High-throughput selection of cells based on accumulated growth and division using PicoShell particles

Abstract

Production of high-energy lipids by microalgae may provide a sustainable energy source that can help tackle climate change. However, microalgae engineered to produce more lipids usually grow slowly, leading to reduced overall yields. Unfortunately, culture vessels used to select cells based on growth while maintaining high biomass production, such as well plates, water-in-oil droplet emulsions, and nanowell arrays, do not provide production-relevant environments that cells experience in scaled-up cultures (e.g., bioreactors or outdoor cultivation farms). As a result, strains that are developed in the laboratory may not exhibit the same beneficial phenotypic behavior when transferred to industrial production. Here, we introduce PicoShells, picoliter-scale porous hydrogel compartments, that enable >100,000 individual cells to be compartmentalized, cultured in production-relevant environments, and selected based on growth and bioproduct accumulation traits using standard flow cytometers. PicoShells consist of a hollow inner cavity where cells are encapsulated and a porous outer shell that allows for continuous solution exchange with the external environment. PicoShells allow for cell growth directly in culture environments, such as shaking flasks and bioreactors. We experimentally demonstrate that Chlorella sp., Saccharomyces cerevisiae, and Chinese hamster ovary cells, used for bioproduction, grow to significantly larger colony sizes in PicoShells than in water-in-oil droplet emulsions (P < 0.05). We also demonstrate that PicoShells containing faster dividing and growing Chlorella clonal colonies can be selected using a fluorescence-activated cell sorter and regrown. Using the PicoShell process, we select a Chlorella population that accumulates chlorophyll 8% faster than does an unselected population after a single selection cycle.

Enabling Clonal Analyses of Yeast in Outer Space by Encapsulation and Desiccation in Hollow Microparticles

“…Of all methods tested here, we found that the vacuum-based desiccation of PicoShells in Novec oil with Pico-Surf caused the least impact on particles and yeast compared to the fresh condition. In this process, particles are fabricated in Novec oil with Pico-Surf according to established protocols [ 15 ], with the addition of 10% trehalose in the dextran phase to aid yeast viability. Particles in Novec oil are strained through a 40 μm filter and washed with additional Novec oil, washing away non-encapsulated cells.…”

“…Specific cell subpopulations in PicoShells can be isolated using commercial flow sorters, with over 100,000 particles screened per sorting experiment [ 15 ]. Flow sorting allows for the isolation of interesting subpopulations based on scattered light and fluorescent readouts [ 11 ].…”

“…In this study, we use PicoShells that are 70 µm in diameter. However, we can achieve PicoShell diameters between 30 and 50 µm to be more compatible with standard nozzle sizes used in flow sorters [ 15 ].…”

“…For example, PEG-maleimide PicoShells crosslinked with DTT or matrix metalloproteinase (MMP)-degradable peptides can be degraded by sodium periodate (NaIO 4 ) or trypsin, respectively. Meanwhile, PicoShells fabricated from PEG-OPSS (ortho-pyridyldisulfide) and crosslinked with DTT can be degraded by reducing agents such as TCEP (tris(2-carboxyethyl)phosphine) or DTT [ 15 ]. While controlled degradation is a useful tool, undesired degradation should be mitigated.…”

“…One 0.5 mL tube filled with PicoShells can more than replace hundreds of plates. Furthermore, cells in PicoShells could be cultured in a fermenter before high-throughput screening, more closely replicating the eventual production environment than microwell plates and potentially accelerating scale up [ 15 ]. If PicoShells became an integral part of the strain engineering workflow, fermentation companies could bring the fabrication process in-house and forgo desiccation, but for academic labs or companies without microfluidics expertise, obtaining desiccated PicoShells from a microfluidics collaborator could be more immediately feasible.…”