Description
Introduction
Liquid biopsy and the use of selected biomarkers from biological fluids, greatly enhance an increasingly patient-centered approach, avoiding invasive tests and tissue biopsies. Extracellular Vesicles (EVs) act as a snapshot of the cells from which they originate and as a repository of crucial information, facilitating the direct extracellular transfer of proteins, lipids, and miRNAs/mRNAs/DNAs. Despite EVs show great potential as powerful biomarkers, their isolation, and characterization remain challenging. Lab-on-Chip (LoC) technologies represent innovative tools to overcome the limits of standard methods and we aim to implement these technologies for EV investigation.
Methods
We design customizable LoC devices based on the experimental needs, starting from fluid dynamic simulations and using microfabrication techniques (micro-milling and 3D printing). We develop two different LoCs exploiting the microfluidic approach and electrochemical detection for EV enrichment and characterization. Large- and small-EVs were isolated by ultracentrifugation and characterized by high-resolution flow cytometry, western-blot, and transmission electron microscopy.
Results
We developed an in-flow device using molded-plastic substrates assembled on a glass slide to create a microfluidic chamber for dynamic cell culture. Oral squamous carcinoma (OECM-1) and neuroblastoma (SH-SY5Y) cell lines were seeded into the device to allow a complete replacement of the medium in the dynamic condition within 2,4,8 and 24h at controlled flow rate (10 µl/min). We observed increased EV production and decreased EV size in dynamic versus static condition. Moreover, we realized a biosensing platform for EVs electrochemical characterization. Functionalized microelectrodes were used for Differential Pulse Voltammetry measurements to build a calibration plot considering different EVs membrane and cargo proteins (Flotilin-1, CD9, CD81, CD63 and Alix). With this method we were able to demonstrate i) the possibility to detect very low concentrations of EVs from cell supernatant and ii) the retention of sample integrity.
Conclusions
The key goals of the LoC technologies are to eliminate high-impact procedures, reduce time and cost, and preserve EV morphology. Our systems allow us to study the release of EVs under dynamic cell culture conditions that mimic the physiological scenario at the cell surface, and to identify arrays of biomarkers associated with EV subclasses. The next step is to integrate microfluidic sorting and electrochemical characterization into a benchtop device that can be customized to meet clinical needs.