High-throughput selection of microalgae based on biomass accumulation rates in production environments using PicoShell Particles

Abstract

Production of high-energy lipids by microalgae may provide a sustainable, renewable energy source that can help tackle climate change. However, microalgae engineered to produce more lipids usually grow slowly, leading to reduced overall yields. Unfortunately, tools that enable the selection of 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 lab often do not exhibit the same beneficial phenotypic behavior when transferred to industrial production. Here we introduce PicoShells, picoliter-scale porous hydrogel compartments, that can enable >100,000 individual cells to be compartmentalized, cultured in production-relevant environments, and selected based on growth and biomass 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 so that nutrients, cell-communication factors, and cytotoxic cellular byproducts can transport freely in and out of the inner cavity. PicoShells can also be placed directly into shaking flasks, bioreactors, or other production-relevant environments. We experimentally demonstrate that Chlorella sp. and Saccharomyces cerevisiae grow to significantly larger colony sizes in PicoShells than in water-in-oil droplet emulsions (P < 0.05). We have also demonstrated that PicoShells containing faster biomass accumulating Chlorella clonal colonies can be selected using a fluorescence-activated cell sorter and re-grown. Using the PicoShell process, we select a Chlorella population that accumulates biomass 8% faster than does an un-selected population after a single selection cycle.

Scalable Fabrication and Use of 3D Structured Microparticles Spatially Functionalized with Biomolecules

“…31 Localization of the binding moieties can further be exploited for the self-assembly of multicell systems. 32 Aside from the crescent-shaped nanovial particles we focus on in this work, this approach can also be applied for scaled fabrication of hollow-shell particles which can be used for studying clonal populations of mammalian cells, microalgae, and bacteria in biologically relevant environments, 13 or act as an immunoprotective layer for allogeneic or xenogeneic cell therapies.…”

“…The copyright holder for this preprint this version posted August 3, 2021. ; https://doi.org/10.1101/2021.07.14.451688 doi: bioRxiv preprint Droplet microfluidics, where single, double, or aqueous two-phase emulsions have been employed to generate shaped microparticles, 11,12 has emerged as a powerful platform to produce uniform microparticles with different functions and properties. In particular, crescent-shaped microparticles 5 or hollow shell particles 13 produced by polymerizing precursors following aqueous two-phase separation (ATPS), possess a sub-nanoliter size cavity which can hold cells, and can template water in oil emulsions for performing single-cell and digital molecular assays. The current approach to manufacture these shaped particles, such as "nanovials", requires precise injection of multiple polymer precursors into flow focusing microfluidic geometries, limiting scalability.…”

Single-cell sorting based on secreted products for functionally defined cell therapies

“…Brower et al demonstrated the preliminary feasibility of sorting single-cell-containing microdroplets using flow cytometry by forming water-oil-water double emulsions where an outer surfactant-stabilized oil layer preserves droplet stability within the aqueous flow of the cytometer 75,76 ; however, the generation of such double emulsions is significantly more complex than that of their single-phase counterparts. Alternative approaches, such as the gelation of aqueous droplets, can provide compatibility with flow cytometry if the gel has secretion capture components 60,77 , but workflows for breaking emulsions and the subsequent recovery of cells or their genetic information from within the microgel are nontrivial 78,79 . Particle-based cell carriers that can isolate cells and capture secretions are ideal candidates for enabling the analysis of single-cell secretions using flow cytometry; however, the microparticle size is often a limiting factor in terms of compatibility with FACS systems.…”

Scalable Fabrication of 3D Structured Microparticles Using Induced Phase Separation

“…Droplet microfluidics, where single, double, or aqueous two-phase emulsions have been employed to generate shaped microparticles, 11, 12 has emerged as a powerful platform to produce uniform microparticles with different functions and properties. In particular, crescent-shaped microparticles 5 or hollow shell particles 13 produced by polymerizing precursors following aqueous two-phase separation (ATPS), possess a sub-nanoliter size cavity which can hold cells, and can template water in oil emulsions for performing single-cell and digital molecular assays. The current approach to manufacture these shaped particles, such as “nanovials”, requires precise injection of multiple polymer precursors into flow focusing microfluidic geometries, limiting scalability.…”