Bio agar

PM-derived TDO were developed as described in Fujii et al. (2016) (link) and Bozzi et al. (2017) (link). TDO were grown in basal cell culture medium consisting of advanced DMEM-F12 (ThermoFisher Scientific) and supplemented with different growth factors (Supplementary Table S2) to mimic different niche factor conditions, as described in Fujii et al. (2016) (link). Incubation was performed at 20% O₂ and 5% CO₂. After expansion, the TDO were cultured in cell culture medium lacking growth factors, which was refreshed every three days. Optimal cell culture medium conditions were determined separately for each organoid culture (Supplementary Table S3).
Organoids were split every 1–2 weeks as follows: they were mechanically removed from the Matrigel by pipetting, incubated in Cell Recovery Solution (Corning) for 1 h at 4°C, washed twice with ice-cold PBS, and seeded as described above.
Aliquots of each organoid culture were frozen or prepared for IHC analyses as follows: samples were fixed in 10% formalin at room temperature for 10 min and embedded into 200 µl Bio-Agar (Bio-Optica). The samples were then cooled at –20°C until solidification. For each sample, sections of 3 µm thickness were obtained.

Sections of formalin-fixed, paraffin-embedded ascites and solid tumor patient-derived organoids were used for histopathological analyses. Organoids were recovered from BME using ice-cold cell recovery solution (Corning, New York, United States) in accordance with manufacturing protocols, fixed in phosphate-buffered 10% formalin, and embedded in 500 μL of Bio-Agar (Bio-Optica Milano Spa, Milano, ITA). Five μm sections were stained with hematoxylin and eosin (H&E) using a Leica ST5020 multistainer and 2 μm sections were cut for IHC analysis. The IHC was performed with an UltraVision LP Detection System HRP DAB kit (Thermo Scientific, Waltham, USA). Heat-induced antigen retrieval was performed using 10 mM citrate buffer pH 6.0. The following antibodies were used to characterize HGOC patients-derived organoids and parental tumors: PAX8 (ProteinTech Group, Germany, EU), WT1 (Abcam, U.K.), and CA-125 (Santacruz Biotechnology, TX).

According to previously reported cytomorphological evidences, microscopic examination of H&E-staining revealed that liver organoids show the typical features of hepatic progenitors and mature hepatocytes [34 (link)]. Organoids were washed with PBS and embedded using Bio-Agar (Bio-Optica, Milano, Italy). Subsequently, the blocks were fixed in 10% neutral buffered formalin overnight and processed for paraffin embedding. Sections of 2.5 µm thickness were used for hematoxylin and eosin (H&E) staining by using Leica ST5020 Multistainer. Mouse liver organoids were evaluated and analyzed untreated and treated with 100, 500, 1000 µM selected PFAS and the induction of morphological changes evaluated at 6, 24 and 48 h by histological analysis.