Composition of corneal implantation, and the use and preparation method thereof

Abstract

The present application provides a composition of corneal implantation, comprising: a collagen film, and renal proximal tubule cells which attached to the collagen film. In addition, the present application further provides a use of the composition of corneal implantation for implanting a patient with damaged cornea endothelium cells and a preparation method of the composition of corneal implantation.

Claims

1. A composition of corneal implantation, comprising: a collagen film, and renal proximal tubule cells, attached to the collagen film.

2. The composition of claim 1, wherein the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.4 cells/cm.sup.2 to 5×10.sup.6 cells/cm.sup.2.

3. The composition of claim 1, wherein the diameter of the collagen film is about 6 mm to 10 mm, the thickness is about 120 μm to 200 μm.

4. The composition of claim 1, wherein the diameter of the collagen film is about 8 mm, the thickness is about 160 μm, and the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.5 cells/cm.sup.2.

5. A use of the composition according to claim 1 for implanting in patients with damaged corneal endothelial cells.

6. The use of claim 5, wherein the renal proximal tubule cells are autologous renal proximal tubule cells of the patients.

7. The use of claim 5, wherein the use is applied in corneal endothelial transplantation.

8. The use of claim 5, wherein the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.4 cells/cm.sup.2 to 5×10.sup.6 cells/cm.sup.2.

9. The use of claim 5, wherein the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.4 cells/cm.sup.2 to 5×10.sup.6 cells/cm.sup.2.

10. The use of claim 5, wherein the diameter of the collagen film is about 8 mm, the thickness is about 160 μm, and the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.5 cells/cm.sup.2.

11. A preparation method of the composition according to claim 1, comprising the following steps: providing renal proximal tubule cells of a patient, seeding the renal proximal tubule cells into a cell culture container with a collagen film placed at the bottom, and cultivating for about 5-10 days to obtain the composition for corneal implantation.

12. The preparation method of claim 11, wherein the renal proximal tubule cells are obtained from the expanded culture of the kidney tissue of the patient.

13. The preparation method of claim 11, wherein about 5×10.sup.4 of the renal proximal tubule cells are seeded into the cell culture container with the collagen film placed at the bottom, and the density of the cells on the collagen film reaches about 5×10.sup.5 cells/cm.sup.2 after cultivating for 7 days.

14. The preparation method of claim 11, wherein the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.4 cells/cm.sup.2 to 5×10.sup.6 cells/cm.sup.2.

15. The preparation method of claim 11, wherein the diameter of the collagen film is about 6 mm to 10 mm, the thickness is about 120 μm to 200 μm.

16. The preparation method of claim 11, wherein the diameter of the collagen film is about 8 mm, the thickness is about 160 μm, and the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.5 cells/cm.sup.2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and other aspects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0022] FIG. 1A shows the result of test 1 of day 0 after implanting.

[0023] FIG. 1B shows the result of test 1 of day 7 after implanting.

[0024] FIG. 1C shows the result of test 1 of day 30 after implanting.

[0025] FIG. 1D shows the result of test 1 of day 60 after implanting.

[0026] FIG. 1E shows the result of test 1 of day 90 after implanting.

[0027] FIG. 2A shows the result of test 2 of day 0 after implanting.

[0028] FIG. 2B shows the result of test 2 of day 3 after implanting.

[0029] FIG. 2C shows the result of test 2 of day 7 after implanting.

[0030] FIG. 2D shows the result of test 2 of day 30 after implanting.

[0031] FIG. 2E shows the result of test 2 of day 60 after implanting.

[0032] FIG. 2F shows the result of test 2 of day 90 after implanting.

[0033] FIG. 3A shows the result of test 3 of day 0 after implanting.

[0034] FIG. 3B shows the result of test 3 of day 3 after implanting.

[0035] FIG. 3C shows the result of test 3 of day 7 after implanting.

[0036] FIG. 3D shows the result of test 3 of day 30 after implanting.

[0037] FIG. 3E shows the result of test 3 of day 60 after implanting.

[0038] FIG. 3F shows the result of test 3 of day 90 after implanting.

[0039] FIG. 4A shows the result of test 4 of day 0 after implanting.

[0040] FIG. 4B shows the result of test 4 of day 3 after implanting.

[0041] FIG. 4C shows the result of test 4 of day 7 after implanting.

[0042] FIG. 4D shows the result of test 4 of day 30 after implanting.

[0043] FIG. 4E shows the result of test 4 of day 60 after implanting.

[0044] FIG. 4F shows the result of test 4 of day 90 after implanting.

[0045] FIG. 5A shows the ZO-1 staining result of test 1.

[0046] FIG. 5B shows the Na—K ATPase staining result of test 1.

[0047] FIG. 5C shows the N-cadherin staining result of test 1.

[0048] FIG. 5D shows the negative control group of test 1.

[0049] FIG. 6A shows the Na—K ATPase staining result of test 2.

[0050] FIG. 6B shows the N-cadherin staining result of test 2.

[0051] FIG. 6C shows the ZO-1 staining result of test 2.

[0052] FIG. 6D shows the GLUT1 staining result of test 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0053] Reference will be made in detail description to the exemplary embodiments and drawings for being more readily understood to the advantages and features of the present invention, as well as the methods of attaining them. However, the present invention may be carried out in many different forms and should not be construed as limited to the embodiments set forth herein. Conversely, these embodiments are provided to render the present disclosure to be conveyed the scope of the present invention more thoroughly, completely, and fully to one having ordinary skill in the art of the present invention. Moreover, the present invention would be defined only by the appended claims. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed components.

[0054] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as generally understood by one having ordinary skill in the art of the present invention. It will be more understandable that, for example, the terms defined in commonly used dictionaries should be understood to have meanings consistent with the contents of the relevant fields, and would not be interpreted overly idealized or overly formal unless clearly defined herein. As described in the present specification, a range of values is used as a shorthand to describe each and every numerical value in the range, and any number within that range may be chosen as the end-value of that range.

[0055] In order to make the disclosed content more concise and easy to understand, the following elements with the same or similar functions will be described with the same symbols, and descriptions of the same or equivalent features will be omitted.

[0056] The present invention provides a composition of corneal implantation, comprising: a collagen film, and renal proximal tubule cells, attached to the collagen film.

[0057] In an embodiment, the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.4 cells/cm.sup.2 to 5×10.sup.6 cells/cm.sup.2.

[0058] In an embodiment, the diameter of the collagen film is about 6 mm to 10 mm, the thickness is about 120 μm to 200 μm.

[0059] In an embodiment, the diameter of the collagen film is about 8 mm, the thickness is about 160 μm, and the density of the renal proximal tubule cells on the collagen film is about 5×10.sup.5 cells/cm.sup.2.

[0060] In addition, the present invention provides a use of the composition of corneal implantation for implanting in patients with damaged corneal endothelial cells. Preferably, the renal proximal tubule cells are autologous renal proximal tubule cells of the patients. Preferably, the use is applied in corneal endothelial transplantation.

[0061] Besides, the present invention provides a preparation method of the composition of corneal implantation, comprising the following steps:

[0062] providing renal proximal tubule cells of a patient,

[0063] seeding the renal proximal tubule cells into a cell culture container with a collagen film placed at the bottom, and

[0064] cultivating for about 5-10 days to obtain the composition for corneal implantation.

[0065] In an embodiment, the renal proximal tubule cells are obtained from the expanded culture of the kidney tissue of the patient.

[0066] In an embodiment, about 5×10.sup.4 of the renal proximal tubule cells are seeded into the cell culture container with the collagen film placed at the bottom, and the density of the cells on the collagen film reaches about 5×10.sup.5 cells/cm.sup.2 after cultivating for 7 days.

Example 1—Acquisition of Renal Proximal Tubule Cells

[0067] Human specimens were obtained from patients who had kidney tumors and were assessed by doctors to undergo nephrectomy. Without affecting the pathological diagnosis, specimens were collected from the kidney tissue during nephrectomy. The renal cortex was selected and the specimen was cut into the size of one cubic centimeter with the surgical scissor and forceps. 3 pieces of specimens were collected, stored in Hanks' Balanced Salt Solution (HBSS), and placed at 4° C. The specimens for animal experiments were obtained from the autologous kidneys of the animals in the same way.

[0068] The specimens were cut into small pieces after washing with PBS, which were put into an Eppendorf then cut into mud with small scissors. The specimens were placed in a 10 cm dish and disintegrated for 30 minutes at 37° C. by 60 mg of collagenase dissolved in 20 ml of Hank's Balanced Salt Solution (HBSS) containing Ca2.sup.+. After using a syringe to disperse pellets, all the liquid was sucked into a 50 ml centrifuge tube, then 30 ml of HBSS was added in and centrifuged at 1000 rpm for 4 minutes. After removing the supernatant, 10 ml of HBSS was added to disperse pellets and filtered through a 250 μm mesh and centrifuged at 1000 rpm for 4 minutes. The supernatant was removed again, and 30 ml of 45% Percoll solution (Pharamacia Biotech) was added to disperse pellets and centrifuged at 1000 rpm, 4° C., for 30 minutes.

[0069] Since the number of renal tubular cell layers is very small, after seeing the cell layering, a drop was taken and observed under the microscope to find the layer where the renal tubules are located. The renal tubule cells were placed in a 15 mL centrifuge tube, 10 mL of HBSS was added, and centrifuged at 800 rpm for 4 minutes. After removing the supernatant, 10 mL of cell culture medium (HPTC) was added to disperse the pellets. The renal proximal tubule cells were seeded in a collagen-coated 10 cm dish and were cultivated an incubator at 37° C., 5% CO.sub.2 for about 10 days to obtain about 7-9×10.sup.6 of renal proximal tubule cells. In addition, the expanded renal proximal tubule cells can be collected and subcultured with 0.05% of trypsin/EDTA.

Example 2—Preparation of Composition of Corneal Implantation

[0070] A collagen film with a diameter of 8 mm and a thickness of about 160 μm was washed twice with PBS, then placed on the bottom of the 48-well plate with an O-ring pressed above to prevent the film from floating. The renal proximal tubule cells obtained by the method of Example 1 at a number of 5×10.sup.4 cells were seeded into the wells containing the collagen films, which was cultivated in an incubator at 37° C., 5% CO.sub.2 for 7 days to obtain a collagen film attached with about 5×10.sup.5 cells/cm.sup.2 of renal proximal tubule cells.

Example 3—Animal Experiments

[0071] The composition of corneal implantation obtained by the method of Example 2 was implanted into the corneas of pigs by the standard operating method of the Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK). FIGS. 1A-1E to FIGS. 4A-4F show the results of tests 1 to 4 respectively, and the results of tests 1 to 4 are assessed the levels of transparency (1 to 4 points, 1 is the most blur, 4 is the most transparent), redness/swelling (1 to 3 points, 1 is the least red and swollen, 3 is the reddest and the most swollen), and attachment (0 to 2 points, respectively, no attachment, partial attachment, and full attachment) after implantation respectively. The assessment results are summarized as the following Table 1:

TABLE-US-00001 TABLE 1 Transparency Redness/Swelling Attachment Test 1 4 1 2 Test 2 4 1 2 Test 3 4 1 2 Test 4 3 1 2

[0072] In addition, the frozen sections of the corneas of Experiment 1 were subjected to immunofluorescence staining of ZO-1, Na/K ATPase, and N-cadherin to examine the viability of the renal proximal tubule cells. As shown in FIGS. 5A to 5C, the renal proximal tubule cells were still alive after 120 days of implantation. Besides, the frozen sections of the corneas of Experiment 2 were subjected to immunofluorescence staining of Na/K ATPase, N-cadherin, ZO-1, and GLUT1. As shown in FIGS. 6A-6D, it was also found that the renal proximal tubule cells were still alive after 120 days of implantation.

[0073] As can be seen from FIGS. 1A-1E to FIGS. 4A-4F and Table 1, the composition of corneal implantation of the present invention has good transparency, low redness and swelling, and can completely attach after implanting to the cornea. As shown in FIGS. 5A-5D to FIGS. 6A-6D, renal proximal tubule cells attached to the collagen film can survive and be active after implantation. At the same time, the collagen film can strengthen the attachment and will be gradually absorbed after implantation, leaving the renal proximal tubule cells to replace the corneal endothelial cells. Furthermore, there is no rejection problem since the autologous renal proximal tubule cells are used. Therefore, the composition of corneal implantation of the present invention can be used as a medical material of an autologous cell membrane to replace the original damaged corneal endothelial cells, thereby restoring the functionality of the corneal endothelial cells.