α minimum essential medium

Hep3B, HepG2, and Huh7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in Dulbecco’s minimum essential medium (Corning, NY, USA), supplemented with 10% fetal bovine serum (FBS; Corning) and 1% penicillin–streptomycin (P/S; Corning). Human NK-92 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in α-minimum essential medium (Corning), which was composed of 12.5% FBS (Corning), 12.5% horse serum (HS; Sigma-Aldrich, St. Louis, MO, USA), 1% P/S (Corning), 0.2 mM Myo-inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), 10 ng/mL Interleukin (IL)-2 (Miltenyi Biotec, Bergisch Gladbach, Germany), and 20 ng/mL IL-15 (Miltenyi Biotec) activating cytokines. Normal human liver THLE-2 cells were obtained from Dr. KP Kim (Asan Medical Center, Seoul, Korea) and were cultured in bronchial epithelial cell growth medium (BEGM Bullet Kit; Lonza, Basel, Switzerland) as per the manufacturer’s protocol. All cells were cultured at 37 °C with 5% CO₂.

MSCs were derived from discarded and de-identified bone marrow filters at the end of bone marrow harvest from normal healthy donors (Age range 18-45) following IRB protocol#2016-0298 at University of Wisconsin Madison. Mononuclear cells (MNCs) were isolated from the bone marrow through a Ficoll density centrifugation gradient. Isolated MNCs were plated onto tissue culture plates containing 1x α-Minimum Essential Medium (Corning, NY, USA) with human platelet lysate (Millcreek Life Sciences, MN, USA) and penicillin/streptomycin/amphotericin B (Hyclone, UT, USA). Non-adherent suspension cells were aspirated and adherent cells washed with phosphate-buffered saline. Cells were maintained by replacing fresh media every 48-72 hours. After the observation of colonies, cells were harvested and reseeded with the density of 3000-5000 cells per centimeter square. Cells were passaged consistently with the maximum confluence of 70% to 80% and cryopreserved until needed for the experiments. MSC identity was confirmed based on cell marker expression as defined in earlier studies (CD45-CD105+CD44+CD90+CD73+) (32 (link), 52 (link)). 48-72 hours prior to the experiment, cryopreserved MSCs are thawed and culture rescued. Experiments were performed with the culture media 1x α-Minimum Essential Medium containing fetal bovine serum and penicillin/streptomycin/amphotericin B.