PROCEDURE FOR LONG TERM CORNEAL CULTURE
First Claim
1. A method for mammalian corneal excision from whole globe eyes, comprising:
- procuring enucleated whole globe mammalian eyes from an abattoir and transporting said eyes on ice in an isotonic buffered saline solution supplemented with an anti-fungal drug;
incubating whole globe eyes in a broad spectrum antiseptic for approximately 2 minutes in a sterile field;
briefly rinsing said eyes with an isotonic buffered saline solution, and then incubating said eyes with an amino glycoside antibiotic in dPBS for 15 minutes;
excising the cornea from the eye with a scalpel using sterile technique, by making an incision 2-3 mm from the cornea into the sclera and cutting at this same distance all around the cornea until the cornea is free from the eye and removing the iris from the cornea with a pair of forceps and discarding said iris;
rinsing the cornea in a series of 12 sterile HBSS baths and storing said corneas in HBSS at room temperature until mounted; and
discarding any unacceptable corneas.
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Accused Products
Abstract
A method for long-term multi-species cornea culture and preservation is provided. Excised mammalian corneas are preserved with structural integrity by filling the endothelial cavity with a plug material. Plugged corneas are sterile-cultured for more than 21 days via an air-interface culture where nutrients from a culture media are supplied to corneal tissue primarily through the plug. Plugged corneas are incubated in culture dishes with a customized media continuously in contact with the ocular sclera and corneal plug, and periodically bathing the corneal surface epithelia. An animal-alternative toxicology assay is also provided using excised porcine corneas capable of assessing ocular injury reversibility within 21 days. Further provided is a method of mammalian corneal preservation for excised human corneas extending their storage life to three to four weeks.
39 Citations
11 Claims
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1. A method for mammalian corneal excision from whole globe eyes, comprising:
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procuring enucleated whole globe mammalian eyes from an abattoir and transporting said eyes on ice in an isotonic buffered saline solution supplemented with an anti-fungal drug; incubating whole globe eyes in a broad spectrum antiseptic for approximately 2 minutes in a sterile field; briefly rinsing said eyes with an isotonic buffered saline solution, and then incubating said eyes with an amino glycoside antibiotic in dPBS for 15 minutes; excising the cornea from the eye with a scalpel using sterile technique, by making an incision 2-3 mm from the cornea into the sclera and cutting at this same distance all around the cornea until the cornea is free from the eye and removing the iris from the cornea with a pair of forceps and discarding said iris; rinsing the cornea in a series of 12 sterile HBSS baths and storing said corneas in HBSS at room temperature until mounted; and discarding any unacceptable corneas. - View Dependent Claims (2)
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3. A method for long-term mammalian corneal storage and preservation for greater than 21 days, comprising:
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preparing, in a sterile manner, a 1.33% agar/gelatin mixture, autoclaving said mixture, and then storing said mixture at approximately 4°
C. until needed;warming a quantity of said 1.33% agar/gelatin mixture to approximately 60°
C., and then cooling it to approximately 40-50°
C., and then adding components to said agar/gelatin mixture to arrive at a plug mixture with the following components and the following final concentrations;
agar/gelatin diluted to 1%;
10×
M199 culture media diluted to 1×
;
2.2 mg/ml NaHCO3;
0.68 mM L-glutamine;
50 μ
g/ml Gentamicin;
1 μ
g/ml Amphotericin B;
100 units/0.1 mg/ml penicillin/Streptomycin; and
tissue culture-grade water;maintaining said plug mixture at approximately 40°
C. until needed;obtaining a quantity of recently excised mammalian cornea; inverting each cornea in a respective well of a well plate filled with HBSS, so that the epithelium is bathed in HBSS below, and the endothelial cavity is exposed; adding the added to, molten plug mixture drop by drop into the exposed endothelial corneal cavity, and then allowing said mixture to cool and congeal to form a plug; placing the plugged corneas plug-side down in a tissue culture dish and filling with a custom corneal culture media to cover the sclera tissue surrounding the corneal tissue, but not the corneal tissue; placing the tissue culture dish with said corneas into an incubator set to 37°
C. and 5% CO2 and periodically causing the custom culture media to flow over the corneas in culture to moisten and provide nutrients to the air-exposed corneal epithelial layer of cells; andchanging the corneal culture media daily using a sterile technique. - View Dependent Claims (4)
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5. A custom preservation tissue culture media, comprising:
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approximately 90% by volume of M199 culture media; approximately 10% by volume of fetal bovine serum; approximately 2.2 g/L sodium bicarbonate; approximately 0.68 mM L-glutamine; approximately 100 units/0.1 mg/ml Penicillin/Streptomycin; approximately 1-3 mg ml Amphotericin B; and approximately 100 μ
g/ml Gentamicin.
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6. A method of preparing a custom preservation tissue culture media by thoroughly mixing approximately 90% by volume of M199 culture media with approximately 10% by volume of fetal bovine serum, and adding and thoroughly mixing approximately each of 2.2 g/L sodium bicarbonate, 0.68 mM L-glutamine, 100 units/0.1 mg/ml penicillin/Streptomycin, 1-3 mg ml Amphotericin B;
- and 100 μ
g/ml Gentamicin, thereafter determining and adjusting the pH of the mixture to a range of 7.0-7.4, and then sterilizing the mixture by passing it through a 0.20 μ
m filter flask
- and 100 μ
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7. A method for toxicological ocular irritation and recovery testing in excised cultured mammalian corneas, comprising:
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obtaining a quantity of excised corneas; placing the excised corneas in a dish of custom culture media; allowing the excised corneas to equilibrate to the custom culture media conditions for approximately 24 hours; periodically examining the culture media microscopically throughout the incubation period for the presence of contamination, and discarding contaminated corneas or treating those corneas with additional antibiotics/antifungal drugs when contamination is evident; removing the cultured corneas from the incubator at the end of the incubation period and transferring them to a sterile field; removing the culture media from the dish; applying 10 μ
l or 20 mg of the toxicological irritant testing material directly to the corneal surface, and allowing the corneal tissue to be dosed with that testing material for 5 minutes;rinsing individual corneas gently to remove the testing material; transferring the rinsed corneas to a new, sterile, tissue culture dish and filling the new culture dish with custin corneal culture media covering the sclera tissue surrounding the corneal tissue, but not the corneal tissue itself; placing the cornea and culture media containing dishes of in an incubator set to 37°
C. and 5% CO2 and periodically causing the custom culture media to flow over the corneas in culture to moisten and provide nutrients to the air-exposed corneal epithelial layer of cells;periodically examining the custom culture media microscopically throughout the procedure for presence of contamination and discarding contaminated corneas or treating those contaminated corneas with additional antibiotics/antifungal drugs if contamination is evident; and determining the toxicity of the test material by measuring cell vitality, death, or impairment in the cultured corneal tissue by one of the following techniques; a) confocal microscopy paired with various vital dyes, whether colored or fluorescent; b) reflective confocal microscopy using no dyes; c) digital imaging techniques having image processing software; and d) visual inspection combined with a vitality or death marker dye such as sodium fluorescein (NaFL) stain. - View Dependent Claims (8, 9)
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10. A corneal culture method for human donor cornea preservation, comprising:
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procuring enucleated whole globe human eyes from an eye bank and transporting said eyes on ice in an isotonic buffered saline solution such as Hank'"'"'s Balanced Salt Solution (HBSS) supplemented with an anti-fungal drug; in a sterile field, incubating the whole globe eyes in a broad spectrum antiseptic, briefly rinsing said eyes with an isotonic buffered saline solution, and then incubating said eyes with an amino glycoside antibiotic; using a sterile technique, excising the cornea from each eye with a scalpel by making an incision 2-3 mm from the cornea into the sclera and cutting at this same distance all around the cornea until the cornea is free from the eye, and removing the iris from the cornea with a pair of forceps and discarding the iris; rinsing the cornea in a series of 12 sterile HBSS baths and storing said corneas in HBSS at room temperature until mounted; discarding any unacceptable corneas; preparing, in a sterile manner a 1.33% agar/gelatin mixture in ultra pure sterile water, autoclaving said mixture, and storing said mixture at approximately 4°
C. until needed;warming a quantity of said agar/gelatin mixture, when needed, to approximately 60°
C. to melt it, and cooling it to approximately 40-50°
C., and then adding to the mixture to arrive at a plug mixture with the following components and the following approximate concentrations;
agar/gelatin diluted to 1%;
10×
M199 diluted to 1×
;
2.2 mg/ml NaHCO3;
0.68 mM L-glutamine;
50 μ
g/ml Gentamicin;
1 μ
g/ml Amphotericin B;
100 units/0.1 mg/ml penicillin/Streptomycin; and
tissue culture-grade water;maintaining said plug mixture at approximately 40°
C. until all corneas are excised and ready to be plugged with the plug mixture;inverting each cornea atop a respective well from a plate filled with HBSS so that the epithelium is bathed in HBSS below, and the endothelial cavity is exposed and able to be filled with the warmed molten added-to agar/gelatin mixture; adding the warmed molten plug mixture drop by drop to the exposed endothelial corneal cavity, and then allowing said mixture to cool and congeal into a plug; placing plugged corneas plug-side down in a tissue culture dish and filling with a customized M199 corneal culture media to cover the sclera tissue surrounding the corneal tissue, but not the corneal tissue; placing culture dish with said corneas into incubator set to 37°
C. and 5% CO2 and periodically causing the customized culture media to flow over the corneas to moisten and provide nutrients to the air-exposed corneal epithelial layer of cells;changing the corneal culture media daily using sterile technique; and maintaining said corneas in said customized culture media incubation for up to four weeks; wherein said customized M119 corneal culture media includes;
approximately 90% by volume of M199 culture media;
approximately 10% by volume of fetal bovine serum; and
approximately of each of 2.2 g/L sodium bicarbonate;
0.68 mM L-glutamine;
100 units/0.1 mg/ml penicillin/Streptomycin;
1-3 mg/ml Amphotericin B; and
100 μ
g/ml Gentamicin. - View Dependent Claims (11)
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Specification