Development of microfluidic devices for long-term cell culture and high-throughput clonal analysis

摘要

Cell cycle and differentiation precisely regulate the size and geometry of tissues and organs, and are of critical importance for tissue regeneration and carcinogenesis. Clonal analysis of progenitor or stem cells is a powerful tool for analysing cell differentiation and proliferation on the single cell level. The aim of this thesis was to develop microfluidic devices for high throughput clonal analysis of non-adherent cells by time-lapse imaging. To this end, a single-pass or recirculating perfusion microbioreactor for clonal analysis of non-adherent cells integrated with microwells, micropumps, valves and gas exchangers was designed and manufactured by multilayer soft lithography. Micropump flow rates and gas exchanger efficiency were characterised. The influence of microwell geometry and flow on cell retention was studied by experimentation and simulated by fluid dynamics. Critical Reynolds numbers for single cell deposition or perfusion culture without cell loss from microwells were determined by experiment. A mechanical equilibrium model was developed to predict docking, rolling, or lifting of a cell in microwells given hydrodynamic forces. The viscous drag and torque forces were calculated from computational fluid dynamic simulation of flow around a cell deposited inside a well. From the biological point of view, material biocompatibility, media degradation and media perfusion flow rate were investigated to optimise long-term perfusion cell culture. KG1a cells were cultured in microwells by media perfusion and continuously imaged at 3 minute intervals for 6 days. Time-lapse movies of 1500 individual KG1a clones were acquired using the device. The stochastic nature of cell growth was demonstrated by manually tracking cell division trees. The cell cycle time distribution was estimated by Kaplan-Meier analysis. This study demonstrates the feasibility of clonal analysis using a microwell array perfusion system and provides practical guidelines for design, manufacture and operation of microwell perfusion bioreactors to study mammalian cell development in a high throughput manner.