Trypsin versene

Vero (African green monkey kidney) cells (ATCC) were adapted and grown using Opti-MEM (Gibco) supplemented with 3% fetal calf serum (Bovogen) in T25 vented flasks (Greiner Bio-One) in a humidified, 37°C incubator with 5% C0₂. Cells were split using 0.5% Trypsin versene (Gibco) and seeded at 50% confluence in either T25 vented flasks or in 96-well cell culture plates (Corning^® Costar^®). Cells were infected with DENV-1, DENV-2, DENV-3, DENV-4 (all East Timor strains corresponding to GenBank entries 440432.1, 440433.1, 440434.1 and 440435.1 respectively) or Kunjin virus (KUNV, a highly attenuated, Australasian variant of West Nile virus) at 0.1 multiplicity of infection in serum-free Opti-MEM for 2 hours at 37°C. After infection, the infection medium was replaced with Opti-MEM supplemented with 2% fetal calf serum. DENV infected cells were cultured for three days while KUNV infected cells were cultured for two days post infection. Cell culture supernatants from T25 flasks were harvested and concentrated (Macrosep Advance Centrifugal Devices, Pall) then used in a sandwich ELISA to detect secreted NS1 while infected cells in 96-well cell culture plates were fixed and used in immunofluorescence assays.

All study procedures were approved by the Institutional Review Board (Taipei Veterans General Hospital). Bone marrow tissues were gathered from 20 healthy donors with their informed consent. After washing the bone marrow sample twice with phosphate buffered saline (PBS, PH = 7.2), density gradient centrifugation (NycoPrep 1.077, Axis-Shield, Oslo, Norway) was possessed and BM-MSCs were isolated. Rinse the BM-MSCs twice in low glucose DMEM (LG-DMEM, 5.5 mM glucose, Invitrogen, Carlsbad, CA) and culture them at 37°C with 5% humidified CO2 in expansion medium consisting of L-DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin/Amphotericin (Biological Industries, Haifa, Israel). The culture medium was replaced every 3 days and the nonadherent cells were removed. When the adherent BM-MSCs were 90–95% confluent (10–15 days), they were subcultured by Trypsin-Versene (Invitrogen). When the third passage BM-MSC reached 80% confluence, it was provided to differentiate into IPCs by culturing in serum-free high glucose DMEM (HG-DMEM, 25 mM glucose) supplemented with 1% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO). 3 days after, the culture medium was replaced with HG-DMEM supplemented with 10% FBS for another 14 days [12 (link)].

Apical and luminal cell fractions were isolated from ALI-PBEC cultures after 19 days as previously described^{26 (link)}. Briefly, cells were washed with calcium-free PBS before adding calcium-free minimum essential medium (MEM, Thermo Fischer Scientific) to both the apical and basolateral compartments to compromise the integrity of the intercellular junctions. Next, the apical medium was substituted for Trypsin Versene (Thermo Fischer Scientific) and incubated for 7–10 min. Detached cells were collected as the luminal fraction while cells still attached to the inserts were harvested as the basal fraction.