Chondroitin sulfate

Primary cultures of normal HCECs were prepared from Descemet membrane dissected from human donor eyes or from FECD patients collected at the time of endothelial keratoplasty. Tissue was stored in Optisol GS (Bausch & Lomb, Bridgewater, NJ) for ≤3 days. Following incubation, tissue was placed in Opti-MEM (Gibco, Waltham MA) with 8% fetal bovine serum (FBS; Gibco) overnight at 37°C, cells were dissociated from the tissue with 0.02% EDTA (Sigma, St Louis, MO) in PBS for 60 min at 37°C, and plated into a single well of a 6-well collagen IV-coated plate (Corning, Tewksbury, MA) containing Joyce’s media [Opti-MEM, 8% FBS, 200 mg/ml CaCl2 (Sigma), 0.08% Chondroitin sulfate (Sigma), 20 μg/ml ascorbic acid (Sigma), bovine pituitary extract 100 μg/ml (Gibco), 5ng/ml EGF (Millipore, St. Louis, MO), 50 μg/mL gentamicin (Sigma), and 1X antibiotic/antimycotic solution (Invitrogen, Waltham, MA)] [14 (link)]. HCECs were allowed to proliferate for 1–4 weeks with media changes every 3–4 days.

MCECs were prepared from 12-week-old mice. Briefly, the globes were aseptically enucleated followed by cornea dissection and corneal endothelium peeling. Then corneal endothelial sheets with attached Descemet's membrane were placed in OptiMEM-I medium (#51985; Thermo Fisher Scientific, Canoga Park, CA, USA) supplemented with 8% heat-inactivated fetal bovine serum (FBS) (#10082139; Thermo Fisher Scientific), EGF 5 ng/mL (#01-107, Millipore, Darmstadt, Germany), pituitary extract 100 μg/mL (Hyclone Laboratories, Logan, UT, USA), calcium chloride 200 mg/L, 0.08% chondroitin sulfate (#G6737; Sigma-Aldrich Corp., St. Louis, MO, USA), gentamicin 50 μg/mL (#15710072; Thermo Fisher Scientific), antibiotic/antimycotic solution diluted 1:100 (#15240062; Thermo Fisher Scientific) and 44 units/mL IFN-γ (#485-MI; R&D Systems, Minneapolis, MN, USA). IFN-γ was used to stimulate the MHC promoter for tsTAg expression. Cells were further selected based on morphology via single-cell cloning for colonies with corneal endothelial hexagonal shape and contact inhibition. Cells were incubated at 33°C with 5% carbon dioxide.
The two lines, Slc4a11^+/+ MCEC and Slc4a11^−/− MCEC, were genotyped by PCR following protocols using QIAamp DNA Mini Kit (Qiagen). Primer sequences used were the same as the primers used for mouse genotyping listed above.

The stripped corneal endothelium was placed in a culture plate coated with FNC Coating Mix^® (Athena Environmental Sciences, Baltimore, MD, USA). The pieces were left undisturbed overnight at 37 °C in a humidified 5% CO₂ chamber in proliferative growth medium, composed of Gibco^TM Opti-MEM-I (Thermo Fisher Scientific) supplemented with 8% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 5 ng/mL human recombinant EGF (PeproTech, Rocky Hill, NJ, USA), 100 μg/mL bovine pituitary extract (Biomedical Technologies, Stoughton, MA, USA), 200 mg/L calcium chloride (Invitrogen, Carlsbad, CA, USA), 0.08% chondroitin sulfate (Sigma-Aldrich), 50 μg/mL gentamicin, and 1× antibiotic/antimycotic solution (Thermo Fisher Scientific). The following day, the culture medium was replaced, and the cells were fed every other day. To minimize endothelial to mesenchymal transition, a previously published dual media was employed [26 (link)]. Here, passage 1 cells were grown to confluency in the described proliferative growth medium and then switched to stabilization medium, composed of Opti-MEM-I supplemented with 4% fetal bovine serum, 50 μg/mL gentamicin, and 1× antibiotic/antimycotic solution. Cells were maintained in stabilization medium for 1 week. Then, the cells were collected for biotinylation and protein extraction or for RNA isolation.

Alginate beads from the resazurin assay were digested with papain (Sigma-Aldrich, Buchs, Switzerland) overnight at 60°C. The samples digested with papain were used for glycosaminoglycan (GAG) and DNA measurement. The GAG content was measured by the modified dimethylmethylene blue (DMMB) assay (pH 1.5)[36 (link), 37 (link)]. The absorbance of the samples added to the DMMB buffer was read at 595 nm with a fluorescence reader (Molecular Devices). GAG concentrations were calculated from a standard curve obtained with chondroitin sulfate (Sigma-Aldrich, Buchs, Switzerland). The amount of DNA in the sample was measured with bisbenzimidol fluorescent dye (Hoechst 33258, Sigma-Aldrich). Fluorescence was again detected with a multi-wavelength fluorescence reader (Molecular Devices). A standard curve was generated with known concentrations of calf thymus DNA (Sigma-Aldrich, Buchs, Switzerland), and the amount of DNA of each sample was calculated from the standard curve.