Particle Uptake in Cell Cultures

Sterilized discs (diameter 6 mm) were punched from transparent Melinex film (Plano, Wetzlar, Germany), treated with 70% ethanol for 30 min, dried, and placed into the wells of a microtiter plate. Cells were seeded (3 × 10⁵ cells/well) in F-12 K medium with 5% serum and cultured on the discs. After 24 h, the medium was replaced by serum-free F-12 K medium containing 100 µg/mL of either PEBCA or PEBCA CBZ. Particle uptake was then continued for 4 and 16 h under cell culture conditions (37 °C, 5% CO₂). Then, the cells were fixed with 2.5% glutardialdehyde in 0.1 M sodium phosphate buffer for 60 min. Cells were washed in PBS, post-fixed in 1% OsO₄, stained en bloc with uranium acetate (1%), and embedded in Epon 812 (Sigma-Aldrich, Taufkirchen, Germany) as described earlier [20 (link)]. Isolated PEBCA particles were embedded in warm agar dissolved in phosphate-buffered saline (PBS) containing 3% bovine serum albumin and cooled on ice. Small pieces of the hardened mixture were embedded in Epon 812 and processed as described for the cells. Thin sections (50–60 nm) of all preparations were cut with a Reichert Ultracut microtome and viewed with a Tecnai G2 electron microscope at 120 kV. Digital images were taken with a Quemesa digital camera (Olympus Soft Imaging Solutions, Münster, Germany).