Dong, Muchen, et al. Journal of Tissue Engineering 10 (2019): 2041731419875876.
Sodium N-lauroyl glutamate (SLG), a mild amino-acid-based detergent, was combined with supernuclease to develop a rapid decellularization method for porcine corneal stroma.
Experimental Protocol: Fresh porcine corneas from 4- to 6-month-old Duroc swine were cut into 500-um lamellae for in vitro work or 300-um lamellae (10 mm diameter) for animal work. Stroma was treated with 0.5% SLG, sodium N-lauroyl sarcosinate, sodium dodecyl sulfate or X-100, each supplemented with 200 U/mL supernuclease in PBS for 2 h at room temperature under shaking at 100 r/min. The SLG method (Method A, about 3 h) was compared with two traditional methods (B, more than 21 h; C, more than 43 h). Characterization used H&E and immunofluorescence staining, TEM, SEM, DNA, GAG and collagen assays and tensile testing; 300-um grafts were implanted in rabbits by lamellar keratoplasty.
Performance Evaluation: SLG plus supernuclease removed DNA from 78.90 plus or minus 1.48 to 1.02 plus or minus 0.07 ng/mg, more than 98% removal, versus 17.83 plus or minus 0.82 (SLS), 20.78 plus or minus 0.89 (SDS) and 34.57 plus or minus 1.25 ng/mg (Triton X-100). Moisture content was 91.80 plus or minus 0.37% and thickening rate 204.90 plus or minus 3.95%. Young modulus reached 10.63 plus or minus 0.54 MPa versus 3.89 plus or minus 0.30 MPa for native stroma. Grafts showed favorable transparency and no immune rejection at 1 month.