N for Endothelial TransplantationFigure 2. Loading and insertion of RAFT into an ex vivo porcine eye using Tan EndoGlideTM. Representative photographs showing the process of loading (A ) of the Tan EndoGlideTM with RAFT construct and insertion (E ) of RAFT into the anterior chamber of an ex vivo porcine eye model. (A) Loading forceps grasp the edge of the RAFT construct from the spatula. (B) RAFT is pulled into the cassette and (C) automatically coils into a double coil configuration. (D) RAFT is fully loaded into the cassette with no upper surfaces touching. (E) Tan EndoGlide TM is inserted into the anterior chamber that is prevented from collapsing using a column of saline via an inserted needle. (F) RAFT is pulled from the cassette (G) into the anterior chamber and positioned centrally before (H) an air bubble 25033180 is inserted to appose RAFT to the posterior cornea. doi:10.1371/journal.pone.0050993.groll was added. A 35 g load was then applied to the system for 15 min to allow compression of the collagen gel with loss of fluid in a confined, upward flow direction through the paper roll. This process yielded a thin collagen construct, that we have NHS-Biotin termed RAFT, which was then either kept in place in a 12 well plate for hCECL culture or trephined using a 8.25 mm trephine (Coronet, Network Medical Products Ltd., Ripon, UK) to obtain small discs for hCEC culture. The trephined discs were transferred to an organ culture dish (Falcon; BD Biosciences, Oxford, UK) and maintained in a small amount of PBS until cell seeding. The thickness of representative RAFT constructs was then measured using optical coherence tomography (OCT) with an anterior segment lens (Spectralis, Heidelberg 
Engineering, Hemel Hempstead, UK). Thickness was measured at 3 positions along the length of a scanned area in the centre of each of three replicate constructs.Seeding of Endothelial Cells onto RAFTRAFT constructs were coated with either FNC coating mix or CS/L and then hCECLs were seeded onto the surface in 12 well plates at a density of 2000?000 cells/mm2 in a MedChemExpress I-BRD9 volume of 2 ml medium. Primary hCECs were seeded onto FNC coated RAFT discs in organ culture dishes at a density of 2000?000 cells/mm2 in a volume of 20 ml. Several hours later, after cells had attached, wells were flooded with endothelial cell culture medium. Dishes were placed in an incubator at 37uC with 5 CO2 in air. Cell culture medium was changed every other day and constructs fixed for staining on day 4 or day 14 for longer-term cultures.Histological Staining 1326631 of Paraffin Embedded SectionsHuman central corneal specimens and RAFT constructs with hCECL on the surface were fixed for 30 min with 4 PFA before processing for paraffin embedding. Tissue sections (6 mm) were cut on a microtome, rehydrated through alcohols to water, stained with haematoxylin and eosin, mounted and coverslipped with DPX. Sections were imaged using a Zeiss 510 Microscope and Axiovison software.Ease of Handling of RAFT for TransplantationPorcine whole globes were obtained from First Link Ltd., Birmingham, UK. Excess tissue was dissected from the scleral globe to clean the eyes. Acellular RAFT 
constructs were prepared as above and 8.25 mm discs were trephined. A RAFT disc was placed onto the donor well of the Tan EndoGlideTM preparation base using a metal spatula. RAFT was then pulled into the cartridge using loading forceps as per manufacturer’s instructions. A 4 mm wound was made in the sclera to allow insertion of the Tan EndoGlideT.N for Endothelial TransplantationFigure 2. Loading and insertion of RAFT into an ex vivo porcine eye using Tan EndoGlideTM. Representative photographs showing the process of loading (A ) of the Tan EndoGlideTM with RAFT construct and insertion (E ) of RAFT into the anterior chamber of an ex vivo porcine eye model. (A) Loading forceps grasp the edge of the RAFT construct from the spatula. (B) RAFT is pulled into the cassette and (C) automatically coils into a double coil configuration. (D) RAFT is fully loaded into the cassette with no upper surfaces touching. (E) Tan EndoGlide TM is inserted into the anterior chamber that is prevented from collapsing using a column of saline via an inserted needle. (F) RAFT is pulled from the cassette (G) into the anterior chamber and positioned centrally before (H) an air bubble 25033180 is inserted to appose RAFT to the posterior cornea. doi:10.1371/journal.pone.0050993.groll was added. A 35 g load was then applied to the system for 15 min to allow compression of the collagen gel with loss of fluid in a confined, upward flow direction through the paper roll. This process yielded a thin collagen construct, that we have termed RAFT, which was then either kept in place in a 12 well plate for hCECL culture or trephined using a 8.25 mm trephine (Coronet, Network Medical Products Ltd., Ripon, UK) to obtain small discs for hCEC culture. The trephined discs were transferred to an organ culture dish (Falcon; BD Biosciences, Oxford, UK) and maintained in a small amount of PBS until cell seeding. The thickness of representative RAFT constructs was then measured using optical coherence tomography (OCT) with an anterior segment lens (Spectralis, Heidelberg Engineering, Hemel Hempstead, UK). Thickness was measured at 3 positions along the length of a scanned area in the centre of each of three replicate constructs.Seeding of Endothelial Cells onto RAFTRAFT constructs were coated with either FNC coating mix or CS/L and then hCECLs were seeded onto the surface in 12 well plates at a density of 2000?000 cells/mm2 in a volume of 2 ml medium. Primary hCECs were seeded onto FNC coated RAFT discs in organ culture dishes at a density of 2000?000 cells/mm2 in a volume of 20 ml. Several hours later, after cells had attached, wells were flooded with endothelial cell culture medium. Dishes were placed in an incubator at 37uC with 5 CO2 in air. Cell culture medium was changed every other day and constructs fixed for staining on day 4 or day 14 for longer-term cultures.Histological Staining 1326631 of Paraffin Embedded SectionsHuman central corneal specimens and RAFT constructs with hCECL on the surface were fixed for 30 min with 4 PFA before processing for paraffin embedding. Tissue sections (6 mm) were cut on a microtome, rehydrated through alcohols to water, stained with haematoxylin and eosin, mounted and coverslipped with DPX. Sections were imaged using a Zeiss 510 Microscope and Axiovison software.Ease of Handling of RAFT for TransplantationPorcine whole globes were obtained from First Link Ltd., Birmingham, UK. Excess tissue was dissected from the scleral globe to clean the eyes. Acellular RAFT constructs were prepared as above and 8.25 mm discs were trephined. A RAFT disc was placed onto the donor well of the Tan EndoGlideTM preparation base using a metal spatula. RAFT was then pulled into the cartridge using loading forceps as per manufacturer’s instructions. A 4 mm wound was made in the sclera to allow insertion of the Tan EndoGlideT.
