Fabrication of 3D concentric amphiphilic microparticles to form uniform nanoliter reaction volumes for amplified affinity assays

Destgeer, Ghulam; Ouyang, Mengxing; Wu, Chueh‐Yu; Carlo, Dino Di

doi:10.1039/d0lc00698j

Cited by 34 publications

(77 citation statements)

References 60 publications

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“…One potential way to address this is to use a coaxial device geometry to reduce the amount of gelled material that sticks to the walls of the device ( 45 ). Use of UV-induced cross-linking mechanisms can also address this problem since gelation would occur downstream of droplet generation ( 29 , 46 , 47 ) unlike pH-induced mechanisms, where mixing of reagents immediately prior to droplet generation often results in gelled material forming in the droplet generation junction over time that disrupts the overall flow. UV cross-linking could also enable PicoShell manufacturing approaches with higher throughput ( 48 ).…”

Section: Discussionmentioning

confidence: 99%

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

Zee

Rutte

Rumyan

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Zee

Rutte

Rumyan

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Use of UV-induced crosslinking mechanisms can solve this problem since gelation would occur downstream of droplet generation 27. 35, 36 , unlike pH-induced mechanisms where mixing of reagents immediately prior to droplet generation often results in gelled material forming in the droplet-generation junction over time that disrupts the overall flow. However, use of UV-induced crosslinking likely creates issues for particular cellular applications, as previously discussed.…”

Section: Discussionmentioning

confidence: 99%

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

Rumyan

et al. 2021

Preprint

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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.

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“…Spherical gel particles have been used previously to template emulsions (28,29), however, the lack of a cavity precluded their use with mammalian cells, and no secretion assays or compatibility with flow cytometry were demonstrated. We have also previously used amphiphilic cavity-containing particles to form uniform emulsions and perform molecular and cellular assays (25)(26)(27); however, these previous systems were not compatible with commercial flow cytometers due to their hydrophobic components.…”

Section: Discussionmentioning

confidence: 99%

“…Nanovials can be analyzed and isolated using commonly available FACS instruments enabling screening at rates >1000 events per second. While other particle systems have been utilized to hold sub-nanoliter volumes (25)(26)(27)(28)(29), ours is the first approach that allows attachment and protection of cells in cavities within particles, unlocking new capabilities to expand the scale of analysis by integration with flow cytometry. Using this easily-adopted nanovial format we conducted a screen of over 1 million events in less than an hour using a commercial FACS instrument, sorting out rare antibody-secreting cells from orders of magnitude more abundant background cells, all within one day.…”

Section: Introductionmentioning

confidence: 99%

Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting

Rutte

Dimatteo

Archang

et al. 2020

Preprint

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Techniques to analyze and sort single cells based on secreted products have the potential to transform our understanding of cellular biology as well as accelerate the development of next generation cell and antibody therapies. However, secretions are rapidly transported away from cells, such that specialized equipment and expertise has been required to compartmentalize cells and capture their secretions. Herein we demonstrate the use of cavity-containing hydrogel microparticles to perform functional single-cell secretion analysis and sorting using only commonly accessible lab infrastructure. These microparticles act as a solid support which facilitates cell attachment, templates formation of uniform aqueous compartments which prevent cross-talk between cells, and captures secreted proteins. Using this platform we demonstrate high-throughput analysis and sorting of Chinese Hamster Ovary cells based on their relative production of human IgG using commercially available flow sorters.Microparticles are easily distributed and used, democratizing access to high-throughput functional cell screening.

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Fabrication of 3D concentric amphiphilic microparticles to form uniform nanoliter reaction volumes for amplified affinity assays

Abstract: Uniform fluid compartments are formed inside shape-coded amphiphilic particles using simple fluid exchange steps. This lab on a particle system enables multiplexed enzymatic reactions without cross talk to democratize cutting-edge biological assays.

Cited by 34 publications

References 60 publications

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

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

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

Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting

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