Conference Registration and Materials Pick-Up

Mid-Morning Coffee Break and Networking in the Exhibit Hall

EVs in Treatment of Lung Diseases and Other Targets
Marcin Jurga, Chief Scientific Officer, EXO Biologics SA

Characterization of Extracellular Vesicles and Other Biological Nanoparticles Using Nanoparticle Tracking Analysis (NTA)
Sven Rudolf Kreutel, Chief Executive Officer, Particle Metrix GmbH and CEO, Particle Metrix Inc., USA

Nanoparticle Tracking Analysis (NTA) has emerged as a fast and vital characterization technology for Extracellular Vesicles (EVs), Exosomes and other biological material in the size range from 30 nm to 1 µm. While classic NTA scatter operation feeds back the size and total particle concentration, the user typically cannot discriminate whether the particle is a vesicle, protein aggregate, cellular trash or an inorganic precipitate. The fluorescence detection capabilities of f-NTA however enables the user to gain specific biochemical information for phenotyping of all kinds of vesicles and viruses. Alignment-free switching between excitation wavelengths and measurement modes (scatter and fluorescence) allow quantification of biomarker ratios such as the tetraspanins (CD63, CD81 and CD9) within minutes. Furthermore, specific colocalization studies using c-NTA gives a deeper understanding of the composition of biomarker on single particle.

Networking Lunch Break in the Atrium -- Network with Colleagues, Visit Exhibitors and View Posters

Large Scale Manufacturing, Cargo Profiling, and Functional Effects of hTERT MSC EVs
Heather Branscome, Senior Scientist, ATCC and Research Assistant, George Mason University

Ocular diseases are a major cause of visual impairment and morbidity. Furthermore, exposure to ionizing radiation (IR) can cause direct damage to the eye. Therefore, there is an urgent need to explore novel ocular therapeutics. Extracellular Vesicles (EVs) from Mesenchymal Stem Cells (MSCs) have demonstrated widespread regenerative properties across multiple pathologies. However, the reparative effects of MSC EVs against ocular damage remains relatively unexplored. Here, we report a large-scale platform for manufacturing of EVs from hTERT-immortalized MSCs and evaluate their reparative properties on retinal cells before and after exposure to IR. Additionally, we evaluate the efficacy of a novel EV lyophilization buffer for improved EV storage, transport, and stability. Physical and biochemical properties of EVs were assayed using various techniques including NTA, western blot, mass spectrometry, RNAseq, and multiplex immunoassays. EV functionality was evaluated in vitro using a combination of assays to assess cell viability, cell migration, cell cycle, and apoptosis before and after exposure to IR. Collectively, our data suggests that hTERT MSC EVs are enriched with unique cargos and that these EVs exert reparative properties on retinal cells in vitro against irradiation-induced damage. Importantly, lyophilization of EVs further extended their shelf life without impacting their function.

Step up your EV Characterization with Leprechaun
Alex Shephard, Market Manager, Unchained Labs

Accurately characterizing extracellular vesicles (EVs) can be challenging, even in highly purified cell culture samples. Interference from lipoproteins, cell debris and protein aggregates make it hard to be confident that you're counting the right stuff. Add in complex biofluids, limited sample volumes, and rare EV subpopulations and the task gets even tougher. Leprechaun lightens the load by isolating EVs on its Luni consumable, before measuring particle size, concentration, and analyzing EV phenotype for up to 4 surface or cargo markers simultaneously, from <25 uL of sample. Leprechaun jives with a range of materials, from crude cell culture media to murine cerebral spinal fluid, without the need for sample purification. With sensitivity down to 5x10^5 particles/mL, single particle analysis and the ability to size EVs as small as 35 nm, Leprechaun is ready to help you step up your EV characterization no matter how rare or small.

Engineering EVs for Cardiovascular Diseases
Eun Ji Chung, Dr. Karl Jacob Jr. and Karl Jacob III Early Career Chair, Associate Professor of Biomedical Engineering, Chemical Engineering and Materials Science, Surgery, and Medicine, University of Southern California

Extracellular vesicles derived from healthy sources contain natural homing properties and therapeutic cargo and can be leveraged as biomimetic carriers for targeted delivery. Additionally, extracellular vesicles represent an endogenous source of nanoparticles and can offer enhanced safety as a nanoparticle platform technology. In this presentation, EVs derived from healthy cells of the vasculature and their ability to be engineered as agents that inhibit vascular calcification and inflammation in atherosclerosis will be included. Strategies to enable EV surface modification and enhance cargo loading will be presented, and the potential of engineered EVs towards clinical applications will be discussed.

Mid-Afternoon Coffee Break and Networking in the Exhibit Hall

Single vs Multi-Parameter EV Isolation Methods
Amber Murray, Senior VP, Application, Exokeryx

The success of extracellular vesicles (EVs) in diagnostics and therapeutics depends on scalable isolation methods that produce high recovery of highly pure EVs.  Current methods for EV isolation exploit one physical property at a time—density for ultracentrifugation, size for size exclusion chromatography, presence of a given surface marker for immunoprecipitation, etc.  As such, isolated EVs exhibit high recovery at the expense of purity for the cruder techniques or high purity (and bias) at the expense of recovery for the more tailored techniques.  In contrast, we introduce a new EV isolation technique called dielectrophoresis that exploits two physical properties at once—particle diameter and particle composition (dipole moment) in the presence of a radio frequency electric field.

Delivery of Mitochondria-Containing Extracellular Vesicles to the BBB
Devika Manickam, Associate Professor, Duquesne University

Extracellular vesicles (EVs) are natural, cell-secreted nanoparticles that have known roles in intercellular communication. Our work has demonstrated that the innate mitochondrial cargo in EVs can be transferred to recipient cells and tissues resulting in increased mitochondrial function. My talk will describe how delivery of innate mitochondrial cargo can be exploited for BBB protection in ischemic stroke.

Networking Prosecco, Beer and Wine Reception - Meet Exhibitors, View Posters, Network with Colleagues

Close of Day 1 of the Conference

Morning Coffee in the Exhibit Hall

Discovering the Secrets of Extracellular Vesicles for Diagnostics and Therapeutics
Fei Liu, Faculty of Medicine, Brigham and Women's Hospital

Extracellular vesicles (EVs), particularly exosomes, play a crucial role in cell-to-cell communication by facilitating the exchange of biological information and materials. EVs are abundantly found in various clinical samples, including blood, urine, saliva, tears, and cerebrospinal fluid. Due to their cargo of diverse biomolecules such as proteins, peptides, lipids, and nucleic acids derived from parent cells, EVs hold great potential as valuable biomarkers for clinical diagnosis and prognosis. In this presentation, I will discuss our research findings on EV isolation techniques, biomarker discovery strategies, detection methods, and translational applications. Firstly, I will introduce the Exosome Total Isolation Chip (EXOTIC) device developed for isolating and identifying EVs from lung cancer patients. This platform enables modular separation and analysis of EV subtypes secreted by different cells along with their respective size distribution. Additionally, I will present the Exosome Detection via the Ultrafast-isolation System (EXODUS) for various applications including characterizing EVs in different biological samples and tracing tissue and cell functions based on urine samples. Next, I plan to introduce iTEARS, which is used to discover the secrets of tear EVs as biomarkers for detecting ocular disorders and systemic diseases. Furthermore, iNEBULA will be discussed for investigating the biological profiles of tear EV subsets with different sizes from healthy individuals and exploring the origins of EV proteins. Finally, I will present the gold nano-dual probe technique (nPES) for quantitative detection of individual plasma exosomes from patients with pancreatic cancer. Simultaneously, a robust acute pancreatitis identification and diagnosis (RAPIDx) method will be demonstrated through proteomic fingerprinting analysis of intact nanoscale EVs from clinical samples. The goal of our work on EVs is to support fundamental research, promote clinical diagnosis, and facilitate the translation of therapeutics.

Accurate Extracellular Vesicle (EV) Size, Concentration, and Payload with Spectradyne’s ARC Particle Analyzer
Lew Brown, Business Development, Spectradyne

Spectradyne’s ARC particle analyzer uses a unique combination of electrical and optical measurement techniques to accurately measure the size, concentration, and internal and external payload of nanoparticles as small as 50 nm in diameter.  Learn how scientists are using the ARC to quantify single-particle encapsulation efficiency for LNPs and characterize subpopulations of extracellular vesicles based on surface marker expression profiles.

High-Efficiency Isolation of Exosomes and Its Application in Diagnosis and Treatment
Johnny Zhuang, Product Application Scientist, EXODUS BIO

Introducing EXODUS, our innovative automatic exosome isolation system, which employs advanced techniques such as periodic negative pressure oscillation and double-coupled harmonic oscillation. This unique integration ensures the high yield and purity of label-free exosomes. Widely utilized in research for disease biomarker discovery, EXODUS also offers a large-scale model, the EXODUS-T, specifically designed to cater to industrial production demands.

Flexible Solutions for Extracellular Vesicle Analysis
Shawn Sternisha, Product Manager, Beckman Coulter Life Sciences

During this presentation, we will discuss some of the current challenges with Flow Cytometry for Extracellular Vesicle (EV) analysis and what it would take to make a purpose-built analyzer. With over 88 years of experience, Beckman Coulter Life Sciences is dedicated to driving innovation in research through improvements in EV analysis. We offer a number of products and solutions, including the CytoFLEX Flow Cytometer and the CytoFLEX SRT benchtop cell sorter, providing the sensitivity and performance you need in an easy-to-use system, for your EV research solutions.

Mid-Morning Coffee Break and Networking in the Exhibit Hall

Multicolor, High-Resolution Exosome Analysis with the Delaware Flow NanoCytometer
Giacomo Vacca, President & CEO, Kinetic River Corp

The Delaware Flow NanoCytometer® is a particle analyzer that can measure from EVs to cells. It has a resolution of 6 nm, it can detect 28-nm gold, 60-nm polystyrene, and 68-nm liposomes. We have characterized HansaBioMed FLuoEVs (~90 nm) by scattering, membrane stain, expressed EGFP, and PE-conjugated tetraspanins (CD9, CD63, and CD81). We have benchmarked the Delaware against Particle Metrix’s ZetaView NTA, with close agreement on fluorescent fractions. The Delaware is designed for multiparameter analysis, with up to 6 channels of fluorescence and three scattering channels.

Comprehensive Single EV Characterization with NanoFCM
Clayton Deighan, North American Sales and Applications Manager, NanoFCM

In this presentation we will review the principles of operation and common protocols for analysis of extracellular vesicles on NanoFCM’s Flow NanoAnalyzer. Example data of scatter based triggering and particle counting, fluorescent detection of surface proteins and nucleic acid cargoes on single vesicles and how this data can inform your research questions will be discussed. Please join us to learn more about nano flow cytometry for extracellular vesicles.

Filamin-A and a4ß1 Integrin are CD47-dependent Cargo Proteins in Extracellular Vesicles
Sukhbir Kaur, Staff Scientist, National Cancer Institute, National Institutes of Health

CD47 is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin-1 and the counter receptor for signal regulatory protein alpha in phagocytes. CD47 is expressed on a subset of extracellular vesicles (EVs) that contain a distinct population of RNAs {Kaur, 2018 #1}. CD47 colocalizes predominantly with CD81 and a4ß1 integrin on Jurkat T cell-derived EVs but not with classical EVs bearing CD63 or CD9 {Kaur, 2022 #2}. CD47 and its cytoplasmic adapter ubiquilin-1 regulate which RNAs are packaged into T cell EVs via physical interactions with components of the exportin-1/Ran nuclear export complex and its known cargos {Kaur, 2022 #3}. Here, we report that disruption of CD47 in Jurkat T lymphoblast and PC3 prostate carcinoma cells impairs the sorting of filamin A and a4ß1 integrin into EVs. Targeted mass spectrometry and coimmunoprecipitation analyses indicate that CD47 indirectly interacts with filamin A either via ubiquilin-1/Exportin-1 or ß1 Integrin. Filamin A may thereby play an important role in CD47-dependent sorting of protein and RNA cargoes into specific subsets of EVs.

Conclusions: CD47 and ubiquilin-1 interact with filamin A, which is known to interact with the cytoplasmic domain of ß1 integrins to regulate integrin function. Less filamin A and a4ß1 integrin sort into EV in the absence of CD47, suggesting that CD47 promotes filamin A and integrin sorting into EV, mediated through ubiquilin1 and/or exportin-1.

Networking Buffet Lunch in the Atrium -- Network with Colleagues, Engage with Exhibitors and View Posters

Round-Table Open Discussion: How does a Scientist Send Their Experiment into Space On-Board International Space Station ? -- Q&A with Mike Roberts and Kristin Kopperud, ISS-National Laboratory

Mid-Afternoon Coffee Break and Networking in the Exhibit Hall

Exploiting an Antiviral Response to Improve Drug Delivery
Tom Anchordoquy, Professor, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences

Early studies in gene delivery observed a period of time (days to weeks) after an initial injection when a repeat injection of lipoplexes had a minimal effect on expression.  This “refractory period” was originally attributed to viral promoters and CpG sequence that elicit the production of inflammatory cytokines which silence expression.  However, subsequent studies with plasmids lacking these components still observed a refractory period.  Ultimately, this effect is responsible for the inability to use repeat administration to achieve progressively greater levels of gene expression and obtain therapeutic efficacy.  While relatively few studies have characterized delivery after repeat injection, our recent experiments have shown that the refractory response involves the inhibition of particle uptake in addition to the silencing of gene expression.  Although this results in reduced delivery to normal tissues that respond to cytokines, the immunosuppressed state of established tumors allows unimpeded delivery upon subsequent injections.  Our data suggest that this effect can be exploited to reduce off-target accumulation and improve delivery to the tumor during repeat injection.

Targeting Viral Genomic RNA by the Delivery of Extracellular Vesicles-Mediated CRISPR Machinery
Houjian Cai, Associate Professor, University of Georgia

Extracellular vesicles (EVs) have recently been co-opted as vehicles for the delivery of therapeutics, including CRISPR-Cas9 (Cas9), and are now being modified for higher gene editing efficiency. N-myristoylation is known to translocate Src kinase to the membrane. We reasoned that fusion of the N-terminal of Src to Cas9 may increase localization with the membrane, and subsequently increase EV-loading and gene editing efficacy in EV-treated recipient cells. Our study demonstrate that fusion of the octapeptide to Cas9 induced N-myristoylation and encapsulation of the mCas9/sgRNA complex into EVs. We provide proof of concept for N-myristoylation as a method to increase EV-mediated delivery of therapeutics. The technology can be applied for targeting oncogenic genes in prostate cancer cells.

Extracellular Vesicle Isolation Methods Identify Distinct HIV-1 Particles Released from Chronically Infected T-cells
Fatah Kashanchi, Professor and Director of Research, Lab of Molecular Virology, George Mason University

In 2022, 1.5 million people acquired Human Immunodeficiency Virus (HIV-1), and an estimated 37.7 million individuals lived with HIV-1 (PLWH) worldwide. While combination antiretroviral therapy suppresses viral replication, it does not silence viral transcription. We have identified presence of HIV-1 products, including non-coding viral RNA and proteins, within extracellular vesicles (EVs). These EVs are not infectious and can be isolated from cell culture supernatants of HIV-1 chronically infected cell lines and biofluids. Here we expanded upon a sequential differential ultracentrifugation (DUC) method by employing higher g-force with longer spin times to recover smaller EVPs (<100 nm) and have found presence of virus in both large and very small EVPs. Furthermore, we modified a virus recovery assay which indicates that these EVPs were infectious, including the novel EVPs under 100 nm. Standard assays for EV characterizations were used for validation.  Data was further validated using filtrations and other methods of EV purification. Viral and EV markers were used to quantify each prep. Collectively, we identified unique, infectious particles smaller than the currently accepted size for HIV-1. This methodology may be employed for other viruses or infectious agents where EVPs may impact disease progression by transmitting highly replicating virulent nucleic acids.

Round-Table Discussion on Viruses and EVs Moderated by Professor Michael Graner

Close of Conference