Patent Description:
It has been reported that approximately <NUM> million people worldwide have both eye blindness, and <NUM> million of them have become blindness due to a corneal disease. The cornea is a structure located at the most anterior part of the eyeball, accounts for approximately <NUM>/<NUM>th of the anterior surface area of the eyeball, and has a size of approximately <NUM> and a thickness of generally <NUM>. The cornea is thinnest in the center and becomes thicker toward the periphery. In addition, the cornea is transparent tissue, which plays a major role in the refraction and transmission of light and responds sensitively to a foreign substance due to well-developed nerves. The cornea consists of five layers such as the corneal epithelium, Bowman's membrane, corneal stroma, posterior limiting membrane (Descemet's membrane) and corneal endothelium. There are no blood vessels in a normal cornea, and oxygen is provided from air via tears, and nutrients are provided from front aqueous humor and limbus behind the cornea.

The corneal epithelium accounts for approximately <NUM>% of the total thickness of the cornea, extends to the limbal and conjunctival epithelium, and consists of <NUM> to <NUM> layers of cells. Bottom cells of the lowermost layer are proliferated and come out of the surface of the cornea, and after <NUM> to <NUM> days, the cells are detached. The Bowman's membrane is a membrane consisting of acellular, colorless and transparent fibrils. When once damaged, this membrane is not regenerated. The corneal stroma accounts for approximately <NUM>% of the corneal thickness, mostly consists of collagen fibers, has a uniform size and direction, and is transparent. In addition, the posterior limiting membrane is a thick basement membrane secreted in endothelial cells, and when a person becomes older, the thickness of this membrane is increased. The corneal endothelial cells are flat cuboidal monolayer cells, which are not regenerated after birth.

The survival of the corneal epithelial cells is maintained by stem cells present in the limbus, and limbal stem cells are monofunctional adult stem cells that can differentiate only into the corneal epithelium. In limbal stem cell deficiency, the reduction in migration of basal corneal epithelial cells to the surface and migration of peripheral corneal epithelial cells to the center and the increase in detachment of the corneal epithelial cells, rather than the proliferation thereof, prevent the regeneration of the corneal epithelium. As a result, the conjunctival epithelial cells penetrate the cornea through the limbus, which is called conjunctivalization. Despite a new environment, conjunctival epithelial cells maintain a unique phenotype, and corneal neovascularization occurs, resulting in corneal opacity and blindness.

When the cornea becomes opaque resulting from an inability to maintain transparency due to trauma, severe inflammation or congenital reasons, although all other functions of the eye including the optic nerve are normal, a patient has serious visual impairment. In the case of difficulty in treatment of such opacity with a medication or laser, the cornea needs to be removed and then replaced with a transparent cornea obtained from a donated eye to facilitate the entry of light into the eye, which is accomplished by corneal transplantation. It can be expected that vision recovery caused by such a lesion of the cornea itself is achieved by corneal transplantation, and in the case of conjunctivalization and opacity of the cornea caused by limbal stem cell deficiency, sight restoration can only be expected by successful transplantation of limbal stem cells. Therefore, in this case, since there is no alternative but stem cell transplantation, the corneal opacity and blindness caused by limbal stem cell deficiency are classified as an intractable disease.

A limbal stem cell-deficient disease is a disease that is caused by extensive damage to the limbus due to genetic factors, or acquired factors such as trauma, infection, UV damage, surgical damage, complications caused by wearing of contact lenses and systemic diseases, and thus deficiency of limbal stem cells that can continuously regenerate the corneal epithelium, and the number of patients with limbal stem cell deficiency is continuously increasing because of unreasonable wearing of contact lenses including unapproved cosmetic colored contact lenses, increased prevalence and severity of dry eye, and increased use of toxic eyedrops, in addition to the above-listed causes.

Autologous or allogeneic limbal tissue transplantation has been used as a method for treating limbal stem cell deficiency. For the autologous limbal tissue transplantation, a method for transplanting the same size of limbal tissue extracted from the opposite eye has been used, but a limbal stem cell-deficient disease may occur in the opposite eye. However, in the case of allogeneic limbal tissue transplantation donated from a corpse, patients are suffering from side effects due to the continuous use of a systemic immunosuppressant, and even when a systemic immunosuppressant is used, there is a frequently-occurring problem in that a considerable proportion of stem cells die due to rejection of limbal grafts. Likewise, the limbal stem cell-deficient disease is continuously increasing, but effective and less complicated treatment methods for patients are not properly developed, and therefore there is a need to develop new technologies that increase the success rate of treatment and achieve optimal therapeutic effects.

Specifically, limbal stem cells present in the limbus, which is the contact site of the cornea and conjunctiva, play a pivotal role in regenerating the corneal epithelium while maintaining and repairing the corneal epithelium. However, since the number of the limbal stem cells is less than <NUM>%, and the cells are buried in the basal membrane of the multilayer limbal epithelium, it is very difficult to isolate the cells. To solve such a problem, methods for isolating multifunctional stem cells from the corneal limbus and culturing the cells have been reported (<CIT>), but there is no effective method that can be used in clinical and practical uses.

<CIT> relates to a tissue system with self-regenerating limbal stem cells, wherein the limbal stem cells are primarily undifferentiated stem cells (USCs).

<NPL> describes a method for the standardized generation of a limbal epithelial stem cell graft.

For this reason, the inventors had continuously conducted a study on an effective method for ex vivo expansion of limbal stem cells, and thus completed the present invention.

Therefore, the present invention is directed to providing a method for effectively increasing the proportion of limbal stem cells in a limbal tissue-derived epithelial cell sheet, so that the success rate of transplantation is maximized by transplanting the limbal tissue-derived epithelial cell sheet into a patient with reduced vision and blindness due to limbal stem cell deficiency.

However, technical problems to be solved in the present invention are not limited to the above-described problems, and other problems which are not described herein will be fully understood by those of ordinary skill in the art from the following descriptions.

To achieve the object of the present invention, the present invention provides a method for culturing limbal stem cells from limbal tissue, comprising: covering a slide glass with an epithelial cell-removed amniotic membrane to obtain a slide glass scaffold for fixation; and culturing limbal tissue on the fixed amniotic membrane, wherein the limbal tissue is cultured in DMEM/F12(<NUM>:<NUM>) supplemented with human serum added at <NUM> to <NUM>%(v/v) of the entire medium, an epithelial cell growth factor (EGF), dimethyl sulfoxide (DMSO), insulin transferrin selenium (ITS) and O-phosphoethanolamine.

In an embodiment of the present invention, each of the width and the length of the amniotic membrane is <NUM> to <NUM>.

In another embodiment of the present invention, epithelial cells of the amniotic membrane were removed using <NUM> to <NUM> urea.

In still another embodiment of the present invention, each of the width and the length of the slide glass is <NUM> to <NUM>.

In yet another embodiment of the present invention, the limbal tissue is cultured for <NUM> to <NUM> days.

In yet another embodiment of the present invention, the limbal tissue-derived epithelial cell sheet is grown to <NUM> to <NUM>% of the area of the scaffold, and then is classified as a transplant for a patient.

In yet another embodiment of the present invention, the human serum is human AB serum.

When an amniotic membrane slide scaffold and a specific culture condition according to the present invention are used, the proportion of limbal stem cells in a limbal tissue-derived epithelial cell sheet can be stable and rapidly increased. Therefore, the success rate of transplantation can be increased when the limbal tissue-derived epithelial cell sheet is transplanted into a limbal stem cell-deficient patient.

In addition, in the present invention, the proportion of limbal stem cells effective for compatibility in transplantation can be confirmed.

The present invention relates to a method for effectively proliferating and culturing the proportion of limbal stem cells in a limbal tissue-derived epithelial cell sheet by culturing limbal tissues on a slide scaffold fixed to an amniotic membrane under a specific culture condition.

The limbus is a circular region having a width of approximately <NUM> between the cornea and the sclera, includes many blood vessels in a matrix underlying multilayer epithelial cells, and is involved in metabolism and integration of the cornea. Particularly, since limbal stem cells (ABCG2 positive and p63α positive) in the limbal epithelium are the source of the corneal epithelial cells, when limbal tissue is transplanted into a patient with a limbal stem cell deficient disease, corneal epithelial cells are differentiated from limbal stem cells distributed in a limbal epithelium basal layer and migrate to the corneal center, thereby forming a multilayer corneal epithelium.

Therefore, the inventors had conducted a study on a method for increasing a transplantation success rate by dividing circular limbal tissue of a donor, which remains after corneal transplantation, into <NUM> pieces, and culturing limbal stem cells in a limbal tissue-derived epithelial cell sheet to contain the limbal stem cells at a predetermined ratio or more from limbal tissue using a slide with an amniotic membrane scaffold, resulting in completion of the present invention.

First, limbal tissue is preferably cultured on an amniotic membrane scaffold, following the division of the circular limbus of a donor into <NUM> pieces by surgery to so as to have a size of <NUM>×<NUM> (major axis × length). The amniotic membrane scaffold is prepared by covering a fixable scaffold with an amniotic membrane. Any plate-type scaffold that can fix the amniotic membrane may be used, and particularly, a slide glass is preferably used.

Here, the amniotic membrane may have a size of <NUM> to <NUM> (width × length), and particularly, an amniotic membrane in which each of the width and length is <NUM> or more is most preferable because, considering the size of a human cornea (approximately <NUM>×<NUM>), limbal tissue with a most suitable size for transplantation can be cultured thereon.

The slide glass having a size of <NUM> × <NUM> (width × length) is most suitable for being stably fixed to the center of a <NUM> culture dish after being covered with the preferable size of amniotic membrane.

The amniotic membrane slide scaffold is located in the center of the <NUM> culture dish, limbal tissue is settled in the middle of the amniotic membrane slide scaffold, and incubated in a culture dish containing a culture medium in a CO<NUM> incubator at <NUM>. Here, the limbal tissue is divided into <NUM> pieces each having a major axis length of <NUM> to <NUM>, which is preferable to increase the proportion of the limbal stem cells in the limbal tissue-derived epithelial cell sheet when being cultured in the middle of the amniotic membrane scaffold.

A culture medium is DMEM/F12(<NUM>:<NUM>) supplemented with human serum added at <NUM> to <NUM>% (v/v) of the entire medium, an epithelial cell growth factor (EGF), dimethyl sulfoxide (DMSO), insulin transferrin selenium (ITS) and O-phosphoethanolamine. Unless particularly described otherwise, the present invention uses DMEM/F-<NUM> (<NUM>:<NUM>) supplemented with EGF, DMSO, ITS, O-phosphoethanolamine and human AB serum (<NUM>%).

A culture medium of the limbal tissue is preferably changed every <NUM> to <NUM> days, and the limbal tissue is grown until a limbal tissue-derived epithelial cell sheet formed through cell division accounts for <NUM> to <NUM>% of the amniotic membrane scaffold to reach <NUM><NUM> to <NUM><NUM>, which is suitable in consideration of the size of the human cornea. A period in which the limbal tissue-derived epithelial cell sheet accounts for <NUM> to <NUM>% of the amniotic membrane scaffold is approximately <NUM> to <NUM> days, and generally a <NUM>-day culture period is needed.

Since the proportion of limbal stem cells present in a limbal tissue-derived epithelial cell sheet is very important for increasing a success rate after transplantation, as a result of confirming the optimal proportion of stem cells in the present invention, it was revealed that at least <NUM>% (<NUM>± <NUM>) stem cells are present in an epithelial cell sheet to be transplanted. Here, the proportion of limbal stem cells may be confirmed as a <NUM>,<NUM>',<NUM>,<NUM>'-tetrachloro-<NUM>,<NUM>',<NUM>,<NUM>'-tetraethylbenzimidazol-carbocyanine iodide (JC-<NUM>) low population, sternness of the limbal stem cells may be identified by colony forming efficiency (CFE) and western blotting for limbal stem cells-positive markers (p63α and ABCG2), but the present invention is not limited thereto.

In addition, when a specific composition of culture medium and the amniotic membrane slide scaffold according to the present invention are used (with slide), compared with other conditions (TW, without PVDF, with PVDF, and with ring), it can be confirmed that a faster cell proliferation rate and a higher proportion of limbal stem cells are exhibited (refer to <FIG>).

Hereinafter, to help in understanding the present invention, exemplary examples will be suggested. However, the following examples are merely provided to more easily understand the present invention, and not to limit the present invention.

Amniotic membrane tissue (<NUM> × <NUM>) that had been provided from a human tissue bank and then cryopreserved at -<NUM> was left at room temperature for <NUM> minutes, and then epithelial cells of the amniotic membrane were removed by treating <NUM> urea for <NUM> minutes. A slide glass having a size of <NUM> × <NUM> is covered with the epithelial cell-removed amniotic membrane, and then the slide glass was put into a <NUM> culture dish and tightly fixed.

To extract limbal tissue from a corpse after corneal transplantation surgery had been finished, the limbal tissue was divided into <NUM> pieces each having a size of approximately <NUM> × <NUM> using a surgical tool, and the peripheral cornea and conjunctiva were dissected and removed. Afterward, the extracted limbus was put on the amniotic membrane slide scaffold prepared in Example <NUM> and cultured for approximately <NUM> days at <NUM> in a CO<NUM> incubator. In the incubation, a culture medium was changed every <NUM> to <NUM> days, and when a limbal tissue-derived epithelial cell sheet accounted for <NUM> to <NUM>% or more of the slide area, it was transplanted into a patient.

Here, the culture medium was a supplemented human epithelium medium (SHEM) which is conventionally and generally used to culture limbal tissue, and its composition includes EGF, DMSO, ITS, O-phosphoethanolamine, CT and FBS in DMEM:F-<NUM>(<NUM>:<NUM>).

To comparatively measure the proportions of limbal stem cells in limbal tissue-derived epithelial cell sheets, cells grown to <NUM>% or more on an amniotic membrane slide scaffold and a transwell (control group) were treated with <NUM>/ml of a dispase II solution at <NUM> overnight. The limbal tissue-derived epithelial cell sheet was isolated from the amniotic membrane using fine forceps, treated with <NUM> of trypLE for <NUM> minutes, and centrifuged at <NUM> rpm for <NUM> minutes, thereby obtaining a cell precipitate. The cell precipitate was suspended in SHEM, and then cells were counted to measure a JC-<NUM> low population and CFE, and seeded in a <NUM>-well plate. To measure the JC-<NUM> low population, the cells were seeded at <NUM> × <NUM><NUM> cells/ml, cultured in SHEM for <NUM> hours and treated with a JC-<NUM> dye for <NUM> hour, followed by measuring a JC-<NUM> low cell population, which are side population cells, using FACS. Meanwhile, to confirm colony formation, the cells were cultured in a CNT50 medium (Cellntec) for <NUM> days, and the number of colonies was counted, thereby confirming CFE.

Consequently, as confirmed in <FIG>, when the amniotic membrane slide scaffold prepared according to the present invention was used, a proliferation rate of the cells was significantly increased, compared to the transwell, which is the control group, and the JC-<NUM> low population, which represents the proportion of stem cells included in the limbal tissue, CFE and expression of stem cell markers (p63α and ABCG2) were also significantly increased.

Conventionally, CT and FBS were included in SHEM which has been generally used to culture limbal tissue. However, the cultured limbal tissue is for use in human transplantation, and therefore, in order to exclude an animal component (FBS) and a toxicity component (CT) in the culture, a culture experiment was carried out with the medium compositions shown in Table <NUM>.

Consequently, as confirmed in <FIG>, in Experimental Group <NUM> from which CT was removed, the proportion of limbal stem cells was slightly reduced, and in Experimental Group <NUM> using <NUM>% human albumin serum (AB serum) instead of <NUM>% animal serum (FBS), the proportion of limbal stem cells and expression of stem cell markers were significantly increased. Meanwhile, in Experimental Group <NUM> in which the ratio of a human AB serum was increased to <NUM>%, compared to the case of adding <NUM>% human AB serum, the proportion of limbal stem cells in a limbal tissue-derived epithelial cell sheet was decreased to the level of the control group. Therefore, it can be seen that the medium condition in Experimental Group <NUM> was most preferable.

To compare the proportions of limbal stem cells in various scaffolds, on day <NUM>, <NUM>, <NUM>, <NUM> and <NUM> of culturing, cell proliferation areas and the proportions of limbal stem cells were measured in the cases of the transwell (TW), the amniotic membrane not attached to a PVDF membrane (w/o PVDF), the amniotic membrane attached to a PVDF membrane (with PVDF), the amniotic membrane fixed with a ring (with ring) as the control groups, and the amniotic membrane attached to the slide glass of the present invention (with slide).

This experiment was carried out in the same manner as used in Example <NUM>, except that the medium condition for Experimental Group <NUM> described in Example <NUM>, instead of SHEM, was used.

Consequently, as confirmed in <FIG>, when the amniotic membrane slide scaffold prepared according to the present invention was used, compared with the control groups, a cell proliferation rate was considerably increased (<FIG>), and as a result of observing a JC-<NUM> low population and flow cytometry for a limbal stem cell marker (p63α(+))(<FIG> and <FIG>), it can be seen that the amniotic membrane slide scaffold was most preferable.

Claim 1:
A method for culturing limbal stem cells from limbal tissue, comprising:
covering a slide glass with an epithelial cell-removed amniotic membrane to obtain a slide glass scaffold for fixation; and
culturing limbal tissue on the fixed amniotic membrane,
wherein the limbal tissue is cultured in DMEM/F12(<NUM>:<NUM>) supplemented with human serum added at <NUM> to <NUM>%(v/v) of the entire medium, an epithelial cell growth factor (EGF), dimethyl sulfoxide (DMSO), insulin transferrin selenium (ITS) and O-phosphoethanolamine.