Isolation and Characterization of Extracellular Vesicles Using Microfluidic Technologies

Abstract

Extracellular vesicles (EVs) are a group of heterogeneous plasma membrane-bound vesicles that are secreted by almost all cell types into extracellular space. EVs contain selected nucleic acids, proteins and metabolites that modulate biological activities after their internalization by recipient cells. Considerable efforts have been devoted to the study of EV-mediated communication among the cells. Such studies have demonstrated the importance of EVs in the spread of diseases. Pathological molecular cargos such as miRNA and proteins are shuttled via EVs into the circulation and are now known to be key players in progression of diseases. Therefore, isolating and subsequently accessing these bioactive vesicles can provide great insights into the diagnosis and prognosis of a patient’s pathological status. A critical caveat to the study of EVs is their extremely small size and the potential rarity of target EVs in a background of off-target EVs. The objective of this thesis is the study on the roles of EV in human disease and development of microfluidic technologies and protocols to advance the isolation and characterization of EVs First, an immuno-affinity based microfluidic device was introduced to study the role of EVs in amyotrophic lateral sclerosis (ALS). The device was adapted to isolate EVs from three types of tissues, namely serum, frontal cortex, and spinal cord from ALS patients and healthy controls. We compared miRNA cargo from diseased tissues including postmortem spinal cord and frontal cortex to healthy controls. As a result, we identified 60 miRNAs that are significantly altered in the patient, suggesting the possibility for use of EV miRNAs as biomarkers for ALS patients. The pathway analysis of miRNA targets also pointed the involvement of EV miRNAs in the ALS. Secondly, a microfluidic device, OncoBean chip is modified and optimized for high throughput EV isolation from cell culture supernatant and human plasma. The OncoBean Chip is a previously reported microfluidic device for isolation of circulating tumor cells (CTCs) with bean-shape microposts functionalized with biotin-conjugated EPCAM antibody. We modified this chip with the antibodies against common EV surface markers to achieve high throughput EV isolation. The high surface area provided by the bean-shaped miroposts and radial flow design facilitate capture of EVs not only at high flow rate (up to 10 ml/hr), but also form larger volumes of media form cell culture supernatant. Through desthiobiotin antibody capture and biotin elution, the device is able to release functional EVs from the device for cell uptake and identification of surface markers. Thirdly, to address the need for technologies for high-resolution analysis, we established a workflow for simultaneous mutation detection and gene expression profiling with low sample-input. We developed the workflow from analyzing circulating tumor cells (CTCs) at a single cell resolution, aiming a future application to EV analysis. By integrating microfluidic technologies including the Labyrinth and Fluidigm C1 system, single CTCs from patients can be isolated and profiled for multiple characteristics. The feasibility of the workflow was validated with six non-small-cell lung cancer (NSCLC) patient diagnosed with epidermal growth factor receptor (EGFR) mutation. The intra- and inter-patient heterogeneity observed in single lung CTCs from patients demonstrated the utility of the proposed workflow.