Papain suspension

Primary neurons were isolated from cortices of E15.5 C57BL6/N mice from in-house facilities. Cortices were dissociated by treatment with Papain suspension (Worthington) and gentle trituration. Cell plating was done in Neurobasal medium supplemented with 2% B27, GlutaMax and 5% FBS (all Gibco), on poly-L-lysine (MW 70,000–150,000; Sigma) coated plastic tissue culture dishes (BD Falcon) at a cell density of 550–650 cells/mm². On DIV1, plating medium was replaced by Neurobasal medium supplemented with 2% B27 and GlutaMax. Cultures were fed for the first time on DIV5/6 and then every 3–4 days.
SH-SY5Y cells (ATCC) were cultured in DMEM (high glucose) supplemented with 10% FBS and GlutaMax (all Gibco). PC12 Tet-Off cells (Clontech, termed PC12 cells for simplicity) were cultured in DMEM (low glucose) supplemented with 10% FBS, GlutaMax and penicillin/streptomycin (all Gibco). HEK293FT cells (ATCC) were cultured in DMEM (high glucose) supplemented with 10% FBS and GlutaMax (all Gibco).
All cells were grown in a humidified incubator (37 °C, 5% CO₂).

Mouse or chick retinas were dissected away from other ocular tissues in Hank’s Balanced Salt Solution (Thermo Fisher Scientific, cat. #14025092) or PBS. The retina was then transferred to a microcentrifuge tube and incubated for 7 min at 37°C with an activated papain dissociation solution (87.5 mM HEPES pH 7.0 (Thermo Fisher Scientific, cat. #15630080), 2.5 mM L-Cysteine (MilliporeSigma, cat. # 168149), 0.5 mM EDTA pH 8.0 (Thermo Fisher Scientific, cat. #AM9260G), 10 µL Papain Suspension (Worthington, cat. #LS0003126), 19.6 µL UltraPure Nuclease-Free Water (Thermo Fisher Scientific, cat. #10977023), HBSS up to 400 µL, activated by a 15 min incubation at 37°C). The retina was then centrifuged at 600 xg for 3 min. The supernatant was removed, and 1 mL of HBSS/10% FBS (Thermo Fisher Scientific, cat. #10437028) was added without agitation to the pellet. The pellet was centrifuged at 600 xg for 3 min. The supernatant was removed, and 600 µL of trituration buffer (DMEM (Thermo Fisher Scientific, cat. #11995065), 0.4% (wt/vol) Bovine Serum Albumin (MilliporeSigma cat. #A9418)) was added. The pellet was dissociated by trituration at room temperature (RT) using a P1000 pipette up to 20 times or until the solution was homogenous.

For cell proliferation, the DNA of cLESCs seeded in SF/G corneal film (100,000 cell/cm²) and cCSSCs seeded in SF/G stromal scaffold (cell concentration 6x10⁶ cells/mL; 80 μl/scaffold) were quantified at days 1, 3, 5, 7, 14, and 28. The samples of each time point were digested with 1.0 mg/mL proteinase K (Worthington Biochemical, USA) in proteinase K buffer (150 mM Tris HCl and 1 mM EDTA pH 8.0) and 20 μl/sample of papain suspension (Worthington Biochemical, USA). After that, all samples were incubated at 60°C for 16 h in a heating block. Centrifugation was performed with 8,000 rpm for 10 min at 4°C to get the supernatant and transferred to a new tube. DNA levels were measured by Qubit Fluorometric Quantification with dsDNA BR assay according to the manufacture’s protocols. Quantitative data was normalized with plain film and scaffold.