Current colony picking technologies post-transfection of cells rely on the imaging of growing colonies on an agar plate and their subsequent removal and inoculation. This standard workflow is limited by the speed of researcher or robotics and by the need to grow cells on a two dimensional agar surface, which may not be representative of the ultimate desired physiological growth environment. On top of this, desired colonies are determined based on the size of the colony on the plate, which prioritizes growth, but may in some instances neglect the expression of a desired metabolic phenotype. By increasing the throughput of colony picking and enabling the selection of cells of varying growth rates, researchers will be able to obtain a population with much greater diversity and improve the outcomes of their studies. To improve the throughput of colony picking workflows, as well as broaden the phenotypes that can be selected for, we have developed PicoShells. PicoShells are polymeric, porous, hollow microparticles that are 40 – 100 µm in diameter, compartmentalize clonal subpopulations of yeast, and enables continuous media and waste exchange. First we encapsulate hundreds of thousands of single cells per hour into polymer-containing aqueous droplets using a microfluidic system. The polymer crosslinks into a hydrogel shell around an aqueous cavity containing the yeast cell, keeping it and its progeny confined. PicoShells are then cultured under normal conditions within an incubator or bioreactor; the porosity and biocompatibility of the shell creates an environment more conducive to the growth of Saccharomyces cerevisiae than comparable aqueous-in-oil microfluidic droplets. The porosity also makes it much easier for stains to enter the compartment, stain cells, and be washed away to enhance the signal-to-noise ratio. Cells grow into monoclonal colonies within the PicoShells, and the colonies expressing high quantities of a protein of interest or increased biomass accumulation under certain environmental pressures can be selected through standard multi-color flow cytometry workflows. Cell viability is maintained through the process, and upon chemical degradation of the shells, downstream population analysis or reculturing can be performed. Since PicoShells can be run through flow cytometers, hundreds of colonies grown in PicoShells can be picked per second based on their light scatter (biomass), their fluorescence (protein expression) or a combination of both. The PicoShell platform represents a culturing system that lends itself to high-throughput, multiplexed colony picking in order to enrich transformed populations, select colonies expressing a particular phenotype, or evolve a more efficient living foundry cell strain. With PicoShells, we have demonstrated the ability to pick colonies that overexpress an intracellular protein, as well as colonies with particular growth rates from an initial population.
