Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS
In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2020-10, Vol.92 (19), p.13262-13270 |
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| creator | Brower, Kara K Khariton, Margarita Suzuki, Peter H Still, Chris Kim, Gaeun Calhoun, Suzanne G. K Qi, Lei S Wang, Bo Fordyce, Polly M |
| description | In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5–25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events ( |
| doi_str_mv | 10.1021/acs.analchem.0c02499 |
| format | Article |
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We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5–25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.</description><identifier>ISSN: 0003-2700</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/acs.analchem.0c02499</identifier><identifier>PMID: 32900183</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Analytical chemistry ; Animals ; Biotechnology ; Cell Encapsulation ; Cell Line ; Cell lines ; Cell size ; Chemistry ; Diameters ; Double emulsions ; Droplets ; Emulsions ; Encapsulation ; Flow Cytometry ; Fluorescence ; Genetic analysis ; Genomes ; Genotypes ; High-throughput screening ; High-Throughput Screening Assays ; Mice ; Microfluidic Analytical Techniques ; Microfluidics ; Morphology ; Nucleic acids ; Particle Size ; Phenotype ; Phenotypes ; Phenotyping ; Robustness ; Screening ; Single-Cell Analysis ; Surface Properties ; Workflow</subject><ispartof>Analytical chemistry (Washington), 2020-10, Vol.92 (19), p.13262-13270</ispartof><rights>Copyright American Chemical Society Oct 6, 2020</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a543t-f72f2cd07803e64f6e206e1c670a85731bfef09ea446f2e3ebce0e74442539423</citedby><cites>FETCH-LOGICAL-a543t-f72f2cd07803e64f6e206e1c670a85731bfef09ea446f2e3ebce0e74442539423</cites><orcidid>0000-0002-9505-0638</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acs.analchem.0c02499$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acs.analchem.0c02499$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,777,781,882,2752,27057,27905,27906,56719,56769</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32900183$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Brower, Kara K</creatorcontrib><creatorcontrib>Khariton, Margarita</creatorcontrib><creatorcontrib>Suzuki, Peter H</creatorcontrib><creatorcontrib>Still, Chris</creatorcontrib><creatorcontrib>Kim, Gaeun</creatorcontrib><creatorcontrib>Calhoun, Suzanne G. 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We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5–25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. 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K</au><au>Qi, Lei S</au><au>Wang, Bo</au><au>Fordyce, Polly M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2020-10-06</date><risdate>2020</risdate><volume>92</volume><issue>19</issue><spage>13262</spage><epage>13270</epage><pages>13262-13270</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><abstract>In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5–25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>32900183</pmid><doi>10.1021/acs.analchem.0c02499</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0002-9505-0638</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Analytical chemistry Animals Biotechnology Cell Encapsulation Cell Line Cell lines Cell size Chemistry Diameters Double emulsions Droplets Emulsions Encapsulation Flow Cytometry Fluorescence Genetic analysis Genomes Genotypes High-throughput screening High-Throughput Screening Assays Mice Microfluidic Analytical Techniques Microfluidics Morphology Nucleic acids Particle Size Phenotype Phenotypes Phenotyping Robustness Screening Single-Cell Analysis Surface Properties Workflow |
| title | Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS |
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