6 cm bacteriological petri dishes

Induction of differentiation was achieved through embryoid body (EB) formation via hanging drop culture following a procedure adapted from De Smedt et al.[26] (link). In brief, stem cells were thawed and a suspension was prepared at a concentration of 3.75×10⁴ cells/ml in ESGRO Complete Basal Medium (EMD Millipore), which does not contain LIP, BMP-4, or GSK3b-I. About 50 drops (each of 20 µl) of the cell suspension were placed onto the inner side of the lid of a 10-cm Petri dish filled with 5 ml phosphate buffered saline (PBS; EMD Millipore) and incubated at 37°C and 5% CO₂ in a humidified atmosphere. After 3 days, EBs formed in the hanging drops (Ø330–350 µm) were subsequently transferred into 6-cm bacteriological Petri dishes (Becton Dickinson Labware, Franklin Lakes, NJ) and were further cultivated for 2 days. On day 5, EBs were plated one per well into 24-well tissue culture plates (Thermo Scientific Nunc, Roskilde, Denmark). During further development of the attached EBs, cells of endodermal, ectodermal and mesodermal origin were obtained in the outgrowths. In EST, differentiation was determined by microscopic inspection of contracting cardiomyocytes in the EB outgrowths on day 10.

Induction of differentiation was achieved through embryoid body (EB) formation in hanging drop culture following a procedure adapted from De Smedt et al. [25 (link)]. In brief, stem cell suspensions were prepared on ice at a concentration of 3.75 × 10⁴ cells/ml in ESGRO Complete Basal Medium (EMD Millipore), which does not contain LIP, BMP-4, or GSK3b-I. About 50 drops (each of 20 µl) of the cell suspension were placed onto the inner side of the lid of a 10-cm Petri dish filled with 5 ml phosphate buffered saline (PBS) (EMD Millipore) and incubated at 37 °C and 5% CO₂ in a humidified atmosphere. After 3 days, EBs formed in the hanging drops were subsequently transferred into 6-cm bacteriological Petri dishes (Becton–Dickinson Labware, Franklin Lakes, NJ) and were exposed to AgNPs or Ag⁺. The EBs had an average diameter of 330–350 μm.