Poly 2 hydroxyethyl methacrylate poly hema

As the BLS implantation model has been demonstrated to be an accurate and effective in vitro assay for use in embryo implantation research, it was employed in these studies, with modifications [21 (link), 22 (link)]. Briefly, BeWo cells were detached with 0.25% trypsin (Gibco BRL/Invitrogen, Carlsbad, CA, USA) after they had reached 80% confluence. The BeWo cell suspensions were then placed in 35 mm² dishes coated with an anti-adhesive polymer, poly-2-hydroxyethyl methacrylate (poly-HEMA; Sigma, St. Louis, MO, USA), to induce the formation of BLSs with diameters ranging from 150 to 200 μm after 48 h of culturing. We cocultured BLSs with hESCs to generate an in vitro model of embryo implantation. Confluent monolayer hESCs were decidualized for 3 days before being exposed to BLSs in 24-well culture plates as indicated in each figure legend. After substandard BLSs were removed using a 0.15 mm filter, several BLSs were transferred per chamber onto the confluent monolayer hESCs under a dissection microscope. The cocultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂ for 2 days. The attached and expanded BLSs were photographed at the indicated time points, and the area of expansion was expressed as a fold increase normalized to that of the untreated group. All cocultures were monitored using a microscope (Leica, Wetzlar, Germany).

The breast (MCF-7 and MDA-MB-231) and the lung (A549 and NCI-H23) cancer cells were maintained according the American Type Culture Collection (ATCC) recommendations. The 3D-rBM assays were performed as previously described^{4 (link)}. For non-adherent 3D culture assay, poly-2-hydroxyethyl methacrylate (polyHEMA, Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in 95% ethanol at 12 mg/ml. Plates were then coated at 0.8 mg/cm² and air dried at 37 °C overnight. Also, 1 × 10⁴ cells/cm² were seeded as single cells, on polyHEMA-coated plates, in Dulbecco’s modified Eagle’s medium supplemented with 2.5% fetal bovine serum. After 48 h (time for 3D structure formation), for the conditions in which the basement membrane effect was evaluated, the media was supplemented with 4% Matrigel^®. The cellular viability in response to cisplatin (60 μM) was initiated 4 days after cell plating.

A549 lung adenocarcinoma, DU-145 and PC-3 prostate cancer as well as MCF-7 breast cancer cell lines were purchased from ATCC and maintained in Roswell Park Memorial Institute 1640 (RPMI) medium (Biosera, Metro Manila, Philippines) complemented with 10% Fetal Bovine Serum (FBS) (EuroClone, Pero MI, Italy), 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA), 0.01% streptomycin and 0.006% penicillin (Biowest, Nuaille, France). Cells were cultured under standard conditions in a 37 °C incubator at 5% CO₂ and 95% humidity.
For 3D cell culture experiments all the solutions were filtered through 0.22 µm membrane filter (Merck, Darmstadt, Germany). U-bottom 96-well plates (Greiner Bio-One, Kremsmünster, Austria) were coated with poly(2-hydroxyethyl methacrylate) (poly-HEMA, Sigma-Aldrich, St. Louis, MO, USA) using 2 times 15 µL of 6 mg/mL poly-HEMA dissolved in 96% ethanol then the plates were left to dry and were UV-sterilized. For spheroid formation 10⁴ cells were seeded into each well of 96-well plates, then cells were centrifuged with 280 g for 5 min and left to grow for a week at 37 °C, 5% CO₂ and 95% humidity in filtered RPMI medium (Corning, Corning, NY, USA) containing 10% FBS (Gibco, ThermoFisher Scientific, Waltham, MA, USA), 2 mM glutamine, 0.01% streptomycin and 0.006% penicillin.

The breast cancer cell lines MCF7, MDA-MB-231, T47D and normal breast epithelial cell line MCF10A were purchased from ATCC (Middlesex, UK). HeCV, the normal human vascular endothelial cell line, [52 (link)] was kindly provided by Prof W Jiang (Cardiff University, UK). Gemcitabine (dFdC), doxorubicin (Dox), paclitaxel (PTX), copper(II) chloride (CuCl₂), copper gluconate (CuGlu), N-acetyl-cysteine (NAC) and poly-2-hydroxyethyl methacrylate (poly-HEMA) were purchased from Sigma (Dorset, UK). Lipo-DS was provided by Prof X Tang (Shenyang Pharmaceutical University, China). The drug loading content of Lipo-DS is 3mg/ml. The cumulative release of DS from liposome in 120h is more than 60%. The pharmacokinetic study of DSF in rat plasma after intravenous administration of a dose of 36 mg/kg has been investigated (t_1/2 =0.1 h and t_1/2d=0.3 h). The comparison of the biological activity between Lipo-DS and conventional DS has been published [53 ].

Mammosphere culture was performed as previously described [32 (link)]. Breast CSC-like cells (CD44⁺CD24^−/low) and non-CSC (CD44^−/lowCD24⁺) cells isolated from BT-549, Hs578T, MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7, T47D, and tumor sample cells were plated in six-well plates coated with 2% poly-2-hydroxyethyl methacrylate (poly-HEMA, Sigma-Aldrich, USA) at a density of 1×10⁴ cells/ml and 5 × 10³ cells/ml in following passages. Cells were cultured in a serum-free DMEM/F12 (Gibco, USA) medium supplemented with B27 (Gibco, USA), 20 ng/ml epidermal growth factor (EGF, Invitrogen), 20 ng/ml basic fibroblast growth factor (bFGF, Invitrogen, USA), 0.4% albumin from bovine serum (BSA, Sigma-Aldrich, USA), 2 μg/ml heparin (Sigma-Aldrich, USA), and insulin-transferrin-selenium (Invitrogen, USA). Mammospheres were incubated at 37 °C with 5% CO₂ and passaged every 7 days. To test the self-renewal capability, the number and size of mammospheres (diameter > 50 µm) were counted manually, and representative images were obtained using an OLYMPUS IX70 microscope (Tokyo, Japan). The percentage of mammosphere-forming efficiency (MFE) was calculated using the following equation: (number of mammospheres per well/number of cells seeded per well) × 100. The average size of mammospheres (N = 30 spheres) was calculated.