F 12 nutrient mixture ham dulbecco s modified eagle s medium

HepG2 and Hep3B cells were maintained in DMEM supplemented with FBS (10%, v/v), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were maintained in 5% CO₂ at 37°C. The confluent cells were passaged with trypsin-EDTA. Untreated medium containing vehicle DMSO was used as a negative control.
Normal liver specimens were collected with the informed consent of the patients, according to Georgetown University Institutional Review Board (Washington, DC) protocols. Normal primary liver human hepatocytes were established as previously reported [17 (link)] with some modifications (Sultan and Albanese; data under publication) to indefinitely extend the life span of primary human keratinocytes, using a Rho-associated kinase (ROCK) inhibitor, Y-27632 [18 (link)], with no feeder cells. Epithelial cells were cocultivated in F medium [3 : 1 (v/v) F-12 Nutrient Mixture (Ham)-Dulbecco's modified Eagle's medium (Invitrogen), 5% fetal bovine serum, 0.4 μg/mL hydrocortisone (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Invitrogen), and 24 μg/mL adenine (Sigma-Aldrich)] with addition of 5 to 10 μmol/L Y-27632 (Enzo Life Sciences).

PDCs were established from residual tumor tissue from primary TNBC after neoadjuvant chemotherapy; the protocol for PDC establishment was as described by Liu et al.^{24 (link)}. Epithelial cells were cocultivated with irradiated (3000 rad) Swiss 3T3 fibroblasts (J2 strain) in F medium [3:1 (v/v) F12 nutrient Mixture (Ham)–Dulbecco’s modified Eagle’s medium (Invitrogen, Waltham, MA, USA), 5% fetal bovine serum (FBS; Gibco; Gaithersburg, MD), 0.4 µg/ml hydrocortisone (Sigma-Aldrich, St. Louis, MO), 5 µg/ml insulin (Sigma-Aldrich), 8.4 ng/ml cholera toxin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (EGF; Invitrogen), and 24 µg/ml adenine (Sigma-Aldrich)] with the addition of 5 to 10 µM/L Y-27632 (Enzo Life Sciences, Seoul, South Korea).

PDX tissues were obtained through the ACC Research Foundation (ACCRF) and Dr. Christopher Moskaluk’s lab where they were initially established. PDX tissues were minced, digested as previously described^{19 (link)} and plated in a modified CR cell media containing 1:3 ACC media and CR media. The CR media consists of 1:3 (v/v) F-12 Nutrient Mixture (Ham)–Dulbecco’s modified Eagle’s medium (Invitrogen, California, USA), 5% fetal bovine serum, 0.4 μg/mL hydrocortisone (Sigma-Aldrich, Missouri, USA), 5 μg/mL insulin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Invitrogen), 100 μg/ml Primocin (Invitrogen), and 10 μM Y-27632 (Enzo Life Sciences). The ACC media contains CR media with the following additional components: 100 ng/ml Noggin (Peprotech, New Jersey, USA), 3 μM SB202190 (Sigma-Aldrich, Missouri, USA), 20 ng/ml rhFGF (R&D Systems, Minnesota, USA), 3 μM CHIR-99021 (Selleckchem, Texas, USA), 20 ng/ml Wnt-3a (R&D Systems). Digested tissue was plated in this modified media with ~400,000 irradiated (40 Gy) J2 mouse fibroblast cells in a red cap T-25 flask (Greiner Bio One, VWR, PA, USA). All cultures were maintained in this media at 37 °C with 5% CO₂ in a humidified chamber.