Penicillin streptomycin mixture

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin mixture were obtained from Millipore Sigma (Merck KGaA, Darmstadt, Germany). Dulbecco’s Modified Eagle’s Medium with no glucose (cat. no. 11966025) was obtained from Gibco (Thermo Fisher Scientific, Inc, Waltham, MA, USA). Thiazolyl blue tetrazolium bromide (MTT; cat. no. M5655), neutral red solution (NR; cat. no. N2889), sulforhodamine B (SRB; cat. no. S1402), 2, 7- dichlorofluorescein diacetate (DCFH-DA; cat. no. D6883), edaravone (cat. no. M70800), and metformin (cat. no. 317240) were obtained from Sigma–Aldrich (Merck, Algés, Portugal). Perampanel (cat. no. 23003) was obtained from Cayman (Tallinn, Estonia).

Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), and Penicillin-Streptomycin mixture were obtained from Millipore Sigma (Merck KGaA, Darmstadt, Germany). Bisbenzimide H33342 trihydrochloride (Hoechst 33342; cat. no. B2261), Thiazolyl Blue Tetrazolium Bromide (MTT; cat. no. M5655), Neutral Red Solution (cat. no. N2889), Sulforhodamine B (cat. no. S1402), propranolol (cat. no. P0884), and isoprenaline (cat. no. I5627) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). paclitaxel (Taxol; cat. no. 1097) and ICI 118,551 (cat. no. 0821) were purchased from Tocris Bioscience (Bristol, UK). Waters Alliance 2695 pump, 3030 Reagent kit^®, Rheodyne loop injector, and the Decade electrochemical detector (ECD) were purchased from, respectively, Waters Corporation (Milford, MA, USA), Chromsystems GmbH (Munich, Germany), and Antec Scientific (Zoeterwoude, The Netherlands).

CHL‐1 cells were cultured in RPMI‐1640 medium (Sigma Aldrich) supplemented with 10% foetal bovine serum (Thermo Fischer) and 50 U/ml of a penicillin/streptomycin mixture (Sigma Aldrich). The cells were grown in 75 cm² flasks (Corning Inc.) and incubated at 37°C with 5% CO².

MnCl₂•4H₂O, L-polylysine, D-Hank’s solution, and penicillin–streptomycin mixture were purchased from Sigma. DMEM-F12 medium was from HyClone. Neurobasal-A medium (without phenol red and serum) and fetal bovine serum (FBS) were purchased from Gibco. Human leukocyte antigen B27 (B27) was obtained from Invitrogen Life Technologies.

Caco-2, the colon adenocarcinoma cell line, was used to evaluate the adhesion ability of E. faecium CM33 to human epithelial cells. RPMI medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin mixture (Sigma Chemical Co., Poole, UK) was used to culture human colon cancer cells. Cells were seeded on 24-well tissue culture plates and incubated at 37°C in 5% CO₂ in a humidified atmosphere. Before the adhesion assay, fresh antibiotic-free RPMI was used to wash wells, which have a monolayer of Caco-2 cells. Then, each well was inoculated with 1 × 10⁷ cfu/ml of E. faecium in a total volume of 1 ml and incubated at 37°C for 3 h in an atmosphere of 5% (v/v) CO₂. The wells were washed thrice with a pre-warmed phosphate buffer saline (Sigma Chemical Co., Poole, UK) to remove non-attached cells of E. faecium. To detach the cells, 1 ml of 1% Triton X-100 (Sigma Chemical Co., Poole, UK) was added to each well with gently stirring for 5–10 min and then the viable human colon cancer cells, Caco-2, were counted. Finally, bacteria and Caco-2 cells were cultured onto MRS agar by the pure plate method and incubated at 37°C anaerobically. The total number of bacteria attached to viable Caco-2 cells expressed as bacterial adhesion and carried out in triplicate.

In order to investigate the biocompatibility of simple and antibiotic-loaded P(3HB-3HV)-CS spheres, the human dermal fibroblasts CCD-1070Sk cell line (ATCC^® CRL-2091™) was employed. Briefly, cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma/Merck, Steinheim, Germany), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin mixture (Sigma/Merck) all throughout the experiment in standard cell culture conditions (5% CO₂, 37 °C). One day prior to spheres treatment, CCD-1070Sk cells were seeded in 12-well or 96-well plates at an initial density of 1 × 10⁵ and 0.1 × 10⁵ cells/well, respectively, and incubated overnight to allow cellular attachment. The next day, the culture medium was discarded and replaced with fresh medium for experimental controls and with simple and antibiotic-loaded spheres at a final concentration of 1 mg/mL. Biocompatibility assays were performed as described below after 24 h and 7 days of treatment.

The cell line selected for this study, HT-29 (ATCC^® HTB-38 ™)—human colorectal carcinoma—was acquired as a frozen item from the American Type Culture Collection (ATCC). For cells’ culture and growth, a specific culture medium was used—McCoy’s 5a modified medium (ATCC^® 30-2007 ™)—which was completed with 10% FBS (fetal bovine serum, Gibco) and a 1% penicillin/streptomycin mixture (Sigma Aldrich, Merck KGaA Darmstadt, Germany). The experiments were performed in accordance with the standard conditions for cell culture, as follows: incubation at 37 °C in 5% CO₂ atmosphere.