Hepes 1m

The human HEPA-RG hepatoma cell line (Thermo Fisher Scientific, Waltham, MA, USA) was cultured using a hepatocyte bullet kit medium added with 10% FBS, depleted of exosomes (Lonza Biowhittaker, Oslo, Norway), and with 1% of 100 mM Antibiotic-Antimycotic (penicillin 10,000 91 u/mL, streptomycin 10,000 u/mL, (Lonza Biowhittaker, Oslo, Norway). The LX2 Human Hepatic Stellate cell line (Millipore, Merck Life Science, Milan, Italy) was cultured using Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA), added with 10% of FBS depleted exosomes, 1% of 100 M Antibiotic-Antimycotic, 2.5% of HEPES 1M (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) (Gibco™, ThermoFisher Scientific, Waltham, MA, USA) and 1% of 100 mM Sodium Pyruvate (Gibco™, ThermoFisher Scientific, Waltham, MA, USA). For all treatments with exosomes, both cell lines were cultured on semi-confluent cell monolayers and were treated with exosomes derived from patients at a concentration of µg exosome proteins/µL of medium, specifically at 20 µg/µL for 24, 48, and 72 h.

Sera or antibody diluted in neuronal differentiation medium was added after about 3 weeks to the co-culture and was incubated for 3 h. For FDSS, medium was replaced by Ca²⁺ buffer (50 ml HBSS 10x (ThermoFisher, cat-# 14065049), 10 ml HEPES 1 M (ThermoFisher, cat-# 15630056), 440 ml dH₂O and 375 µl CaCl₂ (1 M). After 40 min, all plates were read by FDSS/µCELL Functional Drug Screening System (Hamamatsu, cat-# C13299) after injection of 0.5 µM AMPA (Sigma, cat-# A6816). Analysis was done with Hamamatsu software.

Human prostate cell lines were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and maintained at 37  °C in humidified atmosphere at 5% CO₂. RWPE-1 cells (immortalized cells from non-tumorigenic prostate tissue) were cultured in keratinocyte serum free medium (K-SFM, Cat. N. 17005034, Gibco^®, Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant epidermal growth factor. LNCaP cells (malignant prostate cells derived from a lymph node metastasis) were cultured in DMEM 1.0 g/L glucose (Cat. N. P04-01550, Pan Biotech, Aidenbach, Germany,) supplemented with 10% fetal bovine serum (Cat. N. 10270106, Gibco^®), 1% HEPES 1M pH 7.2 and 1% penicillin-streptomycin (Gibco^®). All cells were checked periodically for mycoplasma infection and tested non-infected.