Ex cell advanced cho fed batch medium

All cultivations were carried out in 250-ml flasks with 50 ml filling volume, being shaken on an orbital shaker with a 50 mm shaking diameter. The shaking frequency was set to 140 rpm and the temperature to 36.5°C. Flasks for passaging and manual sampling with subsequent offline analysis were cultured in an incubator (ISF1-X, Kühner AG, Switzerland) at a controlled relative humidity of 70% and a controlled CO₂ concentration of 5%, respectively. Flasks for time-resolved monitoring of the OTR were operated in parallel in an incubator (ISF1-X, Kühner AG, Switzerland) under the same conditions but without CO₂ and humidity control. Instead, these flasks were directly gassed with a gas mixture of 5% CO₂ in synthetic air (19.5% O₂) from a gas bottle (Figure 1A). Cultivations were either carried out in PowerCHO^TM two serum-free chemically defined culture medium, containing HEPES buffer and Pluronic® F68 (Lonza AG, Switzerland) (“medium 1”) or in EX-CELL® Advanced™ CHO Fed-batch Medium (Sigma-Aldrich, United States) (“medium 2”). Both culture media were supplemented with 6 mM L-glutamine from a 200 mM stock solution (Gibco Life Sciences, Thermo Fisher Scientific, United States) before cultivation. Directly before use, culture media and supplements were pre-heated to 36.5°C for about 30 min in a water bath (VWB2 12, VWR, USA).

The OVCAR3 and OVCAR8 OC cell lines, both widely regarded to originate from HGSC, were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions. OVCAR3 cells were cultured in DMEM, OVCAR8 in RPMI (Biological Industries, Beit-Haemek, Israel). The medium was supplemented with 1% L-glutamine, 1% sodium pyruvate, 1% vitamin solution, 1% non-essential amino acids (Biological Industries) and 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in a humidified atmosphere of 95% air and 5% CO2. In cells from which exosomes were extracted, the medium used was EXCELL^® Advanced™ CHO Fed-batch Medium (Sigma-Aldrich). OVCAR3 and OVCAR8 OC spheroids were formed by constant shaking of 400,000 cells per well in 6-well plates for 48 h on a vertical shaker.

Sodium phosphate (Merck) and glycine·HCl (Merck) were purchased from Merck. Tris-HCl and EX-CELL Advanced CHO Fed-batch medium were all purchased from Sigma Aldrich. ACN (Part no: A955-4, Fisher Scientific), PVDF syringe filter (Part no: SLGV013SL, Millex 0.22um PVDF 13 mm sterile syringe filter) were obtained from Merck Millipore (Ireland), whilst centrifugal filters, (Part no: UFC3096, Amicon Ultra Centrifugal Filters) was purchased from Merck Millipore, (USA). Protein A HP Spintrap (28-9031-32) was purchased from GE Healthcare, USA. FAST Glycan Kit (Part no: B94499PTO, SCIEX, USA). Ammonium formate (Part No. 186007081), RapiGest SF (Part No. 186001860), RapiFluor-MS Reagent Solution (Part No. 186008091), and ACQUITY UPLC^® Glycan BEH Amide Column were purchased from Waters Corp.