Roducible method of production and ability to be easily manipulated, particularly

August 29, 2017

Roducible method of production and ability to be easily manipulated, particularly important if they are to be handled by surgeons for transplantation. Here we describe the first use of plastic compressed RAFT as a highly effective, novel carrier for cultured human corneal endothelial cells for transplantation.at 4uC in Optisol GS (Chiron Ophthalmics Inc., Irvine, California) and used within 13 days of enucleation.Isolation and Culture of Human Corneal Endothelial CellsHuman corneal endothelial cells (hCECs) were isolated using a 2-step peel and digest method as previously described [7]. Briefly, corneas were incubated in three washes of antibiotic/antimycotic solution in phosphate buffered saline (PBS; Life Technologies, Ltd., Paisley, UK) and then endothelial layer and Descemet’s membrane were removed for incubation with collagenase A (Sigma-Aldrich Ltd., Dorset, UK) for up to 6 hours at 37uC. Cell clusters were then subjected to TrypLE Express (Life Technologies, Ltd., Paisley, UK) treatment for 5 min at 37uC to disperse the cell clumps. Cells were then seeded onto fibronectin/collagen (FNC) coating mix (US Biologicals, MA, USA) coated 6 cm culture dishes (Falcon; BD Biosciences, Oxford, UK). Cells were cultured in human endothelial culture Licochalcone-A supplier medium based on Engelmann’s F99 medium [13] with slight modifications as previously described [7]. Medium contained Ham’s F12:Medium 199 (1:1), 5 foetal bovine serum, 10 ng/ml bFGF (all Life Technologies, Ltd., Paisley, UK), 20 mg/ml ascorbic acid, 20 mg/ ml bovine insulin, 2.5 mg/ml transferrin and 0.6 ng/ml sodium selenite (all Sigma-Aldrich Ltd., Dorset, UK). Cell culture medium was changed every other day. Cells were sub-cultured after dissociation using TrypLE Express when confluent. Cells at passage 2 or 3 were seeded onto RAFT. Phase contrast images were taken to assess cell morphology using a Nikon TS100 microscope with a Nikon DS-FiI digital camera.Materials and Methods Ethics StatementAll human tissue was handled according to the tenets of the Declaration of Helsinki and written consent was acquired from next of kin of all deceased donors regarding eye donation for research. This study was approved by the institutional review board of the Singapore Eye Research Institute/Singapore National Eye Centre.Culture of the Human 1317923 Corneal Endothelial Cell LineA human corneal endothelial cell line (hCECL) was cultured as per supplier’s instructions (B4G12; DSMZ, Germany). Cells were seeded onto chondroitin sulphate and laminin (CS/L; both SigmaAldrich Ltd., Dorset, UK) coated dishes (Corning Life Sciences, Amsterdam, Netherlands) in culture medium Madrasin manufacturer consisting of human endothelial-SFM (Life Technologies, Ltd., Paisley, UK) supplemented with 10 ng/ml bFGF (Sigma-Aldrich Ltd., Dorset, UK). Cell culture medium was changed every 2 days and cells passaged using 0.05 trypsin solution (Life Technologies, Ltd., Paisley, UK) before reaching confluence. Trypsin was neutralised using protease inhibitor cocktail (Roche Diagnostics, West Sussex, UK) and cells seeded at 2000 cells/mm2.Donor TissueCadaveric donor corneal rims with appropriate written research consent from next of kin were obtained from the Florida Lions Eye Bank (Miami, FL, USA). Three donor cornea pairs were used with donor age ranging from 15?4 years of age. Corneas were storedPreparation of Collagen SolutionCollagen gels were prepared by sodium hydroxide (Sigma Aldrich, Dorset, UK) neutralization of a solution that finally comprised 80 vol/vol.Roducible method of production and ability to be easily manipulated, particularly important if they are to be handled by surgeons for transplantation. Here we describe the first use of plastic compressed RAFT as a highly effective, novel carrier for cultured human corneal endothelial cells for transplantation.at 4uC in Optisol GS (Chiron Ophthalmics Inc., Irvine, California) and used within 13 days of enucleation.Isolation and Culture of Human Corneal Endothelial CellsHuman corneal endothelial cells (hCECs) were isolated using a 2-step peel and digest method as previously described [7]. Briefly, corneas were incubated in three washes of antibiotic/antimycotic solution in phosphate buffered saline (PBS; Life Technologies, Ltd., Paisley, UK) and then endothelial layer and Descemet’s membrane were removed for incubation with collagenase A (Sigma-Aldrich Ltd., Dorset, UK) for up to 6 hours at 37uC. Cell clusters were then subjected to TrypLE Express (Life Technologies, Ltd., Paisley, UK) treatment for 5 min at 37uC to disperse the cell clumps. Cells were then seeded onto fibronectin/collagen (FNC) coating mix (US Biologicals, MA, USA) coated 6 cm culture dishes (Falcon; BD Biosciences, Oxford, UK). Cells were cultured in human endothelial culture medium based on Engelmann’s F99 medium [13] with slight modifications as previously described [7]. Medium contained Ham’s F12:Medium 199 (1:1), 5 foetal bovine serum, 10 ng/ml bFGF (all Life Technologies, Ltd., Paisley, UK), 20 mg/ml ascorbic acid, 20 mg/ ml bovine insulin, 2.5 mg/ml transferrin and 0.6 ng/ml sodium selenite (all Sigma-Aldrich Ltd., Dorset, UK). Cell culture medium was changed every other day. Cells were sub-cultured after dissociation using TrypLE Express when confluent. Cells at passage 2 or 3 were seeded onto RAFT. Phase contrast images were taken to assess cell morphology using a Nikon TS100 microscope with a Nikon DS-FiI digital camera.Materials and Methods Ethics StatementAll human tissue was handled according to the tenets of the Declaration of Helsinki and written consent was acquired from next of kin of all deceased donors regarding eye donation for research. This study was approved by the institutional review board of the Singapore Eye Research Institute/Singapore National Eye Centre.Culture of the Human 1317923 Corneal Endothelial Cell LineA human corneal endothelial cell line (hCECL) was cultured as per supplier’s instructions (B4G12; DSMZ, Germany). Cells were seeded onto chondroitin sulphate and laminin (CS/L; both SigmaAldrich Ltd., Dorset, UK) coated dishes (Corning Life Sciences, Amsterdam, Netherlands) in culture medium consisting of human endothelial-SFM (Life Technologies, Ltd., Paisley, UK) supplemented with 10 ng/ml bFGF (Sigma-Aldrich Ltd., Dorset, UK). Cell culture medium was changed every 2 days and cells passaged using 0.05 trypsin solution (Life Technologies, Ltd., Paisley, UK) before reaching confluence. Trypsin was neutralised using protease inhibitor cocktail (Roche Diagnostics, West Sussex, UK) and cells seeded at 2000 cells/mm2.Donor TissueCadaveric donor corneal rims with appropriate written research consent from next of kin were obtained from the Florida Lions Eye Bank (Miami, FL, USA). Three donor cornea pairs were used with donor age ranging from 15?4 years of age. Corneas were storedPreparation of Collagen SolutionCollagen gels were prepared by sodium hydroxide (Sigma Aldrich, Dorset, UK) neutralization of a solution that finally comprised 80 vol/vol.