Balb 3t3

BALB/3T3 (normal murine fibroblasts) and MCF 10A (human, non-tumorigenic mammary gland epithelial cells) were obtained from the American Type Culture Collection (ATCC; Rockville, Maryland, USA). The BALB/3T3 cell line was cultured in high glucose DMEM (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine (Sigma-Aldrich). The MCF 10A cell line was cultured in Ham’s F12 medium with glutamine (Corning Costar) supplemented with 5% (v/v) FBS, 5% (v/v) horse serum, 10 µg/mL insulin, 0.05 µg/mL cholera toxin, 0.5 µg/mL hydrocortisone, and 20 ng/mL hEGF (all from Sigma-Aldrich). All culture media contained 100 µg/mL streptomycin (Sigma-Aldrich) and 100 U/mL penicillin (Polfa Tarchomin SA, Warszawa, Poland). The cells were grown at 37 °C in a humid atmosphere saturated with 5% CO₂.

Mouse fibroblast BALB/3T3 cells were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The cells were routinely grown in standard Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 25 mM glucose and 10% fetal bovine serum under 5% CO₂ at 37 °C to mimic diabetic conditions. To compare the effect of SF-film prepared with different degumming methods on cell viability, a SF-film of 1 cm in diameter was placed into a well of a 24-well plate, then inoculated with approximately 5 × 10⁵ cells. After incubation for 3 days, cells were treated with the LIVE/DEAD™ Viability/Cytotoxicity Kit (Thermo Fisher Scientific, Waltham, MA, USA) and examined under a fluorescence microscope according to the manufacturer’s instructions. Quantitative analysis of BALB/3T3 fibroblast grown on the SF-films was performed using 1/10 dilution of the purchased alamarBlue reagent (Thermo Fisher Scientific, Waltham, MA, USA) in DMEM for 4 h under 5% CO₂ at 37 °C. The 100% reduced form of alamarBlue reagent, achieved by autoclaving for 10 min, was used as the 100% of cell proliferation (positive control). The fluorescence of the culture supernatant was determined using an excitation and an emission at 570 and 600 nm, respectively. The cell proliferation rate was calculated according to the formula suggested by the dye manufacturer.

Antiproliferative tests were performed on a normal human breast cell line, MCF-10A, and a mouse embryonic fibroblast: Balb/3T3. Both cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). The cell lines were maintained in the Institute of Immunology and Experimental Therapy, Wroclaw, Poland. MCF-10A cells were cultured in the F-12 nutrient mixture (Gibco, Scotland, UK), supplemented with 5% horse serum (Gibco, Scotland, UK), 10 μg/mL of cholera toxin (Vibrio cholerae Pacini), 10 μg/mL of hydrocortisone and 20 ng/mL of human epidermal growth factor (all from Sigma-Aldrich, Chemie GmbH, Steinheim, Germany). Balb/3T3 cells were cultured in Dulbecco medium (Gibco, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM l-glutamine (Sigma-Aldrich, St. Louis, MO, USA). All culture media contained antibiotics: 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/mL streptomycin (Polfa-Tarchomin, Warsaw, Poland). Both cell lines were cultured during the entire experiment in a humid atmosphere at 37 °C and 5% CO₂. Amistar 250 SC (fungicide) with active compound azoxystrobin was provided by the Institute of Soil Science and Plant Cultivation, National Research Institute in Pulawy, Department of Weed Science and Tillage Systems in Wroclaw, Poland in a humid.