Fig 2: Treatments of organoid with fatty acids do not change organoid volume and viability but enhance APOB secretion. (A) 3D spheroids HepG2/LX-2 ratio 24:1 were treated, after 48 h from the seeding with: BSA 1%, a mix of palmitic acid and oleic acid 500 µM (1:2), TGF-ß 10 ng/mL or PDGF 10 ng/mL for 24 or 48 h. (B) Spheroid volumes were calculated measuring their long and short diameter by ZEN 2.3 Lite software (Zeiss) (n = 3). (C) Cellular ATP levels normalized to spheroids volumes remained stable throughout 4 days of culture (n = 3). (D) APOB secretion levels in the media fractions measured by Western blotting were higher in spheroids treated with PAOA while they were lower in spheroids treated with TGF-ß (n = 7). Bars represent mean ± SD. P-value was calculated by Mann-Whitney non-parametric test, (* p < 0.05 vs. BSA 1%). BSA: bovine serum albumin; PAOA: palmitic acid/oleic acid; TGF-ß: Transforming growth factor-ß; PDGF: Platelet-derived growth factor; APOB: Apolipoprotein B; AU: arbitrary unit.

Fig 3: Western blot analysis of EV markers, CD63 and TSG101, and lipoprotein markers, APOB, and APOE. (A) 4 µg of vesicular protein isolated from 200 µl human serum using each method alone or in combination were separated by SDS–PAGE and immunoblotted with anti-human CD63 (H-193 and H5C6), TSG101, APOE and APOB antibodies. Representative images are shown. (B) Graph: Densitometric quantitation of the distribution of CD63, TSG101, APOE and APOB protein expression in each method. Black lines represent medians. N = 3 isolations. Arbitrary Units (AU) = (signal intensity method)/Sum (signal intensity method1–8).

Fig 4: Chitosan-mediated sEV isolation from human plasma. (a) Human plasma (0.25 ml) was diluted 1:4 with PBS and subjected to pre-clearing as described in Figure 1, followed by sEV isolation by scUCF, the addition of the acidic formulation of chitosan to a final concentration of 50 µg/ml, or buffer. Western blot analyses of canonical EV markers CD63, CD9, HSC70, and FLOT1, as well as the non-sEV marker CANX; total cell lysate from MCF-10A cells was used as a positive control for CANX. (b) Western blot analyses of common contaminant plasma proteins APO-A1 and APOB as well as FLOT1, a canonical EV marker; total cell lysate from HEK-293 cells and 5 µl of plasma lysate (undiluted) were used as a positive controls for CANX, APO-A1, and APOB. (c) NTA analysis of sEVs isolated from plasma by scUCF, the addition of the acidic formulation of chitosan to a final concentration of 50 µg/ml, or buffer. Particle concentration (particles/ml) is plotted against size (nm) of particles. Mean plus the standard error of the mean (n = 3) is shown

Fig 5: Co-culture of HepG2 and LX-2 cells enhances the compactness of 3D spheroids. (A) HepG2 cells, which formed loosely aggregated spheroids alone, were co-cultured as multilineage spheroids with LX-2 cells (forming round-shape spheroids quickly) at a 1:1 and 24:1 ratio for 24, 48, 72 and 96 h. Scale bars in bright-field pictures are 100 µm. (B) Spheroid volume was calculated measuring their long and short diameter by ZEN 2.3 Lite software (Zeiss) (n = 4). (C) Cellular ATP levels normalized to spheroids volumes remained stable throughout 4 days of culture (n = 3). (D) Apolipoprotein B (APOB) secretion levels in the media fractions measured by Western blotting are proportioned to the percentage of hepatocytes present in the spheroids (n = 5). Bars represent mean ± SD. P-values were calculated by Mann-Whitney non-parametric test, (* p < 0.05 vs. HepG2). APOB: Apolipoprotein B; AU: arbitrary unit.