On-Demand Droplet Collection for Capturing Single Cells.

Droplet-based microfluidic techniques are extensively used in efficient manipulation and genome-wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single-droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on-demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on-demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell-to-cell variations.

[1]  Mehmet Toner,et al.  Circulating tumor cells: approaches to isolation and characterization , 2011, The Journal of cell biology.

[2]  Ke Xu,et al.  Influenza A Virus Replication Induces Cell Cycle Arrest in G0/G1 Phase , 2010, Journal of Virology.

[3]  A. Folch,et al.  Large-scale single-cell trapping and imaging using microwell arrays. , 2005, Analytical chemistry.

[4]  P. Swain,et al.  Stochastic Gene Expression in a Single Cell , 2002, Science.

[5]  H. Kitano.  Systems Biology: A Brief Overview , 2002, Science.

[6]  A. Abate,et al.  SiC-Seq: Single-cell genome sequencing at ultra high-throughput with microfluidic droplet barcoding , 2017, Nature Biotechnology.

[7]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[8]  V. Lee,et al.  Nuclear Localization of DNAJB6 Is Associated With Survival of Patients With Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells. , 2015, Gastroenterology.

[9]  Mario Roederer,et al.  Single-cell technologies for monitoring immune systems , 2014, Nature Immunology.

[10]  N. Neff,et al.  Quantitative assessment of single-cell RNA-sequencing methods , 2013, Nature Methods.

[11]  Lei Jiang,et al.  Manipulating and dispensing micro/nanoliter droplets by superhydrophobic needle nozzles. , 2013, ACS nano.

[12]  Allon M. Klein,et al.  Single-cell barcoding and sequencing using droplet microfluidics , 2016, Nature Protocols.

[13]  M Paturzo,et al.  Dispensing nano-pico droplets and liquid patterning by pyroelectrodynamic shooting. , 2010, Nature nanotechnology.

[14]  Allon M. Klein,et al.  Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells , 2015, Cell.

[15]  Roland Zengerle,et al.  Single-Cell Printer , 2013, Journal of laboratory automation.

[16]  Jonas Jarvius,et al.  Cytokine detection by antibody-based proximity ligation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Inoki,et al.  TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival , 2003, Cell.

[18]  Dino Di Carlo,et al.  Dynamic single-cell analysis for quantitative biology. , 2006, Analytical chemistry.

[19]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[20]  Russell H. Cole,et al.  Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells , 2017, Proceedings of the National Academy of Sciences.

[21]  S. Bodovitz,et al.  Single cell analysis: the new frontier in 'omics'. , 2010, Trends in biotechnology.

[22]  William H. Grover,et al.  Measuring single cell mass, volume, and density with dual suspended microchannel resonators. , 2013, Lab on a chip.

[23]  Albert D. M. E. Osterhaus,et al.  Detection of Influenza A Viruses from Different Species by PCR Amplification of Conserved Sequences in the Matrix Gene , 2000, Journal of Clinical Microbiology.

[24]  K. Jefimovs,et al.  High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis. , 2014, Analytical chemistry.

[25]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[26]  S. Takeuchi,et al.  Monodisperse Alginate Hydrogel Microbeads for Cell Encapsulation , 2007 .

[27]  A. Singh,et al.  Single-cell protein analysis. , 2012, Current opinion in biotechnology.

[28]  Ruben Godoy-Silva,et al.  Computer simulations of the energy dissipation rate in a fluorescence‐activated cell sorter: Implications to cells , 2008, Biotechnology and bioengineering.

[29]  S. Quake,et al.  A microfabricated fluorescence-activated cell sorter , 1999, Nature Biotechnology.

[30]  S. Au,et al.  Nanoliter dispensing method by degassed poly(dimethylsiloxane) microchannels and its application in protein crystallization. , 2007, Analytical chemistry.

[31]  J. Dual,et al.  Enhanced single-cell printing by acoustophoretic cell focusing. , 2015, Biomicrofluidics.

[32]  M. Vandewoestyne,et al.  Laser capture microdissection in forensic research: a review , 2010, International Journal of Legal Medicine.

[33]  Hongbo Xin,et al.  Single Cell Isolation and Analysis , 2016, Front. Cell Dev. Biol..

[34]  Peng Zhang,et al.  Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms , 2017, Scientific Reports.

[35]  Yuanjin Zhao,et al.  Emerging Droplet Microfluidics. , 2017, Chemical reviews.

[36]  Hans Lassmann,et al.  Clonal Expansions of Cd8+ T Cells Dominate the T Cell Infiltrate in Active Multiple Sclerosis Lesions as Shown by Micromanipulation and Single Cell Polymerase Chain Reaction , 2000, The Journal of experimental medicine.

[37]  Carlo C. Maley,et al.  Clonal evolution in cancer , 2012, Nature.

[38]  A. deMello,et al.  The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. , 2015, Lab on a chip.

[39]  D. Kent,et al.  High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays , 2011, Nature Methods.

[40]  Christoph A. Merten,et al.  Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms. , 2008, Chemistry & biology.

[41]  G. Smyth,et al.  ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. , 2009, Journal of immunological methods.

[42]  Roland Zengerle,et al.  Technologies for Single-Cell Isolation , 2015, International journal of molecular sciences.