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    2020-08-18

    r> 2.3. Culture of human primary pancreatic ductal epithelial LY-500307 and murine organoids
    2.3.1. Human primary pancreatic cells epithelial cells (HPPE)
    Human primary pancreatic cells epithelial cells (HPPE) were pur-chased from Cell Biologics, Inc. (Chicago, IL) and grown in T25 tissue culture flasks pre-coated with gelatin-based coating solution for 2 min and incubated in Cell Biologics’ Culture Complete Growth Medium (H6621, Cell Biologics Inc. IL). Human pancreatic ductal epithelial (HPDE) cell line was kindly provided by Ming-Sound Tsao (University of Toronto, Toronto, Ontario, Canada) and maintained in the kerati-nocyte-SFM supplemented with EGF and BPE (ThermoFisher Scientific, MA).
    2.3.2. Culture of mouse pancreatic ductal organoids
    Normal pancreatic ducts were harvested after enzymatic digestion of pancreas with 0.012% (w/v) collagenase XI (Sigma) and 0.012% (w/
    v) dispase II (Sigma) in DMEM media containing 1% FBS (Gibco). They were seeded in growth factor-reduced (GFR) Matrigel (BD) and cultured in human complete medium, as previously described [6]. In brief, the pancreatic tissue was minced and digested in digestion medium (DMEM supplemented with Penicillin/Streptomycin, 1% FBS, 0.012% (w/v) collagenase XI (Sigma) and 0.012% (w/v) dispase II) at 37 °C with continuous rotation for 20 min. The supernatant was transferred into a clean Petri dish. Pancreatic ducts were dissected and collected into the 15 ml tube and centrifuged at 140g for 5 min at 4 °C. After removal of supernatant, pancreatic ducts were suspended in Matrigel and seeded in 24-well plate. The remain pancreatic tissue were continuously digested and pancreatic ducts were collected after every 10 min incubation. The complete feeding medium (see supplemental data) was added into the 24-well plate where the Matrigel has been solidified after 20 min in-cubation at 37 °C.
    2.4. CRISPR/Cas9 genomic editing with lentiviral infection
    To introduce driver mutations in the HPPE cells, HPPE cells were co-
    infected with Lentiviral Cas9-tdT and Lentiviral-sgRNAs-HDRKrasG12D_donors per protocol [7]. The sgRNA for human Kras mu- tation was designed to change the DNA code GGT that encoded glycine
    (arginine 51) of p16 and Q284 (glutamine 284) of SMAD4. The sgRNA for murine organoid CRISPR/Cas9 engineering was following the pub-lished research design [8] Organoids were broken down into small cell clusters, which were re-suspended in 250 μl of 2 x feeding medium supplemented with 4 μg/ml LY-500307 of polybrene (Sigma-Aldrich, MO) followed by mixing with 250 μl of viral mixture and then transferred into a 48-well culture plate. The plate was centrifuged at 800 rpm at 32 °C for 60 min, followed by another 6 h of incubation at 37 °C. The cells were transferred into 1.5 ml Eppendorf tube and spun down at 800 rpm for 5 min. The supernatant was discarded, and the pellet was re-suspended in 100 μl Matrigel (BD Biosciences) and split into two wells of a 24-well culture plate. After solidification at 37 °C for 60 min, each well was overlaid with 500 μl of infection medium. Media were replaced twice a week, and spheres were collected after 2–3 weeks for passaging. Static images of spheres were collected using an Olympus fluorescence in-verted microscope (Olympus, Japan).
    2.5. AAV vector particle production
    AAV-targeting vectors and AAV2 were generated using the AAVpro™ Helper Free System (AAV2) (Clontech, Mountain View, CA). For pro-duction of the targeting vectors, pRC2-mi342 was engineered by in-sertion of tumor homing peptide sequence. pAAV2-CMV vectors were engineered to generate pAAV2-CMV-GLuc, pAAV2-CMV-eGFP, pAAV2-CMV-FLuc, pAAV2-BIRC5-SPTSTA-GLuc-2A-sr39TK vectors and pAAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK vectors. AAV2RGD-BIRC5-SPTSTA-GLuc-2A-sr39TK vector is a highly-specific PDAC viral vector
    that has been engineered to display RGD peptide on its surface aimed to attach to PDAC cells to deliver GLuc and sr39TK genes into PDAC specifically. Whereas GLuc and sr39TK genes are linked by 2A sequence and controlled by TSTA-enhanced BIRC5 super-promoter, which are therefore only expressed in PDAC cells. GLuc-2A-srTK39 proteins are then into GLuc and srTK39 reporters in PDAC cells.
    For production of AAV2 wild-type (AAV2WT) and AAV2 genetically engineered (AAV2GE), HEK-293FT cells were transfected with plasmids pAAV2, pRC2-mi342, and pHelper vector in an equal molar ratio mixed with PEI. Cells were harvested, pelleted by centrifugation, and lysed post 48 h of transfection. Cell lysates were treated with 50 U/ml Benzonase (Sigma-Aldrich,) at 30 min, 37 °C. Lysates (3,500 g, 20 min, 4 °C) were purified by iodixanol gradient purification for 2 h at 290,000 g in a Beckman 70Ti rotor and harvested from the 40% io-dixanol layer.