Droplet microfluidics: cells, assays and drug combinations

Individualised medicine takes into account a patients genetic, environmental and lifestyle factors when selecting a drug, or drug combination, resulting in improved patient outcomes. The current drug screening methods, utilising automated robotic liquid handling equipment and microplate libraries, are unable to facilitate this advancement due to their time-consuming and costly operation.

This thesis explores a theoretical flexible microfluidic combinatorial rotational instrument for the purpose of combinatorial drug screening. The instrument comprises of forty-eight radial lines and four rings. Along each radial line and on each ring, exists a well, resulting in 192 wells. The flexibility of the instrument allows for any combination of radial lines and rings to be utilised on the same instrument, from a minimum of two radii and two rings; to its maximum, forty-eight radii and four rings. The instrument utilises a novel method of generating aqueous-in-oil droplets, generating at its maximum, 5.4x107 unique droplets, ranging in volume from 200nl-800nl.

An equation for calculating the number of droplets any configuration of the instrument can generate is derived, based on the binomial coefficient. The derivation of equations for a two ring configuration is described and presented, which determine the combinatorial content and positional order of every dispensed droplet in the two-dimensional droplet array from two ring configurations. The feasibility of utilising droplets for cell-based assays is investigated, with droplets containing leukemic cells being fluorescently imaged in real-time over 48 hours to assess cell viability in the incubation line. The results show droplets provide a suitable microenvironment for the culture of suspension cells and assay performance. A comparison is drawn between droplets and microplates, showing that droplets maintain cell viability comparable to microplates. Drug combinatorial screening is completed on two leukemic cell lines, K562 and K562-DR, using the alamarBlue Cell Viability assay in the search for synergy. The K562-DR is a cell line engineered to be resistant to one of the drugs investigated, Imatinib Mesylate, through increasing exposure to the drug. Drug screening was performed through droplets and microplates, with a comparison drawn between the data obtained. It is shown that droplets generate data equivalent to microplates at a 166.6̇ fold decrease in the volume of reagent used, showing encouraging results for the use of droplets in the screening of drugs. The Chou-Talalay Combination Index was chosen as the primary measurement of synergy for this thesis. Synergy was detected between the drugs Imatinib Mesylate and Nilotinib at lower concentrations of the drugs for the drug sensitive cell line, K562. In the drug resistant cell line, K562-DR, all concentrations of the drugs in combination indicated synergy, indicating the use of the drugs in combination to be a powerful tool in combating drug resistant cancer. The results of this thesis show encouraging results in the detection of synergy between drugs in combination within microdroplets for the first known time.