A human pancreatic β-cell line (1.1B4) was maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS), 1% 200 mM L-glutamine and antibiotics - 100 μg/mL streptomycin and 100 units/mL penicillin. Half of the dishes were cultured under NG conditions (11 mM D-glucose), while the second half was grown under long-term (3 passages) HG conditions (35 mM D-glucose). Collected conditioned media were centrifuged to remove cell fragments and debris . Then, supernatants in a volume of 300 mL were concentrated using the 1000 kDa (MWCO - molecular weight cut-off) dialysis cellulose membranes (Spectra/Por Biotech) at low vacuum (−0.3 Bar) to obtain 1 ml of the EV sample. In the next step, the concentrated sample was centrifuged to remove apoptotic bodies. The supernatant was divided into two Eppendorf tubes, which were then subjected to various fractions of EVs: mixed fraction of EVs, ectosomes fraction and exosomes fraction.

Celem pracy było przygotowanie biblioteki widm uzyskanych metodą spektrometrii masowej jonów wtórnych z analizatorem czasu przelotu (ToF-SIMS). Za pomocą widm badano zmiany zawartości wybranych aminokwasów i lipidów w próbkach pęcherzyków zewnątrzkomórkowych (EV). Próbki EVs izolowano z ludzkich komórek β trzustki hodowanych w warunkach normalnych (kontrola) i hiperglikemicznych (stężenie glukozy 35 mM). Do oddzielenia ektosomów i egzosomów zastosowano filtrację niskopróżniową (L-VF) i ultrawirowanie. Objętość 30 µl każdej próbki w stężeniu cząstek 3E9 cząstek/ml nałożono na przygotowane podłoża krzemowe i pozostawiono do przylegania podczas 1-godzinnej inkubacji.

The aim of the work was to prepare a library of spectra obtained by means of time of flight secondary ion mass spectrometry (ToF-SIMS). The changes in the content of selected amino acids and lipids in samples of extracellular vesicles (EVs) were investigated with the help of obtained spectra. EVs samples were isolated from human pancreatic β cells grown under normal (control) and hyperglycemic conditions, with a glucose concentration of 35 mM. Low-vacuum filtration (L-VF) and ultracentrifugation were used to separate ectosomes and exosomes. A volume of 30 µl of each sample at a concentration of 3E9 particles/ml was applied to the prepared silicon substrates and allowed to adhere during the 1-hour incubation.