Metabolic Profiling of MSCs on Nanotopographies

MSC were seeded on nanotopographies at 1000 cells/cm² and allowed to grow for 11 days. Cells were washed and incubated in basal media comprising 50% normal glucose and 50% ¹³C₆-Glucose (Cambridge Isotopes Ltd) for a further 3 days. Extractions were performed as above. LC-MS was performed as previously described⁶⁹. Briefly, the LC-MS platform consisted of an Accela 600 HPLC system combined with an Exactive (Orbitrap) mass spectrometer (ThermoFisher). Two complementary columns were used; the zwitterionic ZIC-pHILLIC column (150 mm × 4.6 mm; 3.5 μm, Merck) and the reversed phase ACE C18-AR column (150 mm × 4.6 mm; 3.5 μm Hichrom) and in both cases sample volume was 10 μl at a flow rate of 0.3 ml/min. Eluted samples were then analysed by mass spectrometry.
LCMS data of ¹³C-labelled extracts were processed to generate a combined PeakML file as described previously^{65 (link)}. Further analysis using mzMatch-ISO in R⁶⁶ generated a PDF file containing chromatograms used to check peak-shape and retention time. A tab-delineated file detailing peak height for each isopotologue was also generated to calculate percentage labelling.