Pinched-flow hydrodynamic stretching of single-cells.

Reorganization of cytoskeletal networks, condensation and decondensation of chromatin, and other whole cell structural changes often accompany changes in cell state and can reflect underlying disease processes. As such, the observable mechanical properties, or mechanophenotype, which is closely linked to intracellular architecture, can be a useful label-free biomarker of disease. In order to make use of this biomarker, a tool to measure cell mechanical properties should accurately characterize clinical specimens that consist of heterogeneous cell populations or contain small diseased subpopulations. Because of the heterogeneity and potential for rare populations in clinical samples, single-cell, high-throughput assays are ideally suited. Hydrodynamic stretching has recently emerged as a powerful method for carrying out mechanical phenotyping. Importantly, this method operates independently of molecular probes, reducing cost and sample preparation time, and yields information-rich signatures of cell populations through significant image analysis automation, promoting more widespread adoption. In this work, we present an alternative mode of hydrodynamic stretching where inertially-focused cells are squeezed in flow by perpendicular high-speed pinch flows that are extracted from the single inputted cell suspension. The pinched-flow stretching method reveals expected differences in cell deformability in two model systems. Furthermore, hydraulic circuit design is used to tune stretching forces and carry out multiple stretching modes (pinched-flow and extensional) in the same microfluidic channel with a single fluid input. The ability to create a self-sheathing flow from a single input solution should have general utility for other cytometry systems and the pinched-flow design enables an order of magnitude higher throughput (65,000 cells s(-1)) compared to our previously reported deformability cytometry method, which will be especially useful for identification of rare cell populations in clinical body fluids in the future.

[1]  H. Amini,et al.  Label-free cell separation and sorting in microfluidic systems , 2010, Analytical and bioanalytical chemistry.

[2]  Klavs F Jensen,et al.  Microfluidics-based assessment of cell deformability. , 2012, Analytical chemistry.

[3]  Keith B Neeves,et al.  Measuring cell mechanics by optical alignment compression cytometry. , 2013, Lab on a chip.

[4]  M. Tanyeri,et al.  Hydrodynamic trap for single particles and cells. , 2010, Applied physics letters.

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

[6]  Chwee Teck Lim,et al.  Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria. , 2005, Acta biomaterialia.

[7]  Daniel A Fletcher,et al.  Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry. , 2008, Lab on a chip.

[8]  R. Tompkins,et al.  Continuous inertial focusing, ordering, and separation of particles in microchannels , 2007, Proceedings of the National Academy of Sciences.

[9]  D. Gossett,et al.  Particle focusing mechanisms in curving confined flows. , 2009, Analytical chemistry.

[10]  M J Brown,et al.  Rigidity of Circulating Lymphocytes Is Primarily Conferred by Vimentin Intermediate Filaments , 2001, The Journal of Immunology.

[11]  Andreas Undisz,et al.  Dynamic deformability of Plasmodium falciparum-infected erythrocytes exposed to artesunate in vitro. , 2012, Integrative biology : quantitative biosciences from nano to macro.

[12]  Steven W Graves,et al.  Inertial manipulation and transfer of microparticles across laminar fluid streams. , 2012, Small.

[13]  Pingfan Meng,et al.  Strategies for Implementing Hardware-Assisted High-Throughput Cellular Image Analysis , 2011, Journal of laboratory automation.

[14]  Chang Lu,et al.  Microfluidic electroporative flow cytometry for studying single-cell biomechanics. , 2008, Analytical chemistry.

[15]  J. Rao,et al.  Nanomechanical analysis of cells from cancer patients. , 2007, Nature nanotechnology.

[16]  Dennis E. Discher,et al.  Physical plasticity of the nucleus in stem cell differentiation , 2007, Proceedings of the National Academy of Sciences.

[17]  A. Deniz,et al.  Multicolor single-molecule FRET to explore protein folding and binding. , 2010, Molecular bioSystems.

[18]  Dino Di Carlo,et al.  Automated cellular sample preparation using a Centrifuge-on-a-Chip. , 2011, Lab on a chip.

[19]  Nathan Cermak,et al.  Characterizing deformability and surface friction of cancer cells , 2013, Proceedings of the National Academy of Sciences.

[20]  Dino Di Carlo,et al.  Hydrodynamic stretching of single cells for large population mechanical phenotyping , 2012, Proceedings of the National Academy of Sciences.

[21]  Stefan Schinkinger,et al.  Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. , 2005, Biophysical journal.

[22]  Dino Di Carlo,et al.  High-throughput size-based rare cell enrichment using microscale vortices. , 2011, Biomicrofluidics.

[23]  Dino Di Carlo,et al.  Microfluidic sample preparation for diagnostic cytopathology. , 2013, Lab on a chip.

[24]  Magalie Faivre,et al.  High-speed microfluidic differential manometer for cellular-scale hydrodynamics. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Dino Di Carlo,et al.  A mechanical biomarker of cell state in medicine. , 2012 .

[26]  Daniel T Chiu,et al.  Controlled rotation of biological micro- and nano-particles in microvortices. , 2004, Lab on a chip.

[27]  Hongshen Ma,et al.  Microfluidic micropipette aspiration for measuring the deformability of single cells. , 2012, Lab on a chip.