Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

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ID 16411 Poster Board 244 Background: One of the difficulties in cancer research is bridging the gap between human and animal tumor models. One proposed method of improving the screening of cancer-promoting toxins and novel cancer therapies is the development of pre-clinical 3D human culture models. We are currently developing 3D human hepatocellular carcinoma (HCC) culture models using the standardized HepG2 liver cancer cell line as a template. Ethanol is being used as a test agent to generate conditioned media (CM) from 2D adherent vs. 3D HepG2 suspension spheroid cultures. The conditioned media is then applied to human monocytes to determine differences in TNF-a (proinflammatory) and IL-10 (anti-inflammatory) cytokine induction.Objectives: The experimental goals were to determine differences in the production of TNF-a and IL-10 between 2D and 3D cultured HepG2 cells, and by THP-1 monocytes treated with conditioned media (CM), +/-ethanol, derived from 2D and 3D HepG2 cells.Methods: HepG2 cells were grown in both 2D (adherent) and 3D (suspension) cultures to produce two different patterns of cell growth (Fig. 1). The cells were then switched to fetal bovine exosome-free media and grown with and without 100 mM ethanol to produce CM. Human monocytes (THP-1 cells) were then cultured in the presence of CM. Cell viability and production of TNF-a and IL-10 were measured using PrestoBlue™ and Lumit™ immunoassays.

The mitochondrial electron transport chain consists of the classical protein complexes (I-IV) that facilitate the flow of electrons and coupled oxidative phosphorylation to produce metabolic energy. The canonical route of electron transport may diverge by the presence of alternative components to the electron transport chain. The following study comprises the bioinformatic identification and functional characterization of a putative alternative oxidase in the smut fungus Sporisorium reilianum f. sp. zeae. This alternative respiratory component has been previously identified in other eukaryotes and is essential for alternative respiration as a response to environmental and chemical stressors, as well as for developmental transitionaoxs during the life cycle of an organism. A growth inhibition assay, using specific mitochondrial inhibitors, functionally confirmed the presence of an antimycin-resistant/salicylhydroxamic acid (SHAM)-sensitive alternative oxidase in the respirasome of S. reilianum. Gene disruption experiments revealed that this enzyme is involved in the pathogenic stage of the fungus, with its absence effectively reducing overall disease incidence in infected maize plants. Furthermore, gene expression analysis revealed that alternative oxidase plays a prominent role in the teliospore developmental stage, in agreement with favoring alternative respiration during quiescent stages of an organism’s life cycle.

Hepatocellular carcinoma cells (HCCs) produce small extracellular vesicles (sEVs or exosomes) to enable tumor survival, including inactivation of anti‐tumor macrophage immune responses. For sEV studies in vitro, 2D adherent culture cells are typically used to manufacture sEVs. In contrast, 3D matrix‐based culture systems closely simulate in vivo tissues but produce difficult to isolate sEVs. To address this issue, we developed a reducible 3D suspension HCC spheroid culture system to generate easily obtainable sEVs for investigations. The objective was to evaluate biophysical and cancer pathway‐focused miRNA content differences between HCC HepG2 sEVs produced in 2D adherent vs. 3D spheroid suspension culture. To summarize methods, sEVs were isolated from 2D and 3D cell culture using differential ultracentrifugation and size and zeta potential biophysical characteristics were determined by nanoparticle tracking analysis. Fold regulation of 2D and 3D derived sEV miRNAs, compared to their source cells and one another, were determined by qRT‐PCR, and statistically significant differences were determined by Student’s t‐test. Results indicated that HepG2 sEVs generated from 2D adherent or 3D spheroid suspension culture do not differ significantly in terms of size (~130 nm) and zeta potential (< ‐30 mV) characteristic of sEVs. A comparison of 2D vs. 3D sEVs revealed differences in the enrichment of let‐7a‐5p (decreased in HCC in vivo), miR‐21‐5p (enables HCC drug resistance), and miR‐126‐3p (impairs HCC tumor volume in vivo). let‐7a‐5p and miR‐21‐5p were downregulated, while miR‐126‐3p was upregulated in 3D sEVs compared to 2D sEVs (control) respectively. Overall, similarities and differences between 2D and 3D sEV miRNA content were observed relevant to HCC pathogenesis. In conclusion, the 3D HepG2 spheroid suspension model provides an additional reducible level of HCC sEV investigation to augment traditional 2D sEV studies while better simulating an in vivo 3D source of easily obtainable HCC sEVs. Our 3D model could be used in conjunction with typical 2D EV culture systems to streamline the identification of candidate sEV‐based biomarkers and therapeutic targets for HCC and other cancers.