Poster C4 - Interrogating Extracellular Vesicle Production as a Function of Matrix Properties

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Josiah I. Garan (he/him/his)

Undergraduate Research Assistant
Harvey Mudd College
Claremont, California, United States
Caiya Coggshall (she/her/hers)

Undergraduate Research Assistant
Harvey Mudd College, United States

Primary Investigator(s)

Steven Santana

Joseph B. Platt Chair in Effective Teaching; Assistant Professor, Engineering
Harvey Mudd College
Claremont, California, United States

Presenting Author(s)

JT

Jonathan Tabb

Undergraduate Research Assistant
Harvey Mudd College, United States

Introduction: Extracellular vesicles (EVs) are cell-secreted lipid-bilayer enclosed structures. EVs contain functional molecules such as nucleic acids, lipids, and proteins to recipient cells throughout organisms. As such, they are important mediators of cell-cell communication and afford opportunities for insight into cellular function in physiological and pathological states. Because of these features, EVs have attracted interest as potential biomarkers and therapeutics.
Since EVs are produced by virtually all cell types, understanding their biogenesis is critical. EV production is responsive to the extracellular microenvironment cues. In this study, synthetic hydrogels are engineered with varying stiffnesses to deliver mechanical microenvironmental cues. Characterizing EV cargoes and composition in these environments provides initial clues as to their biogenesis in response to these cues. For each condition, in addition to miRNA expression, we examine EV size distribution and concentration. The analysis of these biological characteristics affords deeper insights into the impact of environmental conditions on EV production and, in the future, the signaling pathways implicated in their biogenesis.

Materials and

Methods: U87-MG cells are cultured in standard tissue culture polystyrene (TCPS) flasks in EMEM (Eagle’s Minimum Essential Medium (EMEM; ATCC, Cat# 30-2003 ™) with 10% (v/v) Fetal Bovine Serum (FBS; ATCC, Cat# 30–2020 ™) and 1% (v/v) Penicillin-Streptomycin (P/S; ATCC, Cat# 30-2300 ™). TCPS is coated with poly(ethylene glycol) (PEG) hydrogels formed via thiol-ene click chemistry. Hydrogels of unique moduli are characterized using shear rheology on a TA Instruments Discovery Hybrid Rheometer (DHR3). The reported hydrogel properties are within the linear viscoelastic region (LVR).
U87-MG cells are seeded within flasks, with and without hydrogel coatings, and are cultured in each condition until 80% confluent. Cells are then triple washed with phosphate buffered saline and cultured in serum-free media for 10-15 hours. EVs are collected from conditioned media. EV size distribution and concentration are characterized using a nanoparticle tracking analysis (NTA) using a Nanosight NS300. EV-derived miRNAs are isolated using a Qiagen exoRNeasy kit prior to next generation sequencing.

Results, Conclusions, and Discussions: EV size distribution and concentration are characterized via NTA as shown in Figure 1. These results are consistent with typical EV size distributions.
Young's modulus is characterized for each hydrogel formulation, as shown in Figure 2. The modulus is directly related to the crosslinking density within the hydrogel network. The modulus measured for formulation A corresponds to the modulus of scarred tissues whereas formulation B corresponds to the modulus of healthy tissues, both in the central nervous system.
Our current results characterize the size distributions and concentrations of EVs from U87 MG cells. We aim to study EV composition and cargo, including proteins and nucleic acids, in response to biomimetic matrix properties. Future work includes comparison of these features across culture conditions to interrogate their differential impact on EV biogenesis and packaging.

Acknowledgements (Optional): Thank you to our sources of funding: the National Science Foundation (Award #2233193), Harvey Mudd College Engineering Department, the Engman Fellowship, the Fletcher Jones Fund, and the Norman F. Sprague III Fund.