STEM-CELLBANKER® is preferred due to the high ability to maintain the cell survival rate and cell density after preservation. Bambanker® hRM is particularly preferable due to the high ability to maintain the cell survival rate and cell density after preservation and the high ability to grow cells in culture thereafter. Bambanker® hRM comprises about 1% (w/w) of albumin, about 10% (w/w) of dimethyl sulfoxide, and other medium components. Those skilled in the art can appropriately modify the medium components. It is possible to confirm whether a medium comprising a modified medium component is within the scope of the present disclosure by using any approach that is explicitly disclosed herein or known in the art, preferably by testing the component is clinically applicable after preservation.
(Cell Formulation)
In another aspect, the present disclosure provides a cell formulation comprising corneal endothelial cells, albumin, and dimethyl sulfoxide. In some embodiment, the cell formulation of the present disclosure can be provided in a frozen state. The cell formulation of the present disclosure can comprise a therapeutically effective amount of corneal endothelial cells. Since a high cell survival rate and/or cell density is maintained even in a frozen state, a therapeutically effective amount of clinically usable normal corneal endothelial cells can be administered after thawing. Therefore, a cell formulation provided in a frozen state can be thawed immediately before administration and administered to a subject without a subsequent culturing step, such that the formulation is provided in a “ready to use” state. The cell formulation of the present disclosure can be for treating or preventing a corneal endothelial disease, disorder, or symptom of a subject. Examples of corneal endothelial disease, disorder, or symptom include, but are not limited to, Fuchs' endothelial corneal dystrophy, post-corneal transplant disorder, corneal endotheliitis, trauma, post-ophthalmic surgery disorder, post-ophthalmic laser surgery disorder, aging, posterior polymorphous dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, cytomegalovirus corneal endotheliitis, and the like.
As for constituent elements contained in a cell formulation (albumin, dimethyl sulfoxide, metal ion, saccharide, ROCK inhibitor, and the like), one or more embodiments described above (composition) can be appropriately used.
(Preservation Method)
In another aspect, the present disclosure provides a method for preserving ocular cells (e.g., corneal endothelial cells), comprising suspending the ocular cells in the composition described above to provide a suspension, and freezing the suspension without pre-freezing. Typically, cells are suspended in a cell preservation medium (composition of the present disclosure) in a container for low temperature preservation (tube, plate, dish, or the like) at about 1×103 to about 1×108 cells/ml, and this is preserved at about −80° C. or in liquid nitrogen. If the preservation is for a short period of time, the temperature can be about −4° C. to about −20° C. Ocular cells can be suspended in a cell preservation medium, dispensed into a plurality of containers for cell preservation, and cryopreserved without pre-freezing. Upon preservation, the temperature of cells suspended in the cell preservation medium can be gradually reduced with a programmable freezer or the like, or rapidly reduced directly. Regarding recovery of cryopreserved cells, cells preserved at a low temperature are melted in a thermostatic vessel at room temperature or 37° C., and the cells and the cell preservation medium are separated by an operation such as centrifugation using a centrifuge to recover the cells. The separated cells can be cultured by a common method after washing with a suitable culture medium or the like.
In a preferred embodiment, one advantageous feature of the present disclosure can be in being ready-to-use, where the cryopreserved cells can be directly administered after recovery to a patient. A method of thawing and/or recovery for direct administration in such an embodiment can be any approach, but the following is a representative approach. Specifically, cells in a frozen state can be administered by thawing according to a conventional method, and performing a simple operation such as exchanging the composition of the present disclosure with another composition by means such as centrifugation as needed without culturing cells for a certain period of time in order to grow and redifferentiate the cells, and administrating directly or by suspending the cells in a solution for administration (e.g., saline or phosphate buffered saline (PBS)). It is understood that this is within the scope of direct administration.
(Treatment/Prevention)
In another aspect, the present disclosure provides a method for treating or preventing an ophthalmic (e.g., corneal endothelial) disease, disorder, or symptom in a subject, comprising administering a therapeutically effective amount of ocular cells (e.g., corneal endothelial cells) preserved in accordance with the aforementioned preservation method of the present disclosure to the subject.
The following procedure is provided as a representative approach.
1) preserve ocular cells (e.g., corneal endothelial cells) using the composition for preservation of the present disclosure and cryopreserve as needed;
2) thaw the ocular cells (e.g., corneal endothelial cells) preserved in 1) as needed upon treatment, and subsequently suspend the cells in an intraocular injection solution, centrifuge the suspension, and resuspend the cells into the injection solution;
3) add a ROCK inhibitor as needed; and
4) inject the composition comprising the ocular cells (e.g., corneal endothelial cells) into an eye of a subject in need thereof.
The preferred embodiments described in other parts of the present specification such as each of the section of (Composition), (Preservation method), (Cell formulation), (Cells), and (Use) can be appropriately combined and used as needed as the technologies used in treatment/prevention of the present disclosure.
(Cells)
In another aspect, the present disclosure provides ocular cells (e.g., corneal endothelial cells) for treating or preventing an ophthalmic (e.g., corneal endothelial) disease, disorder, or symptom in a subject, wherein the ocular cells (e.g., corneal endothelial cells) are corneal endothelial cells preserved in accordance with the aforementioned preservation method of the present disclosure.
The preferred embodiments described in other parts of the present specification such as each of the section of (Composition), (Preservation method), (Cell formulation), (Treatment/prevention), and (Use) can be appropriately combined and used as needed as the technologies used in the cells of the present disclosure.
(Use)
In another aspect, the present disclosure provides use of corneal endothelial cells in the manufacture of a drug for treating or preventing a corneal endothelial disease, disorder, or symptom in a subject, wherein the ocular cells (e.g., corneal endothelial cells) are ocular cells (e.g., corneal endothelial cells) preserved in accordance with the aforementioned preservation method of the present disclosure.
The preferred embodiments described in other parts of the present specification such as each of the section of (Composition), (Preservation method), (Cell formulation), (Treatment/prevention), and (Cells) can be appropriately combined and used as needed as the technologies used in the use of the present disclosure.
The present disclosure has been described while showing preferred embodiments to facilitate understanding. The present disclosure is described hereinafter based on the Examples. The above descriptions and the following Examples are not provided to limit the present disclosure, but are provided for the sole purpose of exemplification. Thus, the scope of the present disclosure is not limited to the embodiments and Examples that are specifically disclosed herein and is limited only by the scope of claims.
The present disclosure is more specifically described hereinafter based on the Examples. It is understood that the specifically shown reagents as well as those available from Sigma-Aldrich, BASF Japan Ltd., or the like can be used as the various reagents used in the Examples.
(Cell Culture Example)
The summary of exemplary cell culture in this Example is shown in
Donor corneas were obtained from SightLife™. Corneas of healthy donors who were 40 years old or older were used. The corneas preserved in Optisol (Chiron Vision) at 4° C. for a preservation period of 14 days or less were used in the experiments. Descemet's membranes containing the corneal endothelium were stripped from the donor corneas, and the Descemet's membranes were digested for 12 hours at 37° C. with 1 mg/mL collagenase A (Roche Applied Science, 10 103 586 001). After washing three times with OptiMEM™-I (invitrogen, 31985-088), they were seeded in one well of a 48-well plate coated with laminin 511 E8 (iMatrix-511; Nippi, 892012). The culture medium was prepared as follows. NIH-3T3 cells treated for 2 hours with 4 μg/mL of mitomycin C (Kyowa Hakkko Kirin, 4231400D1031) were seeded on a culture dish at a cell density of 1.35×104 cells/cm2. NIH-3T3 cells were cultured with a basal medium for 24 hours, and the basal medium was collected. The collected basal medium was filtered through a 0.22-μm filtration unit (Millipore, R7NA75775), and used for culturing corneal endothelial cells after adding 5 ng/mL of epithelial growth factor (invitrogen, PHG0311), 0.1 mM ascorbic acid (SIGMA-Aldrich, PHR1008), 10 μM of 5B203580 (Cayman, 13067), and 1 μM of 5B431542 (WAKO, 192-16541) upon use. Cultured human corneal endothelial cells at passages 5 through 10 and with a cell density of 1000 to 1500 cells/mm2 were used for the screening experiments.
The following basal medium was used.
Basal medium: Opti-MEM®-I (invitrogen, 31985-088)+8% FBS (Thermo, 5H30084.03)+5 ng/mL epithelial growth factor (invitrogen, PHG0311)+20 μg/mL ascorbic acid (SIGMA-Aldrich, PHR1008)+200 mg/L calcium chloride (SIGMA-Aldrich, 449709)+0.08% chondoroitin sulfate (WAKO, 032-14613)+50 μg/mL gentamicin (invitrogen, 15710-064).
The medium was removed from the culture dish where human corneal endothelial cells were being cultured, and 1×PBS(−) (Nippon Flour Mills Co., Ltd., 05913) preheated to 37° C. was added to wash the cells. This was repeated twice. 1×PBS(−) was added again, and the cells were incubated for 3 minutes at 37° C. (5% CO2). After removing the PBS (−), TrypLE™ Select Enzyme (10×) (Thermo Fisher Scientific, A1217701) was added, and the cells were incubated for 15 minutes at 37° C. (5% CO2). The cells were then suspended in a medium. The cells were collected by centrifugation for 3 minutes at 280 G. After counting the cells, human corneal endothelial cells were centrifuged for 3 minutes at 280 G. After removing the supernatant, various cryopreservation reagents (1 ml for 5×105 cells) were added. The human corneal endothelial cells were then frozen at −80° C. by using a BICELL bio freezing vessel (Nihon Freezer Co., Ltd., BICELL) for 24 hours, and preserved in liquid nitrogen (−196° C.) for 13 more days. After 14 days of cryopreservation, human corneal endothelial cells were re-cultured from the frozen stock. Briefly, cryotubes preserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. The human corneal endothelial cells were then seeded at a density of 800 cells/mm2 in a culture plate. The prepared preservation medium was used as the cryopreservation medium. Opti-MEM®-I+10% DMSO+10% FBS was used as a control for the cryopreservation medium.
(Example of Observation with Phase Contrast Microscope)
An exemplary method of observing cells in this Example is described.
Cryotubes preserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. The human corneal endothelial cells were then seeded at a density of 800 cells/mm2 in a well coated with laminin-511 E8 and an uncoated well after adding Y-27632 (Wako Pure Chemical Industries, Ltd., 253-00513) to the suspension. The cell morphology and cell adhesion were observed after 24 hours under a phase contrast microscope.
(Results)
(Example of Measuring Viable Cells)
A typical method of measuring viable cells in this Example is described.
Cryotubes cryopreserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. The human corneal endothelial cells were then seeded at a density of 800 cells/mm2 in a well coated with laminin-511 E8 and an uncoated well after adding Y-27632 to the suspension. To find the viable cell count after 24 hours, the viable cell count was measured with Cell Titer-Glo Luminescent Cell Viability Assay by the following procedure. First, a human corneal endothelial cell suspension was prepared for calibration curves. The medium in the plate was removed. The cells were washed with Opti-MEM®-I preheated to 37° C., and then Opti-MEM®-I was added at 50 μl/well. Human corneal endothelial cells used only for calibration were seeded in an empty well at 0 cells/well, 5000 cells/well, 10000 cells/well, and 15000 cells/well. Cell Titer-Glo Luminescent Cell Viability Assay solution (Promega, G7572) was added at 50 μl/well so that medium:solution would be 1:1. The operation hereafter was performed with the light shielded. A shaker was shaken thoroughly for 2 minutes at about 120 minutes−1 and incubated for 10 minutes. After incubation, 80 μl was transferred to an Assay plate (Corning, 3912, Assay plate 96 well, white polystyrene), and absorbance was measured using GloMax-Multi Detection System (Promega, E7051).
(Results)
In this Example, cryopreservation media suitable for cryopreservation of corneal endothelial cells were screened.
(A Cell Survival Rate)
Cryotubes preserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Dead cells were then stained for 10 minutes with 0.5%-Trypan Blue Stain solution (Nacalai Tesque, 29853-34). The viable cell count and the dead cell count were determined using a hemocytometer. The cell survival rate was calculated by dividing the viable cell count by the preserved cell count (5×105 cells) in cryopreserved cells. This was calculated by dividing the viable cell count by the sum of dead cell count and viable cell count in cells without cryopreservation. CELLBANKER®2 (Nippon Seiyaku Kogyou Co., Ltd., CB031), Bambanker (Nippon Genetics Co., Ltd., CS-02-001), KM Banker (Kohjin Bio Co., Ltd., 16092005), STEM-CELLBANKER® (Nippon Seiyaku Kogyou Co., Ltd., CB047), Bambanker hRM (Nippon Genetics Co., Ltd., CS-07-001), and ReproCryo DMSO Free RM (ReproCELL, RCHEFM003) were used as the preservation medium. Opti-MEM®-I+10% Dimethyl sulfoxide (DMSO; Nacalai Tesque, 13408-64)+10% FBS was used as a control for the cryopreservation reagent.
(Results)
(B Examination of Ability to Grow Cells with Cell Titer Glo)
Cryotubes cryopreserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was added to the cell suspension, and the cells were seeded on a 96-well plate coated with laminin-511 E8. To find the ability to grow cells after 1 day, 7 days, 14 days, and 28 days, the viable cell count was measured with Cell Titer-Glo Luminescent Cell Viability Assay by the following procedure. First, a human corneal endothelial cell suspension was prepared for a calibration curve. The medium in the plate was removed. The cells were washed with Opti-MEM®-I preheated to 37° C., and then Opti-MEM®-I was added at 50 μl/well. Human corneal endothelial cells for the calibration curve were seeded in empty wells. Cell Titer-Glo Luminescent Cell Viability Assay solution (Promega, G7572) was added at 50 μl/well so that medium:solution would be 1:1. The operation hereafter was performed with the light shielded. A shaker was shaken thoroughly for 2 minutes at about 120 minutes−1 and incubated for 10 minutes. After incubation, 80 μl was transferred to an Assay plate (Corning, 3912, Assay plate 96 well, white polystyrene), and absorbance was measured using GloMax-Multi Detection System (Promega, E7051).
(Results)
(C Cell Density)
Cryopreserved cryotubes were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 800 cells/mm2 on a culture plate coated with laminin-511 E8. After washing the cells that have reached confluence on day 28 after seeding twice with 1×PBS(−), the cells were incubated for 3 minutes at 37° C. (5% CO2). After washing the cells that have reached confluence on day 28 after seeding twice with 1×PBS (−), the cells were incubated for 3 minutes at 37° C. (5% CO2). A picture was then taken using a phase contrast microscope at a 200× magnification. The cell density was measured by using a measurement software, ImageJ software (National Institutes of Health). First, the taken phase contrast microscope picture was uploaded into the software, and the cells were counted using the counting function. Analysis was performed when 100 cells were counted to measure the cell density. Dunnett's multiple comparison test was used for testing. The results are represented as mean±standard deviation.
(Results)
This Example studied the suitable amount of cryopreservation medium used in cryopreservation.
(A Cell Survival Rate)
The medium was removed from the culture dish where human corneal endothelial cells were being cultured, and 1×PBS(−) (Nippon Flour Mills Co., Ltd., 05913) preheated to 37° C. was added to wash the cells. This was repeated twice. 1×PBS(−) was added again, and the cells were incubated for 3 minutes at 37° C. (5% CO2). After removing the PBS (−), TrypLE™ Select Enzyme (10×) (Thermo Fisher Scientific, A1217701) was added, and the cells were incubated for 15 minutes at 37° C. (5% CO2). The cells were then suspended in a medium. The cells were collected by centrifugation for 3 minutes at 280 G. After counting the cells, the suspension was dispensed in each 15 ml centrifuge tube and centrifuged for 3 minutes at 280 G. After removing the supernatant, 0.5 ml, 1 ml, and 1.5 ml of cryopreservation reagents were added so that each would have a concentration of 5×105 cells per 1 ml. The human corneal endothelial cells were then frozen at −80° C. by using a BICELL bio freezing vessel (Nihon Freezer Co., Ltd., BICELL) for 24 hours, and preserved in liquid nitrogen (−196° C.) for 13 more days. After 14 days of cryopreservation, human corneal endothelial cells were re-cultured from the frozen stock. Briefly, cryotubes preserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Dead cells were then stained for 10 minutes with 0.5%-Trypan Blue Stain solution (Nacalai Tesque, 29853-34). The viable cell count and the dead cell count were determined using a hemocytometer. The cell survival rate was calculated by dividing the viable cell count by the preserved cell count (5×105 cells) in cryopreserved cells. This was calculated by dividing the viable cell count by the sum of dead cell count and viable cell count in cells without cryopreservation. Bambanker hRM was used as the preservation medium. Dunnett's multiple comparison test was used for testing. The results are represented as mean±standard deviation.
(Results)
(B Cell Density)
Cryopreserved cryotubes were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 800 cells/mm2 on a culture plate coated with laminin-511 E8. After washing the cells that have reached confluence on day 28 after seeding twice with 1×PBS(−), the cells were incubated for 3 minutes at 37° C. (5% CO2). A picture was then taken using a phase contrast microscope at a 200× magnification. The cell density was measured by using a measurement software, ImageJ software (National Institutes of Health). First, the taken phase contrast microscope picture was uploaded into the software, and the cells were counted using the counting function. Analysis was performed when 100 cells were counted to measure the cell density. Dunnett's multiple comparison test was used for testing. The results are represented as mean±standard deviation.
(Results)
When cells were suspended in 0.5, 1.0, and 1.5 ml of Bambanker hRM, the cell densities were all at the same level.
(C Phase Contrast Microscope Image)
Cryotubes cryopreserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 800 cells/mm2 in a culture plate coated with laminin-511 E8. The cell morphology and cell adhesion were observed after 1 day, 7 days, 14 days, and 28 days under a phase contrast microscope.
(Results)
(A Cell Survival Rate)
To evaluation the feasibility of a cryopreservation protocol, cells of clinically usable quality with a cell density of 2000 cells/mm2 or greater and at passage 3 to 5 were used as the clinically used cells to study the effect of Bambanker hRM on cell preservation. The medium was removed from the culture dish where human corneal endothelial cells were being cultured, and 1×PBS(−) (Nippon Flour Mills Co., Ltd., 05913) preheated to 37° C. was added to wash the cells. This was repeated twice. 1×PBS (−) was added again, and the cells were incubated for 3 minutes at 37° C. (5% CO2). After removing the PBS(−), TrypLE™ Select Enzyme (10×) (Thermo Fisher Scientific, A1217701) was added, and the cells were incubated for 15 minutes at 37° C. (5% CO2). The cells were then suspended in a medium. The cells were collected by centrifugation for 3 minutes at 280 G. After counting the cells, human corneal endothelial cells were centrifuged for 3 minutes at 280 G. After removing the supernatant, cryopreservation reagents were added (1 ml for 5×105 cells). The human corneal endothelial cells were then frozen at −80° C. by using a BICELL bio freezing vessel (Nihon Freezer Co., Ltd., BICELL) for 24 hours, and preserved in liquid nitrogen (−196° C.) for 13 more days. After 14 days of cryopreservation, human corneal endothelial cells were re-cultured from the frozen stock. Briefly, cryotubes preserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Dead cells were then stained for 10 minutes with 0.5%-Trypan Blue Stain solution (Nacalai Tesque, 29853-34). The viable cell count and the dead cell count were determined using a hemocytometer. The cell survival rate was calculated by dividing the viable cell count by the preserved cell count (5×105 cells) in cryopreserved cells. This was calculated by dividing the viable cell count by the sum of dead cell count and viable cell count in cells without cryopreservation. The statistical significance (p value) of the mean value in a two-sample comparison was determined by Student's t test. The results are represented as mean±standard deviation. Bambanker hRM was used as the preservation medium.
(Results)
The cell survival rate after cryopreservation was 83.2%. A significant decrease was not found in comparison to the 91.0% cell survival rate of cells without cryoperservation.
(B Cell Density)
Cryopreserved cryotubes were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 2500 cells/mm2 on a culture plate coated with laminin-511 E8. After washing the cells that have reached confluence on day 28 after seeding twice with 1×PBS(−), the cells were incubated for 3 minutes at 37° C. (5% CO2). A picture was then taken using a phase contrast microscope at a 200× magnification. The cell density was measured by using a measurement software, ImageJ software (National Institutes of Health). First, the taken phase contrast microscope picture was uploaded into the software, and the cells were counted using the counting function. Analysis was performed when 100 cells were counted to measure the cell density. Dunnett's multiple comparison test was used for testing. The results are represented as mean±standard deviation.
(Results)
(C Phase Contrast Microscope)
Cryotubes cryopreserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 2500 cells/mm2 on a culture plate coated with laminin-511 E8. The cell morphology and cell adhesion were observed after 1 day, 7 days, 14 days, and 28 days under a phase contrast microscope.
(Results)
(D Immunostaining)
Cells cultured on a 48-well plate were washed three times with 1×PBS (+) and fixed in 0.5% paraformaldehyde (PFA: WAKO, 163-20145) for 45 minutes. After removing the PFA, the cells were washed three times with 1×PBS (−). The cells were then permeabilized with 1% Triton X-100 (Nacalai Tesque, 287229-25) for 5 minutes. After removing the 1% Triton X-100, the cells were washed three times with 1×PBS (−). The cells were incubated with 2% bovine serum albumin (BSA: SIGMA-Aldrich, A9418-5G) for 30 minutes at 37° C. for blocking. The cells were shaken with primary antibodies for 45 minutes at 37° C. at 25 min−1. ZO-1 (1:200, invitrogen, 617300), N-cadherin (1: 300, BD Biosciences, 610921), and Na+/K+-ATPase (1:200, Merck Millipore, 05-369) were used as the primary antibodies. After removing the primary antibodies, the cells were washed three times with 1×PBS(−) and shaken for 5 minutes each at 25 min−1. Either Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, A21206) or Alexa Fluor® 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, A-11005) was diluted at 1:500 in BSA, and the samples were shaken with the secondary antibodies for 45 minutes at 37° C. at 25 minutes−1. Actin in samples was shaken with a 1:200 diluted Alexa Fluor® 546-conjugated Phalloidin (invitrogen, A22283), and nuclei in samples were shaken with 1:1000 diluted 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Dojindo Laboratories, D523) for 45 minutes at 37° C. at 25 minutes−1. After removing the secondary antibodies, the cells were washed three times with 1×PBS (−) and shaken for 5 minutes each at 25 min−1. After removing the 1×PBS(−), an antifading agent was added, and the samples were examined with a fluorescence microscope (Keyence, BZ-9000).
(Results)
In view of the above, the composition of the present disclosure was found to be capable of preservation without compromising the cell survival rate or cell density even at a cell density at a clinically applicable level (e.g., 2000 cells/mm2 or greater) of cell density.
0.36 g of sodium bicarbonate (Nacalai Tesque, 31213-15), 60 g of D-(+)-Glucose ≥99.5% (GC) (SIGMA-ALDRICH, G7021-100G), 0.72 g of HEPES (Nacalai Tesque, 17514-02), and 19.29 g of PBS (−) powder (Nippon Flour Mills Co., Ltd., 05913) were weighted using an analytical balance (SHIMADZU, AUW220D), and dissolved in ultrapure water to prepare a 500 ml solution. This is used as reagent (1).
Likewise, 0.5 g of calcium chloride (Nacalai Tesque, 06729-55) and 0.5 g of magnesium chloride hexahydrate (Nacalai Tesque, 20909-55) were each weighted using an analytical balance, and dissolved in ultrapure water to prepare a 500 ml solution. This is used as reagent (2).
Reagent (1) and reagent (2) were sterilized for 20 minutes at 121° C. with an autoclave. 12.5 ml of reagent (1) and 27.5 ml of sterilized water (Nacalai Tesque, 06442-95) were measured out and admixed. 5 ml of DMSO (Nacalai Tesque, 13408-64) was then added and admixed. Furthermore, 5 ml of reagent (2) was added and admixed. 0.5 g of bovine serum albumin (SIGMA-ALDRICH, A9418-5G) weighted with an analytical balance was added and admixed. Finally, the cell preservation medium was adjusted by filtration with Millex-GV, 0.22 μm and PVDF, 33 mm (Millipore, R7NA75775). The following Table 1 shows the reagents used.
(A Observation with Phase Contrast Microscope)
The medium was removed from the culture dish where human corneal endothelial cells were being cultured, and 1×PBS(−) (Nippon Flour Mills Co., Ltd., 05913) preheated to 37° C. was added to wash the cells. This was repeated twice. 1×PBS (−) was added again, and the cells were incubated for 3 minutes at 37° C. (5% CO2). After removing the PBS (−), TrypLE™ Select Enzyme (10×) (Thermo Fisher Scientific, A1217701) was added, and the cells were incubated for 15 minutes at 37° C. (5% CO2). The cells were then suspended in a medium. The cells were collected by centrifugation for 3 minutes at 280 G. After counting the cells, human corneal endothelial cells were centrifuged for 3 minutes at 280 G. After removing the supernatant, cryopreservation reagents (1 ml for 5×105 cells) were added. The human corneal endothelial cells were then frozen at −80° C. by using a BICELL bio freezing vessel (Nihon Freezer Co., Ltd., BICELL) for 24 hours, and preserved in liquid nitrogen (−196° C.) for 13 more days. Cryotubes cryopreserving human corneal endothelial cells were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 800 cells/mm2 on a culture plate coated with laminin-511 E8. The cell morphology and cell adhesion were observed after 1 day, 7 days, 14 days, and 28 days under a phase contrast microscope.
(Results)
(B Cell Density)
Cryopreserved cryotubes were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected in a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were suspended in the medium. Y-27632 was then added to the cell suspension, and the cells were seeded at a density of 800 cells/mm2 on a culture plate coated with laminin-511 E8. After washing the cells that have reached confluence on day 28 after seeding twice with 1×PBS(−), the cells were incubated for 3 minutes at 37° C. (5% CO2). A picture was then taken using a phase contrast microscope at a 200× magnification. The cell density was measured by using a measurement software, ImageJ software (National Institutes of Health). First, the taken phase contrast microscope picture was uploaded into the software, and the cells were counted using the counting function. Analysis was performed when 100 cells were counted to measure the cell density. The test used the Tukey-Kramer multiple comparison test. The results are represented as mean±standard deviation.
(Results)
In this Example, corneal endothelial cells preserved in accordance with the method of the present disclosure were transplanted into rabbit bullous keratopathy model.
(Materials and Methods)
(Culture)
Human corneal endothelial cells obtained from donor corneas provided by SightLife™ were used in the following experiment. All corneas were placed in Optisol (Chiron Vision) before use in the experiment and preserved at 4° C. for less than days. Descemet's membranes containing the corneal endothelium were stripped from the donor corneas, and the Descemet's membranes were digested for 12 hours at 37° C. with 1 mg/mL collagenase A (Roche Applied Science, 10 103 586 001). After washing three times with OptiMEM™-I (invitrogen, 31985-088), they were seeded in one well of a 48-well plate coated with laminin 511 E8 (iMatrix-511; Nippi, 892012). The culture medium was prepared as follows. Briefly, NIH-3T3 cells treated for 2 hours with 4 μg/mL of mitomycin C (Kyowa Hakkko Kirin, 4231400D1031) were seeded on a culture dish at a cell density of 2×104 cells/cm2. NIH-3T3 cells were cultured with a basal medium for 24 hours, and the basal medium was collected. The collected basal medium was filtered through a 0.22 μm filtration unit (Millipore, R7NA75775), and used for culturing human corneal endothelial cells after adding 5 ng/mL of epithelial growth factor (invitrogen, PHG0311), 0.1 mM ascorbic acid (SIGMA-Aldrich, PHR1008), 10 μM of 5B203580 (Cayman, 13067), and 1 μM of 5B431542 (WAKO, 192-16541) upon use.
Basal medium: Opti-MEM®-I (invitrogen, 31985-088)+8% FBS (Thermo, 5H30084.03)+5 ng/mL epithelial growth factor (invitrogen, PHG0311)+20 μg/mL ascorbic acid (SIGMA-Aldrich, PHR1008)+200 mg/L calcium chloride (SIGMA-Aldrich, 449709)+0.08% chondoroitin sulfate (WAKO, 032-14613)+50 μg/mL gentamicin (invitrogen, 15710-064).
(Cryopreservation of human corneal endothelial cells) The medium was removed from the culture dish where human corneal endothelial cells were being cultured, and 1×PBS(−) (Nippon Flour Mills Co., Ltd., 05913) preheated to 37° C. was added to wash the cells. This was repeated twice. 1×PBS (−) was added again, and the cells were incubated for 3 minutes at 37° C. (5% CO2). After removing the PBS (−), TrypLE™ Select Enzyme (10×) (Thermo Fisher Scientific, A1217701) was added, and the cells were incubated for 15 minutes at 37° C. (5% CO2). The cells were then suspended in a medium. The cells were collected by centrifugation for 3 minutes at 280 G. After counting the cells, human corneal endothelial cells were centrifuged for 3 minutes at 280 G. After removing the supernatant, Bambanker hRM (Nippon Genetics Co., Ltd., CS-07-001) (1 ml for 5×105 cells) were added. The human corneal endothelial cells were then frozen at −80° C. by using a BICELL bio freezing vessel (Nihon Freezer Co., Ltd., BICELL) for 24 hours, and preserved in liquid nitrogen (−196° C.) for 5 days. The cells were cryopreserved for 6 days.
(Preparation of Cells for Transplantation)
Human corneal endothelial cells were cryopreserved for 6 days using Bambanker hRM. Cryopreserved cryotubes were incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells were collected by pipetting twice with a basal medium preheated at 37° C. The human corneal endothelial cells were then centrifuged at 190 G for 5 minutes. The supernatant was removed, and the human corneal endothelial cells were resuspended in Opti-MEM®-I at a concentration of 5.0×105 cells/300 μl. Y-27632 (Wako Pure Chemical Industries, Ltd., 253-00513) was added so that the final concentration would be 100 μM.
(Transplantation Method)
Cryopreserved and cultured corneal endothelial cells were used for cultured corneal endothelial transplant using a rabbit bullous keratopathy model. A corneal endothelium of a rabbit was mechanically stripped with a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan) to create a bullous keratopathy model. 5.0×105 cultured human corneal endothelial cells initiated from a frozen state from the post-cryopreservation cultured human corneal endothelial cells created above, which were suspended in 300 μl of Opti-MEM®-I with an addition of Y-27632 resulting in the final concentration of 100 μM, were injected into the anterior chamber of a bullous keratopathy model. The model was laid face down for three hours.
(Histological Test)
Corneal tissue extracted from a rabbit was fixed in 4% paraformaldehyde (PFA: WAKO, 163-20145) for 10 minutes at room temperature (RT). The cells were then permeabilized with 1% Triton X-100 (Nacalai Tesque, 287229-25) for 5 minutes and blocked with 2% bovine serum albumin (BSA: SIGMA-Aldrich, A9418-5G) for 1 hour. To study the phenotype of the regenerated corneal endothelial tissue, ZO-1 and N-cadherin were used as markers related to the barrier function of cells, and Na+/K+-ATPase was used as a marker related to the pump function as the primary antibodies. For ZO-1, N-cadherin, and Na+/K+-ATPase staining, ZO-1 (1:300, Thermo Fisher Scientific, 33-9100), N-cadherin (1:300, BD Biosciences, 610921), and Na+/K+-ATPase (1:300, Merck Millipore, 05-369) were used, respectively. As the secondary antibodies, Alexa Fluor® 594-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, A-11005) was diluted to 1:1000 in BSA and used. For actin, a 1:400 diluted Alexa Fluor® 488-conjugated Phalloidin (invitrogen, A12379) was used. For nuclei, 1:1000 diluted 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI: Dojindo Laboratories, D523) was used. The samples were then examined with a confocal microscope (Leica Microsystems, DMI4000 B).
(Results)
This Example provides a cell formulation of corneal endothelial cells provided based on the present disclosure.
(Materials and Methods)
Human corneal endothelial cells are cryopreserved for 30 days using Bambanker hRM. Cryopreserved cryotubes are incubated in a 37° C. water bath for 1 to 2 minutes. Human corneal endothelial cells are collected by pipetting twice with a basal medium (e.g., Opti-MEM®-I or the like) preheated at 37° C. The human corneal endothelial cells are then centrifuged at 190 G for 5 minutes. The supernatant is removed, and the human corneal endothelial cells are resuspended in Opti-MEM®-I at a concentration of 5.0×105 cells/300 μl. Y-27632 (Wako Pure Chemical Industries, Ltd., 253-00513) is added so that the final concentration would be 100 μM.
(Use)
The cell formulation prepared in this Example can be used directly, and clinically applied after exchanging a solution with phosphate buffered saline.
This Example performs transplant therapy using a cell formulation of corneal endothelial cells of the present disclosure.
This Example uses the technology of Examples 1-5 or the cell formulation prepared as in Example 6.
A corneal endothelium of a bullous keratopathy patient is mechanically stripped with a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan) in the range of about an 8 mm diameter. The cryopreserved cultured human corneal endothelial cells prepared in Example 6 are injected in the anterior chamber using a 26 G needle, and laid face down for three hours.
The effect of the cultured human corneal endothelial cells is studied using any indicator that is known in the art. For example, the effect can be confirmed using an approach such as measurement of the cell density or vision examination.
As disclosed above, the present disclosure is exemplified by the use of its preferred embodiments. However, it is understood that the scope of the present disclosure should be interpreted based solely on the Claims. It is also understood that any patent, any patent application, and any references cited herein should be incorporated herein by reference in the same manner as the contents are specifically described herein.
The present application claims priority to Japanese Patent Application No. 2018-163238 filed on Aug. 31, 2018. The entire content thereof is incorporated herein by reference.
A composition for preserving corneal endothelial cells, or culturing the cells after preservation, is provided. The composition of the present disclosure can preserve ocular cells at a high cell survival rate and/or cell density. Since ocular cells preserved in such a manner can be used in cell transplant or the like, ocular cells can be utilized in the field of drug development or the like.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-163238 | Aug 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/034176 | 8/30/2019 | WO | 00 |
