Controlled microfluidic production of alginate beads for in situ encapsulation of microbes

The development and refinement of a microfluidic-based alginate bead generator system for bacterial encapsulation is presented. The resulting microgels have application for the encapsulation of single cells, and can allow for small scale, clonal expansion of thousands of isolated cells in parallel. PDMS based microfluidic chips were fabricated using conventional lithography techniques to produce both externally gelled and directly gelled alginate microspheres using a controlled, water-in-oil emulsion system. The production of directly gelled beads, formed by the in-chip mixing of aqueous alginate and calcium chloride solutions dispersed within an organic carrier flowstream is qualitatively compared to a system, which produces beads and relies on diffusion of a crosslinking agent from the carrier fluid to cause gelation (external gelation). While the direct gelation scheme allows the use of biocompatible oils as the organic carrier, it also has a detrimental effect on device stability often resulting in clogging and gel-streaming at the microfluidic interface of these solutions. A design for the continuous production of directly gelled beads was evaluated in terms of the threshold flow conditions and reagent concentrations that did not result in clogging or streaming. Monodisperse alginate microgels of 30 mum diameter were produced at frequencies of over 500 beads per second. The beads could be completely dispersed into aqueous media using an off-chip washing protocol to remove the organic phase. The microgels effectively encapsulated individual or small numbers of GFP-expressing Escherichia. coli, which could be subsequently clonally expanded. The described microfluidic platform is a robust front-end sample preparation technology that shows strong potential for use in drug delivery systems, biosensors, and other cell-based microcompartmentalization applications. The co-culturing of microbial colonies in a large population of alginate beads will allow for functional sorting and genetic analyses at the single cell level.