Untargeted metabolomics analysis of RPE cells during six month in culture

Primary RPE cell cultures were established with human fetal RPE cells acquired from ScienCell (Cat. No, 6540) seeded at passage 3 (P3) in 12-well Transwell^® inserts (Corning Inc., Cat. No. CLSS3460) coated with 2% v/v Geltrex^® matrix (Thermo Fisher Scientific; Cat. No. A1413202). Cells were cultured for a total time of 6 months (25 weeks), following the same protocol as previous works (16,59). Samples were acquired for protein immunolocalization, transcriptomic and metabolomics analysis from independent cell cultures along the total 6 months culture time (specifically at 4, 12, 17 and 25 weeks in culture), collecting 3 biological replicates per type of analysis and time point. Cell metabolites were quenched and extracted using MeOH:H₂O (80:20, -20ºC) containing a spiked solution of isotopically enriched low molecular mass internal standard. Quality control (QC) samples were prepared by creating a pool of equal volumes from each biological replicate and were analyzed after a blank solution every fifth sample to monitor the performance, stability, and reproducibility of the LC-MS run. A liquid chromatography (LC) system 1290 Infinity II (Agilent Technologies) was coupled to an Agilent 6560B Ion Mobility quadrupole-time-of-flight mass spectrometer (IM-QTOF-MS) equipped with an Agilent G1607A dual jetstream ESI source and the MassHunter WorkSation 11.0. A reference solution containing purine and hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazene for mass correction was added using the second ESI source. Chromatographic, ion source and MS conditions were optimized using QCs and selected parameters are shown in Supporting Information. MS analysis was conducted with an untargeted approach, operating the instrument in both positive and negative ionization modes. All samples were analysed in a randomized order. Raw data was processed using the software Profinder B10.00 (Agilent) and refined data were exported as CEF files. Data were converted to mzml files using MS convert.