SINGLE ADHESIVE CELL PROTEOMIC SECRECTION ANALYSIS BY USING MICROFLUIDICS
Chia-Hung Chen1,2, Ming Wang1, Ee Xien Ng1, Liang Leo Hwa1, Chwee Teck Lim1,3,
1National University of Singapore, Singapore
2Singapore Institute for Nuerotechnology, Singapore
3Mechanobiology Institute, Singapore
Secreted enzymes in individual adhesive cells are associated with the cascades of cellular signaling that determine their biochemical states and fates. Morphologically identical cells with the same genotypes can exhibit strikingly distinct enzyme profiles due to attached matrix stiffness and chemical environment. At present, most single adhesive cell assays are based on microwells, dielectrophoresis (DEP), or hydrodynamic trappings, which are unable to effectively quantify single adhesive cell enzyme activities. Recent studies have shown that droplet technology has the potential for high throughput single cell enzyme secretion measurement. Despite the advantages of this approach, the challenge in adhesive cell measurement remain, which entails the essential step of flowing suspended cells through an aqueous solution during droplet encapsulations. In this study, we introduced a novel approach involving the encapsulation of single-cell-adhered hydrogel particles, coupled with high throughput continuous time-point analysis of the adherent cancer cells with single cell precision. Monodispersed gelatin hydrogel particles of 30 μm in diameter were first prepared using the droplet-based technology. After demulsification, these gelatin particles were suspended in aqueous phase. This process was then followed by chemical crosslinking of the particles with genipin to form a robust gelapin (genipin crosslinked gelatin) shell that is stable at 37˚C during cell incubation. The capability to control extracellular matrix for individual cells and to monitor their enzymatic secretions effectively make this integrative system a potentially powerful platform to address the intricate interaction between matrix properties and biochemical signals at the single cell level. The system also enables standardization of single adhesive cell measurement for systematic analysis and benchmarking cell phenotypes in epithelial mesenchymal transition (EMT) by monitoring different parameters, such as integrin/cadherin adhesion, enzymatic activities, or matrix stiffness.