ISOLATION OF A NEW POPULATION OF LACTICASEIBACILLUS CASEI BL23 EXTRACELLULAR VESICLES BY HYDROSTATIC FILTRATION?.

CECILIA D´ANTONI; ANA PAULA DOMINGUEZ RUBIO; MARCELA MORETTON; DIEGO CHIAPPETTA; MARIANA PIURI; OSCAR E PÉREZ

Mar del Plata

Encuentro; Reunión Anual de Sociedades de Biociencias; 2022

Sociedad Argentina de Investigación Clínica (SAIC), la Sociedad Argentina de Inmunología (SAI) y la Sociedad Argentina de Fisiología (SAFIS).

Microbiota-derived extracellular vesicles (EVs) are naturally produced delivery systems with potential therapeutic purposes. The traditional method to isolate EVs is ultracentrifugation (UC). However, this method has many disadvantages, like EV lysis and high rates of contamination with bacterial proteins. The aim of this work was to compare the alternative method hydrostatic filtration (HF) for the isolation of EVs released by Lacticaseibacillus casei BL23 with the UC traditional method1. To this end, a 48 h-L. casei BL23 culture was centrifuged at 4500 g, and the cell-free supernatant was filtered using a 0.45 μm pore size. The filtered supernatant was poured inside a cellulose ester dialysis membrane (MWCO=1000 kDa). After 24h, the filtrate was dialyzed in PBS for 3 h. Dialyzed samples were concentrated using a Centricon ultrafiltration system (MWCO=100 kDa). We compared SDS-PAGE protein profiles of samples obtained by UC, by HF, and by HF followed by UC. For each sample, we determined the particle size distribution.The protein profile was similar among samples. The size distribution for samples obtained by HF (n=4) showed a bimodal distribution (peaks: 15.9 nm, 91.1 nm) unlike the resuspended pellet obtained by UC, in which a single size population was observed (peak: 62.3 nm). A bimodal distribution was also observed in the UC supernatant after HF (peaks: 17.06 nm, 84.46 nm). By using transmission electron microscopy (TEM), the presence of the larger size EV population was confirmed, and there were elements with a diameter below 30 nm.HF allowed for the isolation of two populations, while a single population is recovered in the UC pellet and one population remains in the supernatant. We described here the presence of a small population that may have implications in the development of natural or engineered nanoparticles for the delivery of small RNAs, proteins and drugs, with promising applications in nanomedicine.1 Domínguez Rubio AP, Martínez JH, Martínez Casillas DC, Coluccio Leskow F, Piuri M, Pérez OE. Lactobacillus casei BL23 Produces Microvesicles Carrying Proteins That Have Been Associated with Its Probiotic Effect. Front Microbiol. 2017 Sep 20;8:1783. doi: 10.3389/fmicb.2017.01783. PMID: 28979244; PMCID: PMC5611436.