Method for Culturing Limbal Stem Cells by Using Amniotic Membrane Slide Scaffold

Abstract

The present invention relates to a method for culturing limbal tissues on an amniotic membrane slide scaffold, thereby enabling the proportion of limbal stem cells in a limbal tissue-derived epithelial cell sheet to be effectively increased, and the same to be cultured. According to the present invention, the proportion of limbal stem cells in in a limbal tissue-derived epithelial cell sheet can be stably and rapidly increased, and thus the success rate can be increased when in limbal tissue-derived epithelial cell sheets are transplanted into a patient with limbal stem cell deficiency.

Claims

1. A method for culturing limbal stem cells, comprising: covering a slide glass scaffold with an epithelial cell-removed amniotic membrane for fixation; and culturing limbal tissue on the fixed amniotic membrane.

2. The method according to claim 1, wherein each of the width and length of the amniotic membrane is 28 to 35 mm.

3. The method according to claim 1, wherein epithelial cells of the amniotic membrane are removed using 4 to 6 M urea.

4. The method according to claim 1, wherein each of the width and length of the slide glass is 18 to 28 mm.

5. The method according to claim 1, wherein the limbal tissue is cultured in DMEM/F12(1:1) supplemented with human serum, an epithelial cell growth factor (EGF), dimethyl sulfoxide (DMSO), insulin transferrin selenium (ITS) and 0-phosphoethanolamine.

6. The method according to claim 5, wherein the human serum is a human albumin serum, and added at 4 to 6% (v/v) of the entire medium.

7. The method according to claim 1, wherein the limbal tissue is cultured for 10 to 14 days.

8. The method according to claim 7, wherein, when the limbal tissue is grown to 85 to 95% of the area of the scaffold, the limbal tissue is classified as a transplant for a patient.

Description

DESCRIPTION OF DRAWINGS

[0022] FIGS. 1A to 1E show cell morphology (FIG. 1A), a cell proliferation area (FIG. 1B), a JC-1 low population, which is the proportion of limbal stem cells, (FIG. 1C) and colony forming efficiency (CFE) (FIG. 1D), and a western blotting result for limbal stem cell markers (p63(+) and ABCG2(+)) (FIG. 1E) in an experimental group in which limbal tissues are cultured on an amniotic membrane slide scaffold prepared according to an exemplary embodiment of the present invention using a supplemented human epithelium medium (SHEM) prepared by adding EGF, DMSO, ITS, 0-phosphoethanolamine, cholera toxin (CT) and fetal bovine serum (FBS) to DMEM:F-12(1:1), and a control group in which limbal tissues are cultured on a transwell.

[0023] FIGS. 2A to 2C shows the comparison of a JC-1 low population, which is the proportion of limbal stem cells, (FIG. 2A), CFE (FIG. 2B), and a western blotting result for limbal stem cell markers (p63(+) and ABCG2(+)) (FIG. 2C) in Experimental Groups 1 to 3 in which limbal tissues are cultured using SHEM containing human albumin sera (5% and 10%) except CT and FBS on an amniotic membrane slide scaffold prepared according to an exemplary embodiment of the present invention (Control Group: SHEM used in FIG. 1 of the present invention).

[0024] FIGS. 3A to 3C show a cell proliferation area (FIG. 3A) and a JC-1 low population (FIG. 3B), and a flow cytometry result for a limbal stem cell marker (p63(+)) (FIG. 3C) in an experimental group (with slide) in which limbal tissues are cultured on an amniotic membrane slide scaffold prepared according to an exemplary embodiment of the present invention using SHEM (Experimental Group 2) containing 5% human albumin serum except CT and FBS, which is used in FIG. 2 of the present invention; and control groups in which limbal tissues are cultured on a transwell (TW), an amniotic membrane unattached to a PVDF membrane (w/o PVDF), an amniotic membrane attached to a PVDF membrane (with PVDF) and an amniotic membrane fixed with a ring (with ring).

MODES OF THE INVENTION

[0025] Hereinafter, the present invention will be described in detail.

[0026] 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.

[0027] The limbus is a circular region having a width of approximately 1 mm 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 p63 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.

[0028] 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 12 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.

[0029] First, limbal tissue is preferably cultured on an amniotic membrane scaffold, following the division of the circular limbus of a donor into 12 pieces by surgery to so as to have a size of 23 mm (major axislength). 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.

[0030] Here, the amniotic membrane may have a size of 28 to 35 mm (widthlength), and particularly, an amniotic membrane in which each of the width and length is 20 mm or more is most preferable because, considering the size of a human cornea (approximately 1212 mm), limbal tissue with a most suitable size for transplantation can be cultured thereon.

[0031] The slide glass having a size of 20 mm26 mm (widthlength) is most suitable for being stably fixed to the center of a 30 mm culture dish after being covered with the preferable size of amniotic membrane.

[0032] The amniotic membrane slide scaffold is located in the center of the 30 mm 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.sub.2 incubator at 37. Here, the limbal tissue is divided into 12 pieces each having a major axis length of 2 to 3 mm, 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.

[0033] A preferable culture medium is DMEM or DMEM/F-12 (1:1) supplemented with nutritional serum, for example, a serum or serum-based solution (e.g., knock-out serum or heat-inactivated human serum) providing nutrients effective in maintenance of growth and survival of cells, in addition to a growth factor. Here, the growth factor refers to a protein that binds to a cell surface receptor as a primary consequence of cell proliferation and differentiation activation. The growth factor that is used to culture limbal tissue is preferably selected from the group consisting of EGF, basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), insulin, sodium selenite, human transferrin, human leukemia inhibitory factor (hLIF), and a combination thereof. However, any suitable culture medium known to those of ordinary skill in the art may be used without limitation.

[0034] Unless particularly described otherwise, the present invention uses DMEM/F-12 (1:1) supplemented with EGF, DMSO, ITS, O-phosphoethanolamine and human albumin serum (5%).

[0035] A culture medium of the limbal tissue is preferably changed every 2 to 3 days, and the limbal tissue is grown until a limbal tissue-derived epithelial cell sheet formed through cell division accounts for 85 to 95% of the amniotic membrane scaffold to reach 250 mm.sup.2 to 300 mm.sup.2, 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 85 to 95% of the amniotic membrane scaffold is approximately 10 to 14 days, and generally a 12-day culture period is needed.

[0036] 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 30% (38.73.52) stem cells are present in an epithelial cell sheet to be transplanted. Here, the proportion of limbal stem cells may be confirmed as a 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1) low population, stemness of the limbal stem cells may be identified by colony forming efficiency (CFE) and western blotting for limbal stem cells-positive markers (p63a and ABCG2), but the present invention is not limited thereto.

[0037] 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 FIGS. 3A to 3C).

[0038] 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.

EXAMPLES

Example 1: Preparation of Amniotic Membrane Slide Scaffold

[0039] Amniotic membrane tissue (30 mm30 mm) that had been provided from a human tissue bank and then cryopreserved at 70 was left at room temperature for 30 minutes, and then epithelial cells of the amniotic membrane were removed by treating 5M urea for 5 minutes. A slide glass having a size of 20 mm26 mm is covered with the epithelial cell-removed amniotic membrane, and then the slide glass was put into a 35 mm culture dish and tightly fixed.

Example 2: Culture of Limbal Stem Cells Using Amniotic Membrane Slide Scaffold

[0040] To extract limbal tissue from a corpse after corneal transplantation surgery had been finished, the limbal tissue was divided into 12 pieces each having a size of approximately 2 mm3 mm 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 1 and cultured for approximately 12 days at 37 in a CO.sub.2 incubator. In the incubation, a culture medium was changed every 2 to 3 days, and when a limbal tissue-derived epithelial cell sheet accounted for 85 to 90% or more of the slide area, it was transplanted into a patient.

[0041] 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, 0-phosphoethanolamine, CT and FBS in DMEM:F-12(1:1).

Example 3: Comparative Analysis of Proportions of Limbal Stem Cells on Amniotic Membrane Slide Scaffold and Transwell

[0042] To comparatively measure the proportions of limbal stem cells in limbal tissue-derived epithelial cell sheets, cells grown to 90% or more on an amniotic membrane slide scaffold and a transwell (control group) were treated with 2 mg/ml of a dispase solution at 4 overnight. The limbal tissue-derived epithelial cell sheet was isolated from the amniotic membrane using fine forceps, treated with 1 ml of trypLE for 5 minutes, and centrifuged at 1000 rpm for 5 minutes, thereby obtaining a cell precipitate. The cell precipitate was suspended in SHEM, and then cells were counted to measure a JC-1 low population and CFE, and seeded in a 6-well plate. To measure the JC-1 low population, the cells were seeded at 1.010.sup.5 cells/ml, cultured in SHEM for 24 hours and treated with a JC-1 dye for 1 hour, followed by measuring a JC-1 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 14 days, and the number of colonies was counted, thereby confirming CFE.

[0043] Consequently, as confirmed in FIGS. 1A to 1E, 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-1 low population, which represents the proportion of stem cells included in the limbal tissue, CFE and expression of stem cell markers (p63a and ABCG2) were also significantly increased.

Example 4: Comparison of Efficiencies According to Medium Composition

[0044] 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 1.

TABLE-US-00001 TABLE 1 Experimental Control Group 1 Experimental Group 2 Experimental Group 3 SHEM SHEM without SHEM with human SHEM with human CT albumin serum albumin serum (5%) without CT (10%) without CT

[0045] Consequently, as confirmed in FIGS. 2A to 2C, in Experimental Group 1 from which CT was removed, the proportion of limbal stem cells was slightly reduced, and in Experimental Group 2 using 5% human albumin serum (AB serum) instead of 5% animal serum (FBS), the proportion of limbal stem cells and expression of stem cell markers were significantly increased. Meanwhile, in Experimental Group 3 in which the ratio of a human albumin serum was increased to 10%, compared to the case of adding 5% human albumin 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 2 was most preferable.

Example 5: Comparative Analysis of Proportions of Limbal Stem Cells in Various Scaffolds

[0046] To compare the proportions of limbal stem cells in various scaffolds, on day 5, 7, 9, 12 and 14 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).

[0047] This experiment was carried out in the same manner as used in Example 3, except that the medium condition for Experimental Group 2 described in Example 4, instead of SHEM, was used.

[0048] Consequently, as confirmed in FIGS. 3A to 3C, 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. 3A), and as a result of observing a JC-1 low population and flow cytometry for a limbal stem cell marker (p63(+))(FIGS. 3B and 3C), it can be seen that the amniotic membrane slide scaffold was most preferable.

[0049] It should be understood by those of ordinary skill in the art that the above description of the present invention is exemplary, and the exemplary embodiments disclosed herein can be easily modified into other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the exemplary embodiments described above should be interpreted as illustrative and not limited in any aspect.

INDUSTRIAL APPLICABILITY

[0050] A method for culturing limbal stem cells using an amniotic membrane slide scaffold according to the present invention may allow the proportion of the limbal stem cells in a limbal tissue-derived epithelial cell sheet to be stably and rapidly increased, and thus this method is expected to be useful in treatment of a patient with limbal stem cell deficiency through limbal stem cell transplantation.