High-throughput single-cell isolation, pairing and analysis on microchips

Abstract

Cells are the most fundamental structural and functional units of life. It was first discovered and named by Robert Hooke in 1665, since when numerous biologists have been dedicated to uncover its mysteries. In most conventional cellular bioassays, such as Western blotting, real-time PCR, ELISA, etc., cell samples containing numerous individual cells are normally treated as homogenous and analyzed in bulk level. However, in recent decades, advances in different fields of biology have found that cellular heterogeneity widely exists in all cell populations. Analysis in bulk assays may not accurately reflect true biology of individual cells. The studies in single-cell level help to reveal critical secrets concealed in conventional population-averaged measurements and provide valuable insights into many unsolved problems in fundamental biology and human diseases. To date, the rapid development of many high-precision tools such as high-resolution microscopes, patch-clamp techniques, micropipettes, optical tweezers etc., has enabled the single-cell manipulation and exploration. However, those techniques are typically low throughput, of which the sampling volume is far below statistical level. To extract meaningful statistics and accurately assess heterogeneities among cell populations, the number of single cells for analysis should be large enough, which requires high-throughput single-cell analysis techniques. To date, many novel techniques with greatly enhanced throughput have been established. Among them, flow cytometry is a very powerful tool pioneering in high-throughput single-cell studies, which is able to analyze many thousand single cells per second. In recent years, microfluidic platforms have also been widely utilized for high-throughput single-cell analyses by leveraging the well-designed structures with sizes matching well with cells. With optimized geometries, these platforms have many advantages over conventional methods, such as high throughput, high sensitivity, improved parallelization and low cost. In the past four years, my studies were basically focused on developing convenient and effective microchip-based methods for high-throughput single-cell isolation, and combining different techniques for single-cell quantitative analysis, including fluorescence microscopy and mass spectrometry. On the basis of developed single-cell isolation technique, I also explored its applications in single-cell pairing, which exhibited great potentials in the study of cell-cell interactions and controllable cell fusion. In the first chapter, an overview of single-cell studies including their significance, current advances and challenges are present. Then a brief introduction about single-cell pairing is given. In the rest chapters, I present the research projects completed during my PhD study, including centrifugation-assisted single-cell trapping (CAScT) in a truncated cone-shaped microwell array (TCMA) chip for the real-time observation of cellular apoptosis, fast single-cell patterning for study of drug-induced phenotypic alterations of HeLa cells using time-of-flight secondary ion mass spectrometry and cell pairing and polyethylene glycol (PEG)-mediated cell fusion using two-step centrifugation-assisted single-cell trapping.

Date of Award	2018
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology