Centriprep centrifugal filter

Human PSCs (hPSCs) and mouse PSCs (mPSCs) were grown on culture flasks until 70% to 80% confluent and then cultured in serum-free medium (SFM) for 24 hours at 37°C. Conditioned medium (CM) was collected separately from hPSCs and mPSCs and stored at −80°C in multiple rounds. Human and mouse collections were separately pooled. PSCs were maintained in culture with fresh media added twice weekly. CM and culture medium was concentrated using Millipore centriprep centrifugal filters (EMD Millipore, Billerica, MA) with 10k cut off and centrifuged at 2000 rpm, 4°C to concentrate. The concentrated CM and culture medium was quantified for the protein concentration and the aliquots were stored at −80°C. In the following experiments, PSC-CM and culture medium protein was diluted with serum-free medium (SFM) as indicated to evaluate the effect of PSCs secretions on the mouse PanIN and human PDAC cells.

Bioneedles (15 mm long and 1 mm wide, internal volume of 5 μl) were obtained from the Bioneedles Technologies Group. Influenza A/PR/8/34 whole inactivated virus was produced by Intravacc. The process was based on egg virus propagation and β-propiolactone virus inactivation [12] (link). Split and subunit vaccines were produced by solubilization of WIV with n-octyl-β-D-glucopyranoside (Sigma-Aldrich) as described previously [8] (link). Virosome vaccine was produced as described previously [13] (link). All vaccines were concentrated with Centriprep centrifugal filters (Millipore) with a molecular weight cut-off (MWCO) of 10 kDa, and formulated in HBS (20 mM HEPES, 125 mM NaCl, 9 mM CaCl₂, 5 mM MgCl₂). Vaccine formulations for bioneedles contained 2.5% (w/w) D-trehalose dihydrate (Sigma-Aldrich) as a stabilizer. Influenza vaccine containing bioneedles (subunit, split, virosomal and WIV vaccine) were prepared by filling bioneedles with 5 μL of 1 mg/mL (HA content) liquid vaccines from the hollow back of the bioneedle using specially designed filling apparatus, and frozen on a metal plate at minus 50°C [11] (link). Next, bioneedles were freeze-dried using a Zirbus Sublimator 3×4×7 (Zirbus Technology). Lyophilized bioneedles were stored in glass vials with rubber stoppers under ambient air and relative humidity.

For lentiviral packaging, the pLV-hHTR2Apro-ZsGreen1 plasmid containing the HTR2A promoter sequence was co-transfected with the Lentiviral High Titer Packaging Mix (Takara, Shiga, Japan) using Fugene HD (Promega, Madison, WI) into LentiX-293 T cells (Z2180N; Takara). The 48-h–cultured supernatant was filtrated through 0.45-μm pore cellulose acetate filters (Millipore) and concentrated using Centriprep Centrifugal Filters (Millipore). Viral titration was determined by flow cytometric quantification of ZsGreen1-positive cells. Lenti-HTR2A-ZsGreen1 particles were transduced into hiPSCs at a multiplicity of infection (MOI) of 10 (Fig. S1). Flow cytometry was performed to measure HTR2A promoter activity that induced ZsGreen1 expression in hiPSCs. The lentivirus was transduced into hiPSCs at DIV 22 and hiPSCs were collected at DIV 41 and 51. As a negative control of HTR2A, the lentivirus was transduced into hiPSCs at DIV-10 and hiPSCs were collected at DIV-5. ZsGreen1-positive cells were sorted using the SH800 cell sorter (Sony, Tokyo, Japan). Dead cells were excluded by staining with Fixable Viability Dye eFlour 450 (Thermo Fisher Scientific) before fluorescence-activated cell sorting analysis.

Anti-PC and anti-MDA were extracted as previously described^{15 ,26 (link)}. Briefly, PC-BSA and MDA-HAS, 1 mg/mL was coupled to Hitrap NHS column (GE Healthcare, Sweden). Human IgM (Sigma Aldrich, Israel) was passed through Sepharose column coupled with PC-BSA and MDA–HSA. Unbound IgM considered as non-anti-MDA or non-anti-PC (mentioned as flow through, FT) was collected by washing the columns with binding buffer followed by elution with 0.1 mol/L glycine–HCl, elution buffer. The eluted antibodies were desalted in PD-10 columns (GE Healthcare, UK) and concentrated by a Centriprep centrifugal filter (Millipore, Ireland). After filtration through a 0.22-lm filter (Sarstedt, Germany), extracted antibodies were stored at −20 °C.

HEK293T cells were co-transfected with the indicated AAV vectors and pHelper and AAV1.0 (serotype2/9) capsids vectors. Seventy-two hours later, transfected HEK293T cells were collected, lysed, and mixed with 40% polyethylene glycol and 2.5 M NaCl, and centrifuged at 2000 × g for 30 min. The cell pellets were resuspended in HEPES buffer (20 mM HEPES; 115 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM KH₂PO₄) and an equal volume of chloroform was added. The mixture was centrifuged at 400 × g for 5 min, and concentrated three times with a Centriprep centrifugal filter (Millipore) at 1,220 × g for 5 min each and with an Amicon Ultra centrifugal filter (Millipore) at 16,000 × g for 10 min. Before titering AAVs, contaminating plasmid DNA was eliminated by treating 1 μl of concentrated, sterile-filtered AAVs with 1 μl of DNase I (Sigma-Aldrich) for 30 min at 37 °C. After treatment with 1 μl of stop solution (50 mM ethylenediaminetetraacetic acid) for 10 min at 65 °C, 10 μg of protease K (Sigma-Aldrich) was added and AAVs were incubated for 1 h at 50 °C. Reactions were inactivated by incubating samples for 20 min at 95 °C. The final virus titer was measured by quantitative reverse transcription-PCR (qRT-PCR) detection of EGFP sequences and subsequent reference to a standard curve generated using the pAAV-U6-EGFP plasmid. All plasmids were purified using a Plasmid Maxi Kit (Qiagen GmbH).