Epithelial growth factor egf

MCF10A, MDA-MB-231, MDA-MB-468, Hs578T, and three mouse breast cancer cell lines 4T1, EMT6, EO771 were purchased from the American Type Culture Collection (ATCC). 293A, MDA-MB-231, MDA-MB-468, and EO771 cells were cultured in DMEM supplemented with 10% FCS (Sigma-Aldrich). Hs578T cells were grown in DMEM with 10% FCS and 0.01 mg/ml human insulin (Sigma-Aldrich). MCF10A normal breast epithelial cells were grown in DMEM/F12 medium supplemented with 20 ng/ml epithelial growth factor (EGF; Thermo Fisher Scientific), 100 ng/ml cholera toxin (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), 0.5 mg/ml hydrocortisone (Sigma-Aldrich), and 5% horse serum (Thermo Fisher Scientific). 4T1 cells were cultured in Roswell Park Memorial Institute 1640 medium with 10% FBS. EMT6 cells were grown in Waymouth MB 752/1 Medium with 2 mM L-glutamine and 15% FBS. All the cell lines were tested to verify that they were free of mycoplasma contamination.

Porous silk films were prepared using 1% w/v silk solution and 0.05% w/v polyethylene oxide (PEO, MW = 900,000, Sigma-Aldrich) in deionized water cast on 12 mm glass coverslips (Electron Microscopy Science, Hatfield, PA)^{28 (link)}. Films for epithelial growth were stamped using 12 mm polydimethylsiloxane (PDMS) (Fisher Scientific Co. Fair Lawn, NJ) molds dipped in stamping solution composed of 50 μL (4 mg/mL) type I collagen (rat-tail tendon, BD, Franklin Lake, NJ), 100 ng/mL keratinocyte growth factor (KGF) (Sigma), 100 ng/mL hepatic growth factor (HGF) (Sigma), 200 ng/mL epithelial growth factor (EGF) (Thermo Fisher, Waltham MA) and NGF (200 ng/mL) (R&D Systems, Minneapolis, MN). Stamped films were placed in a desiccator to water anneal at 25 mmHg for 2 hrs, UV-sterilized, and incubated in 10 µg/mL poly-D-lysine (PDL) overnight at 4 °C. Larger, patterned silk films for keratocyte growth were cast by using 1.6 cm² polydimethylsiloxiane (PDMS) molds with aligned microgrooves at a density of 600 lines/mm followed by water annealing, PEO leaching and UV-sterilization. Patterned films were functionalized with Arginine-Glycine-Aspartic acid-Serine (RGD) peptides (Bachem, Torrance, CA) and used within one week of functionalization.

The hPSCs were directly differentiated into hIECs via generation of hIEC progenitors according to a previously reported procedure.^{26 (link)} The hIECs progenitors were maintained with hIEC differentiation medium 1 (DMEM/F-12, 1% P/S, 1% HEPES, 1% NEAA, 1% L-glutamine, 1% N2 supplement, 1% B-27 supplement, 2% FBS, 100 ng/mL epithelial growth factor [EGF], 100 ng/mL R-spondin1, and 5 μg/mL insulin [Thermo Fisher Scientific]). To further differentiate functional IECs, 1.34 × 10⁵ hIEC progenitor cells/cm² were seeded onto 1% Matrigel coated transwell plates (Corning) and treated with hIEC differentiation medium 1 containing 10 μM Y-27632. After 2 d, the cells were moved to hIEC differentiation medium 2 (DMEM/F-12, 1% P/S, 1% HEPES, 1% NEAA, 1% L-glutamine, 1% N2 supplement, 1% B-27 supplement, 2% FBS, 100 ng/mL EGF, 2 μM Wnt-C29 [Selleckchem, Houston, TX, USA], 1 mM VPA [Stemgent, Houston, TX, USA]). The medium was changed every other day. After 14 d, the bacterial co-culture experiment was performed under aerobic and anaerobic conditions. All experiments were performed at least two biological replicates.