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Pipette tips
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12 and 24-well cell culture plates (Greiner, Cellstar, catalog numbers: 665180 and 662160)
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Screw Cap Urine Cup (Sarsted, catalog number: 75.9922.721)
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Plastic base mould (Simport, catalog number: HIS0221)
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Parafilm M (Amcor, catalog number: PM996)
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2 ml tube (Sarstedt, catalog number: 72.693.105) and 5 ml tube (Sarstedt, catalog number: 62.558.201)
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Sterile filter 0.2 μm (Corning, catalog number: 431219)
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Petri dish 100 mm diameter (Corning, catalog number: CLS43016)
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Cotton swab (autoclaved) (purchased from local supermarket)
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Porcine eye globes (Local abattoir or butcher)
Note: For this protocol, age and strain of animals could not be controlled. This may also vary between countries.
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Corneal stromal cells from human donor tissue (described in Fernández-Pérez and Ahearne, 2019b)
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Corneal epithelial cells from human limbal biopsies, or an immortalized human corneal epithelial cell line (EverCyte, catalog number: hTCEpi)
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Povidone-Iodine antiseptic 10% w/w (Ecolab, Videne, catalog number: HAS051P)
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OCT embedding cryoembedding matrix (Thermo Fisher Scientific, catalog number: 12678646)
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Liquid nitrogen held in small insulated container
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Phosphate buffered saline (PBS) without CaCl2 and MgCl2 (Sigma-Aldrich, catalog number: D8537)
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Decellularization solution. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4390) and Triton X-100 (Sigma-Aldrich, catalog number: T8787)
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Magnesium chloride (Sigma-Aldrich, catalog number: M0250)
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Calcium chloride solution (Sigma-Aldrich, catalog number: 21115)
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DNase (Sigma-Aldrich, catalog number: DN25)
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RNase (Sigma-Aldrich, catalog number: R5000)
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Autoclaved de-ionized water
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Antimicrobials, 2% v/v Penicillin-streptomycin (Gibco, catalog number: 15140122) and 0.25 μg/ml amphotericin B (Sigma-Aldrich, Merck, catalog number: A2942) in PBS or cell culture medium.
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Teflon (polytetrafluoroethylene) cut into 12 mm diameter discs (Chemours, 2 mm thick sheet)
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Rat tail collagen ~10 mg/ml (Corning, catalog number 354249)
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Dulbecco’s modified Eagle media (DMEM) low glucose (Hyclone, catalog number: 10529702)
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DMEM/F12 1:1 (Hyclone, catalog number: SH30023.01)
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Foetal bovine serum (FBS) (Gibco, catalog number: 10270106)
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Ascorbic acid (Sigma-Aldrich, catalog number: A8960)
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ITS 100× (Gibco, catalog number: 41400045)
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Keratinocyte growth medium (PromoCell, catalog number: C20011)
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Manual cell counter and hemocytometer (Blaubrand, catalog number: BR717805)
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10× PBS without CaCl2 and MgCl2 (Corning, catalog number: 46-013-CM)
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Phenol Red, sodium salt (Sigma-Aldrich, catalog number: P5530)
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1 M NaOH (Honeywell, catalog number: CCS8045)
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Decellularization solution (see Recipes)
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MgCl2 Buffer (see Recipes)
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DNase & RNase solutions (see Recipes)
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10× PBS with phenol red (see Recipes)
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Stromal cell media (see Recipes)
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Keratocytic media (see Recipes)
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Dissection and manipulation tools (Sterile forceps, scissors, scalpels including a 2 mm wide spatula bent to a L-shape (Witig, catalog number: WITG7.412.051)
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Biopsy punch 10 mm diameter (Acuderm, Acu-Punch, catalog number: P1050)
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Cryostat (Leica, model number: CM1850UV)
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-80 °C freezer (Thermo Fisher, Forma Model 906)
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Orbital shaker (Witeg SHO-1D)
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Stirring plate (Henry Troemner, VWR, Advanced series 4x4)
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Centrifuge (Hettich, model: 320R)
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37 °C oven (Memmert, UF55)
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Biosafety cabinet (Faster S.r.l., SafeFAST Classic, 212)
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Humidified CO2 incubator (Thermo Scientific, Forma Steri-Cycle, i790)
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Vortexer (Fisherbrand, Mini-E, catalog number: 15212985)
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Benchtop mini centrifuge (StarLab, catalog number: SLN2631-0007)
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Pipettes (Brand, Transferpette, catalog number: 705891)
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Cooling tray. Expanded Polystyrene container with melting ice or pre-cooled metallic beads (Lab Armor Beads)
This protocol is divided into 5 main procedures. Firstly, we describe how to excise the cornea from the porcine eye globes (A). After that, we explain how to obtain the decellularized sheets (B). Third, the process of preparing cell-laden collagen hydrogels is detailed (C). We then outline the procedure of assembling the constructs with the decellularized sheets and the hydrogels (D). Lastly, the process of adding epithelial cells to the construct is described (E).
The research leading to these results has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement no. 637460) and from Science Foundation Ireland (15/ERC/3269).
Graphical abstract created with BioRender.com.
The authors report no conflicts of interest.
Porcine eye globes were provided postmortem as a byproduct of food supply. Human cells came from tissue remnants following corneal transplantation via the Eye Tissue Bank of the Irish Blood Transfusion Service, Dublin, after appropriate research consent of the donor family. Ethics approval for the use of human tissue to obtain cells was approved by the School of Medicine research ethics committee at Trinity College Dublin.