Methods and compositions for growing corneal endothelial and related cells on biopolymers and creation of artifical corneal transplants

A method of making an artificial cornea comprising:

• a) a base biopolymer;
• b) molding the biopolymer into a desired shape;
• c) coating the biopolymer with an attachment mixture comprising laminin, fibronectin, RGDS, bFGF conjugated with polycarbophil and EGF conjugated with polycarbophil;
• d) incubating the reagent with the biopolymer at approximately 4° C. for a sufficient period of time to improve adherence of corneal endothelial cells;
• e) removing the attachment mixture;
• and f) seeding of corneal endothelial cells onto the biopolymer.

A method of making an artificial cornea comprising:

• a) a base biopolymer;
• b) molding the biopolymer into a desired shape;
• c) coating the biopolymer with a BCE-ECM coating comprising the steps of:
• 1) seeding onto the biopolymer at low density, a population of bovine corneal endothelial (BCE) cells in a culture media suitable for their growth;
• 2) allowing the BCE cells to grow to confluence;
• and 3) aspirating the media and treating the biopolymer with ammonium hydroxide for a sufficient period of time to remove the cells;
• d) washing the biopolymer with a suitable buffer;
• and e) seeding corneal endothelial cells onto the biopolymer and growing to confluence.

A method of making an artificial cornea comprising:

• a) a base biopolymer;
• molding the biopolymer into a desired shape;
• c) coating the biopolymer with Diamond-Like Carbon using a suitable process;
• d) washing the biopolymer with a suitable buffer;
• and e) seeding corneal endothelial cells onto the biopolymer and growing to confluence.

A method of growing endothelial cells suitable for use in a cornea comprising:

• a) a base biopolymer;
• b) molding the biopolymer into a desired shape;
• c) coating the biopolymer an adhesion factor mixture comprising a sufficient quantity of laminin, fibronectin, RGDS, and collagen IV in a suitable biological buffer;
• d) applying the biopolymer to the corneal button;
• and e) seeding corneal endothelial cells onto the biopolymer and growing to confluence.

A method of growing endothelial cells suitable for use in a cornea comprising:

• a) creating a base biopolymer in contact with an adhesion factor mixture comprising a sufficient quantity of laminin, fibronectin, RGDS, and collagen IV in a suitable biological buffer and a growth factor mixture comprising a sufficient quantity of bFGF, EGF and polycarbophil in a suitable biological buffer;
• b) molding the biopolymer into the shape of a cornea;
• c) applying the biopolymer to the corneal button;
• and d) seeding corneal endothelial cells onto the biopolymer and growing to confluence.

An attachment mixture comprising:

• a) 10 μg to 500 μg/ml of fibronectin in PBS;
• b) 10 μg/ml to 500, μg/ml of laminin in PBS;
• c) 1 μg/ml to 100 μg/ml RGDS in PBS;
• d) 10 μg to 1000 μg of collagen type IV in 0.1 M acetic acid;
• e) 1 ng/ml to 500 ng/ml b-FGF in PBS;
• and f) 1 ng/ml to 500 ng/ml EGF in PBS.

An artificial full-thickness corneal transplant support comprising:

• a) a base biopolymer having a thickness of approximately an average cornea;
• b) incorporating into the biopolymer during its synthesis an attachment reagent comprising one or more of the following:
• laminin, fibronectin, RGDS, bFGF conjugated with polycarbophil, EGF conjugated with polycarbophil, and heparin sulfate;
• and c) molding the biopolymer into a desired shape of a cornea.

An artificial full-thickness corneal transplant comprising:

• a) a base biopolymer having a thickness of approximately an average cornea;
• b) incorporating into the biopolymer during its synthesis an attachment reagent comprising one or more of the following:
• laminin, fibronectin, RGDS, bFGF conjugated with polycarbophil, EGF conjugated with polycarbophil, and heparin sulfate;
• c) molding the biopolymer into the shape of a cornea;
• d) seeding HCEC onto the biopolymer and growing to confluence.

An artificial half-thickness corneal transplant support comprising:

• a) a base biopolymer having a thickness of approximately one half the thickness of an average cornea;
• b) incorporating into the biopolymer during its synthesis an attachment reagent comprising one or more of the following:
• laminin, fibronectin, RGDS, bFGF conjugated with polycarbophil, EGF conjugated with polycarbophil, and heparin sulfate;
• and c) molding the biopolymer into the shape of a cornea.

An artificial half-thickness corneal transplant comprising:

• a) a base biopolymer having a thickness of approximately one half the thickness of an average cornea;
• b) incorporating into the biopolymer during its synthesis an attachment reagent comprising one or more of the following:
• laminin, fibronectin, RGDS, bFGF conjugated with polycarbophil, EGF conjugated with polycarbophil, and heparin sulfate;
• c) molding the biopolymer into the shape of a cornea;
• d) seeding HCEC onto the biopolymer and growing to confluence.

A method of repairing a damaged cornea comprising the steps of:

• a) obtaining an artificial full-thickness cornea;
• b) overlaying said corneal surface with a biopolymer having confluent HCEC on it;
• c) implanting the artificial full-thickness cornea of step a onto a damaged cornea;
• d) securing said cornea by surgical or other means.

A method of repairing a damaged cornea comprising the steps of:

• a) obtaining an artificial half-thickness cornea;
• b) overlaying said corneal surface with a biopolymer having confluent HCEC on it;
• c) implanting the artificial half-thickness cornea of step a onto a damaged cornea;
• d) securing said cornea by surgical or other means.

A method for making retinal pigment epithelial (RPE) cells suitable for transplantation into a retina comprising the steps of:

• a) obtaining a biopolymer having a top and a bottom surface and having a thickness between about 10 to 100 m in thickness;
• b) placing said biopolymer in a medium suitable for the growth of RPE cells in vitro;
• c) seeding RPE cells onto the top surface of said biopolymer sheet at a certain density and allowing the RPE cells to grow to confluence;
• and d) removing said sheet and cutting to a desired size.