Introduction: One of the biggest challenges for the extracellular vesicles (EVs) at this stage is to understand the heterogeneity within EV populations. Increasing evidence shows that within populations of EVs, important properties including morphology, molecular composition, content, and biological properties vary substantially, various subpopulations may exist even with EVs originated from the same parental cells. The separation and detection of similar-sized EVs as well as from similar sized cells can be a challenge, and the size ranges of EVs may overlap with protein aggregates. The emerging microfluidic technologies may offer new solutions to EV heterogeneity research.
Methods: We fabricate microwells of different sizes ranging from20μm to 70μm using microfabrication techniques. We characterise the single cell loading efficiency by using microscopy counting and localisation. Glass substrates modified with EV-specific antibodies are used to capture EV secreted from single cells. Immuno-sandwich assays combining with high-resolution fluorescent imaging are used to characterise surface protein expressions.
Results: We have characterised the performance of single-cell arrays by trapping human colon cancer cells (HT29) in microwells. We have found that single cell loading efficiency shows a strong correlation with the well size. The 20 μm-diameter wells match the size of individual cells, showing the maximum around 30% single-cell yield. The single-cell arrays can help quickly analyse EV secretion heterogeneity over a large scale. Compared with microplates-based culture approaches, the single cell chip arrays can improve the throughput of analysis and reduce the sample volume to a million times with the reduction of the overall size of sample reservoir. Excellent spatiotemporal control can be achieved, facilitating highly parallelized analysis for the understanding of complex biological heterogeneity issues.
Summary/Conclusion: Microfluidic technologies hold potential in investigating the EV heterogeneity at the single cell level. Developing microfluidic-based single cell heterogeneity analysis will allow for more in-depth and precise studies on the underlying complexity of EV heterogeneity and accelerate the development of EV-based diagnostics and therapeutics.
Funding: This work is supported by NHMRC (GNT1160635).