Fig 1: Characterization of the HDL removal from the fractionated EV and yield/quality of the isolated EV.a Schematic of the EV fractionation and immunocapture of HDL. The diluted plasma (sample I) runs through the asymmetric nanoporous membrane to remove other particles with a size-exclusion mechanism, thus obtaining the EV fraction (sample II). Due to the dominating amount of HDL, a small portion of HDL remained in the EV fraction, so it was incubated with anti-ApoA1 and anti-ApoB antibodies, and then magnetic nanobeads conjugated with anti-rabbit IgG antibodies and run through the MNM device to remove the HDL, and the flow-through was collected (sample III). b Lipoproteins (cholesterol) remnant at different stages of purification from samples I, II, III in a), and (inset) EV concentration before and after MNM immunocapture (sample II and III) (n = 5 measurements for each sample). c Size distribution of EV samples with NTA before (sample II) and after MNM immunocapture (sample III) showing minimal EV loss or lysis/coalescence. (inset) CD63 and CD9 concentration before and after MNM immunocapture (sample II and III) measured by ELISA (n = 2 and 3 measurements for CD63, CD9, respectively). Error bars indicate the standard deviation (SD) in each plot.
Fig 2: Characterization of MNM immunocapture of specific EV, i.e., EV with EGFR.a Schematic of the EV fractionation and immunocapture of EV with EGFR. The DiFi cell culture medium was run through the asymmetric nanoporous membrane to obtain the EV fraction by size separation (sample I), which was then incubated with the anti-EGFR antibodies and then magnetic nanobeads conjugated with anti-human IgG antibodies. Then the samples passed through the MNM device, with the captured sample on the magnetic membrane mixed with lysing buffer (sample II), and the flow-through was collected (sample III). b Hm-miR-21 expression level of different DiFi exosome fractions from samples I, II, III in (a) (n = 3 measurements for each sample). (inlet) The total amount of EGFR was measured for I and III, indicating most EGFR-positive EVs from the ANM-isolated EVs are captured by MNM (n = 7 measurements for each sample). c CD63 and CD9 concentration of ANM isolate (sample I) and MNM EGFR isolate (sample II) measured by ELISA. (n = 3 measurements for each sample) d Expression level of Hm-miR-21 in the EGFR exosomes before and after 8 × dilution of the DiFi samples and the Ct value of their qRT-PCR results (n = 5 measurements for each sample). e Western blot of the isolated EVs from the DiFi cell line. After the Marker Lane then (1) ANM-isolated sEV; (2) MNM bead captured sEV. Each well was loaded with 40 µg protein. Odyssey exposure (bottom: 10 min, top: overexposed). The top panel is blotted by EGFR and the bottom by syntenin-1, respectively. Error bars indicate the standard deviation (SD) in each plot.
Fig 3: Evaluation of restoration capacity of EV characteristics after lyophilization. (A) Restoration of EV concentration lyophilized with PBS and TS 2%, 4%, and 8% compared to fresh EV (n = 12). (B) Number-based size distribution of fresh EV and lyophilized EVs with TS 2%, 4%, and 8%. (C) Cryo-transition electron microscopy (TEM) images of lyophilized EVs compared to fresh EVs. The scale bar indicates 100 nm. EV contents were investigated by (D) total protein detected using BCA protein assay (n = 8), (E) total RNA concentration (n = 6), (F) CD63 quantification conducted by ELISA (n = 3), and (G) fold change of miRNA levels calibrated with fresh EV (1; red line). The vascularization efficacy of lyophilized EVs was investigated using HUVEC tube formation assay. Each group was evaluated using (H) the number of loops (n = 5) and (I) the images of each group compared to the mock group. The scale bar indicates 200 μm. *P < 0.05 and **P < 0.001 were calculated by one-way ANOVA.
Fig 4: Development of a potency matrix to enhance consistency of MSCs as cell therapy product. (A–D) Quantification of intracellular IDO1 expression (A), or secretion of M-CSF (B), CD63 (C), or CCL2 (D) by MSCs from 8 different donors. Data are means ± SD. (E) Potency matrix whereby 0 to 3 points are assigned to each MSC donor for each potency factor based on quartiles. (F) Weighted potency matrix whereby 2 points per quartile are assigned for IDO1 and EV scores and ½ point per quartile for CCL2 scores.
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