Treatment with 1 µg/mL bergamot PF extract reduces intracellular lipid content in 3D spheroids. (A) HepG2 + LX2 cells were cultured as 3D spheroids for a total of 96 h. Initially, 24 h after seeding cells, the media was replaced every 48 h. Objective 20X. (B) HepG2 + LX2 cells were cultured as 3D spheroids for a total of 96 h. Initially, 24 h after seeding, the media was supplemented with 1 µg/mL bergamot PF extract and the media was replaced every 48 h. Objective 20X. (D) The average volume was calculated by measuring their long and short diameter using the ZEN 2.3 Lite software (Zeiss). (E) For both spheroids, cellular ATP levels remained stable between the experimental groups. (C) Intracellular lipid content was measured by Oil red-O staining; the results showed a significant reduction after incubation with bergamot PF extract. Data are shown as mean ± SD of three independent experiments. Statistical analysis was as follows: Mann–Whitney non-parametric test vs. 0. p = 0.03. Abbreviations are as follows: PF, polyphenols fraction; RLU, relative fluorescence unit; 3D, three dimensional; SD, standard deviation.