METHOD FOR PREPARING CORNEAL TISSUE

Abstract

The present invention relates to a method for preparing corneal tissue for applications predominantly in transplantation, and to the use of a solution for decellularizing corneal tissue. The present invention further relates to transplant corneal tissue

Claims

1. A method for preparing corneal tissue, comprising the following steps: decellularizing the tissue by way of a detergent that is gentle on the tissue in the presence of a polyalcohol.

2. The method according to claim 1, characterized in that the detergent for decellularization comprises a cyclic lipopeptide.

3. The method according to claim 1 or 2, characterized in that the detergent comprises surfactin, daptomycin, caspofungin, arthrofactin, an echinocandin, an iturin, a syringomycin, a syringopeptide and/or a polymyxin.

4. The method according to any one of the preceding claims, characterized in that the decellularization is carried out in the presence of a second decellularizing agent.

5. The method according to any one of the preceding claims, characterized by comprising the following step: treating the decellularized tissue with a DNase in the presence of a polyalcohol.

6. The method according to any one of the preceding claims, characterized by comprising the following step: treating the decellularized tissue with -galactosidase in the presence of a polyalcohol.

7. The method according to claim 6, characterized in that the -galactosidase is from green coffee beans or from Cucumis melo.

8. The method according to any one of the preceding claims, characterized in that the polyalcohol is selected from the group comprising or consisting of glycerol, 1,2-propanediol, 1,3-propanediol, polyethylene glycol, polypropylene glycol, ethylene glycol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,2,3-butanetriol, 1,2,4-butanetriol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 1,2,5-pentanetriol, 1,3,4-pentanetriol, 1,3,5-pentanetriol, 1,2,3,4-butanetetrol, preferably meso-1,2,3,4-butanetetrol, and further alditols including a linear C5 to C8 carbon chain.

9. Use of a solution comprising at least one cyclic lipopeptide and a polyalcohol as detergents for decellularizing corneal tissue the method comprising providing corneal tissue in need of decellularization, and exposing the corneal tissue to the solution.

10. The use according to claim 9, characterized in that the solution comprises glycerol as the polyalcohol.

11. Corneal tissue, obtainable by a method according to claims 1 to 8.

12. Transparent decellularized corneal tissue, comprising a polyalcohol.

13. The transparent decellularized corneal tissue according to claim 12, which on the surface is free of, or approximately free of -Gal epitopes.

14. The transparent decellularized corneal tissue according to claim 12 or 13, which is free of, or approximately free of, DNA.

Description

[0036] The invention shall be described in more detail hereafter based on exemplary embodiments according to the invention and by way of figures, and compared to samples not treated according to the invention.

[0037] FIG. 1 shows the comparison between the DNA content of native corneal tissue and corneal tissue that was subjected to a tissue preparation described herein.

[0038] The residual DNA content of the corneal tissue in (g/cornea) after tissue preparation is plotted on the y-axis of the diagram in FIG. 1. The samples of the original DNA content prior to the tissue preparation and after the tissue preparation according to a method proposed herein were compared. The DNA content forms a direct measure of the removal of cellular components from the biological tissue. As is apparent from FIG. 1, a tissue preparation according to the method proposed herein, carried out by way of example with surfactin, deoxycholic acid and a DNase here, results in a removal of DNA from the corneal tissue.

[0039] FIG. 2 shows corneal tissue (a) that was decellularized by a method according to the invention in the presence of a polyalcohol proposed herein. The corneal samples have an extremely high degree of transparency, which allows printed manner on the surface beneath the cornea sample to be identified without difficulty and to be read.

[0040] FIG. 3 shows corneal tissue (b) that was decellularized by a method not according to the invention in the absence of a polyalcohol proposed herein. The tissue is very cloudy and so opaque that printed matter on the surface beneath the cornea sample cannot even vaguely be identified.

[0041] FIG. 4 shows the use of -Gal epitopes on native tissue, decellularized tissue, and decellularized tissue treated with galactosidase. The information native is used for the concentration of -Gal epitopes on untreated tissue, while the information M86 initial represents the threshold value for the presence of no -Gal epitopes. In this way, a measuring system is predefined, which is bounded by the absorption values of a minimal/non-existent amount of -Gal epitopes (M86 initial) and a maximal amount of -Gal epitopes (native). It is apparent that the decellularization proposed herein can be used not only to produce transparent tissue, but also to achieve a significant reduction, or almost complete removal, of -Gal epitopes, whereby the likelihood of an immune response to transplanted cornea is minimized.

EXAMPLE 1

[0042] Lenticels (which is to say pieces of corneal tissue) are stored in cell culture medium, for example comprising or consisting of DMEM ((Dulbecco's Modified Eagle's Medium) or similarly acting solutions.

[0043] Afterwards, the cell culture medium is removed, and the lenticels are rinsed 3 times with 5 ml physiological NaCl solution comprising 40 wt. % glycerol at room temperature and under slight movement.

[0044] Thereafter, the samples are decellularized by treating them with the following solution for approximately 20 hours at 37 C.: 0.06 wt. % surfactin, 0.5 wt. % sodium deoxycholate and 40 wt. % glycerol in physiological NaCl.

[0045] The samples are rinsed 5 times with 5 ml NaCl plus 40 wt. % glycerol at room temperature and under slight movement and then stored in the refrigerator.

EXAMPLE 2

[0046] In one refinement of the present invention, samples from Example 1 are cleaned further enzymatically in a further treatment step. For this purpose, the lenticels are treated for 16 hours at 37 C. with 40 U/ml DNase I and 0.1 U/ml green coffee bean (GCB) -galactosidase in 50 mM HEPES (pH 6.0), 2 mM CaCl, 5 mM MgCl comprising 40 wt. % glycerol.

[0047] The samples are subsequently rinsed 5 times with 5 ml NaCl comprising 40 wt. % glycerol at room temperature and under slight movement.

[0048] For the determination of the DNA content of the samples, these are enzymatically treated with 15 U proteinase K in DPBS and finally measured.

[0049] For the determination of the remaining -Gal epitopes on the surface of the samples, these were determined with the aid of an M86 antibody-based immuno assay.

COMPARATIVE EXAMPLE

[0050] Lenticels are stored in a cell culture medium. The samples are divided. Individual samples are treated with 15 U/ml proteinase K in DPBS for analytical purposes and then subjected to a DNA determination.

[0051] Afterwards, the storage solution is removed from further samples, and the samples are rinsed 3 times with 5 ml physiological NaCl solution. Thereafter, the samples are decellularized for approximately 20 hours with 5 ml 0.06 wt. % surfactin and 0.5 wt. % deoxycholic acid. The comparative example was thus carried out in the absence of a polyalcohol as described herein.

[0052] For further analysis, the samples are treated with 15 U/ml proteinase K in PBS, rinsed 5 times with 8 ml NaCl, and furthermore treated for 16 hours with 40 U/ml DNase I.

[0053] The DNA content in the untreated lenticels was determined to be 2.42+/0.38 g/cornea. Treated lenticels, in contrast, no longer contained any measurable DNA.

[0054] The exemplary embodiments described here are intended to illustrate the invention. The number and/or design of the rinsing steps (in particular the concentrations and composition of the solution for rinsing or of the buffer solution) may be varied by a person skilled in the art within the scope of this person's expert knowledge.