Decellularization and Quantification of Lenticules

Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and GAG contents. The lenticules were lyophilized in a freeze dryer (FD5512, IlShin, Gyeonggi-do, Korea) and weighed. The DCLs were digested in 0.2 M sodium phosphate buffer (pH 6.4) containing 125 μg/mL papain (Sigma), 10 mM cysteine hydrochloride (Sigma), 0.1 M sodium acetate (Junsei, Tokyo, Japan), and 2 mM EDTA (Sigma) for 3 h at 65°C as described previously [25 (link)]. The DNA content was measured by using a DNA quantitation kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Briefly, 5 μL solution of dissolved lenticules (10 mg/mL) was added to 1 mL TEN buffer (100 mM Tris, 2 M NaCl, 10 mM EDTA, pH 7.4) containing Hoechst 33528 (1 μg/mL). Fluorescence intensity was read using a 460 nm emission filter with a microplate reader (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany). The DNA contents were calculated from a standard curve determined by using calf thymus DNA.
Sulfated GAG in the DCL was measured with a Blyscan™ glycosaminoglycan assay kit (Biocolor, County Antrim, UK). GAG quantification was performed according to the manufacturer's protocol. Absorbance was measured using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 656 nm.