Chapter 5 —— 123 —— culture medium. The HEK293T cell were detached at 5 days post-transfection by using a cell scrapper and collected into 50-ml tubes together with the conditioned medium. This material was then centrifuged at 1,000 ×g for 10 min at 4°C and the resulting supernatants and cell pellets were separately recovered and stored at -80℃ until further processing. After thawing, 25 ml of a 40% (w/v) polyethylene glycol 8000 solution (PEG 8000; Sigma-Aldrich; Cat. No.: P2139) was added per 100 ml of supernatant with this mixture being first gently stirred for 1 hour at 4°C and subsequently stayed overnight at 4℃ without stirring for particle precipitation. Next, the supernatant-PEG8000 mixtures were subjected to 2,820 ×g for 15 min at 4℃ in 50-ml tubes after which the pellets were resuspended in 7 ml of PBS (pH 7.4) and mixed with 10 ml of clarified cell lysates to yield 17 ml of vector suspensions. The clarified cell lysates were generated by resuspending the producer-cell pellets in 10 ml of PBS (pH 7.4), subjecting the resuspended cells to three rounds of freezing and thawing using liquid N2 and 37℃ water baths, respectively, and eliminating cell debris via centrifugation at 3,220 ×g for 15 min at 4°C. The 17-ml AAV vector suspensions were then exposed to 50 U ml-1 of Benzonase (Millipore; Cat. No.: UFC910024) for 1 hour at 37℃ and subsequently centrifuged at 2,420 ×g for 10 min at 4℃. Recombinant AAV purification and characterization The clarified supernatants harboring the AAV particles were then placed onto Iodixanol-OptiPrep (Progen; Cat. No.: 1114542) cushions of 15%, 25%, 40% and 60% in Quick-Seal round-top polypropylene tubes (Beckman; Cat. No.: 342414). The AAV vectors were purified through iodixanol gradient ultracentrifugation at 69,000 RPM in a 70Ti rotor (Beckman Coulter) at 16°C in a Beckman Coulter Optima XE-90 centrifuge. The ultracentrifuge tubes were pierced with a needle (18G needle BD Microlance™; Cat. No.: 304622) for recovering AAV vector particles in the 40% iodixanol cushion. The collected material was then subjected to buffer exchange using Amicon Ultra15 100K MWCO filters (Millipore; Cat. No.: UFC910024) and Dulbecco’s Phosphate-Buffered Saline (DPBS; Thermo Fisher Scientific; Cat. No.:
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