High-content single-cell analysis on-chip using a laser microarray scanner.

High-content cellomic analysis is a powerful tool for rapid screening of cellular responses to extracellular cues and examination of intracellular signal transduction pathways at the single-cell level. In conjunction with microfluidics technology that provides unique advantages in sample processing and precise control of fluid delivery, it holds great potential to transform lab-on-a-chip systems for high-throughput cellular analysis. However, high-content imaging instruments are expensive, sophisticated, and not readily accessible. Herein, we report on a laser scanning cytometry approach that exploits a bench-top microarray scanner as an end-point reader to perform rapid and automated fluorescence imaging of cells cultured on a chip. Using high-content imaging analysis algorithms, we demonstrated multiplexed measurements of morphometric and proteomic parameters from all single cells. Our approach shows the improvement of both sensitivity and dynamic range by two orders of magnitude as compared to conventional epifluorescence microscopy. We applied this technology to high-throughput analysis of mesenchymal stem cells on an extracellular matrix protein array and characterization of heterotypic cell populations. This work demonstrates the feasibility of a laser microarray scanner for high-content cellomic analysis and opens up new opportunities to conduct informative cellular analysis and cell-based screening in the lab-on-a-chip systems.

[1]  Pradeep S Rajendran,et al.  Single-cell dissection of transcriptional heterogeneity in human colon tumors , 2011, Nature Biotechnology.

[2]  A. Manz,et al.  Lab-on-a-chip: microfluidics in drug discovery , 2006, Nature Reviews Drug Discovery.

[3]  C. Dominguez,et al.  High-Content Screening Analysis of the p38 Pathway: Profiling of Structurally Related p38α Kinase Inhibitors Using Cell-Based Assays , 2006 .

[4]  N. Gadegaard,et al.  Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. , 2011, Nature materials.

[5]  Donald Wlodkowic,et al.  Microfluidic cell arrays in tumor analysis: new prospects for integrated cytomics , 2010, Expert review of molecular diagnostics.

[6]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[7]  M. E. Ruaro,et al.  Design, fabrication and evaluation of nanoscale surface topography as a tool in directing differentiation and organisation of embryonic stem-cell-derived neural precursors , 2009 .

[8]  Rong Fan,et al.  A Clinical Microchip for Evaluation of Single Immune Cells Reveals High Functional Heterogeneity in Phenotypically Similar T Cells Nih Public Access Author Manuscript Design Rationale and Detection Limit of the Scbc Online Methods Microchip Fabrication On-chip Secretion Profiling Supplementary Mater , 2022 .

[9]  Claudia Fischbach,et al.  Microfluidic culture models of tumor angiogenesis. , 2010, Tissue engineering. Part A.

[10]  Megan L. McCain,et al.  Ensembles of engineered cardiac tissues for physiological and pharmacological study: heart on a chip. , 2011, Lab on a chip.

[11]  J. Philip McCoy,et al.  High-content screening: getting more from less , 2011, Nature Methods.

[12]  R. Cheong,et al.  Using a Microfluidic Device for High-Content Analysis of Cell Signaling , 2009, Science Signaling.

[13]  Christophe Antczak,et al.  Live-Cell Imaging of Caspase Activation for High-Content Screening , 2009, Journal of biomolecular screening.

[14]  S. Bhatia,et al.  An extracellular matrix microarray for probing cellular differentiation , 2005, Nature Methods.

[15]  Stephen R. Quake,et al.  Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria , 2006, Science.

[16]  D. Ingber,et al.  Reconstituting Organ-Level Lung Functions on a Chip , 2010, Science.

[17]  W. Saltzman,et al.  Biodegradable meshes printed with extracellular matrix proteins support micropatterned hepatocyte cultures. , 2009, Tissue engineering. Part A.

[18]  Yousef Al-Kofahi,et al.  Laser scanning cytometry and its applications: a pioneering technology in the field of quantitative imaging cytometry. , 2011, Methods in cell biology.

[19]  Bingcheng Lin,et al.  Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device. , 2010, Lab on a chip.

[20]  Z. Darżynkiewicz,et al.  Laser scanning cytometry: principles and applications. , 2006, Methods in molecular biology.

[21]  M. Harnett,et al.  Laser scanning cytometry: understanding the immune system in situ , 2007, Nature Reviews Immunology.

[22]  Anne E Carpenter,et al.  Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software , 2011, Bioinform..

[23]  Stephen R Quake,et al.  Whole-genome molecular haplotyping of single cells , 2011, Nature Biotechnology.

[24]  High-content imaging , 2010, Nature Biotechnology.

[25]  P Ravi Selvaganapathy,et al.  Microfluidic devices for cell based high throughput screening. , 2010, Lab on a chip.

[26]  K. Cheung,et al.  Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing. , 2010, Lab on a chip.

[27]  F. Tay,et al.  Studying tumor extravasation using a microfluidic CHIP , 2007 .

[28]  Anne E Carpenter,et al.  CellProfiler: free, versatile software for automated biological image analysis. , 2007, BioTechniques.

[29]  B. Lin,et al.  Cell-based high content screening using an integrated microfluidic device. , 2007, Lab on a chip.

[30]  M. Roederer,et al.  11-color, 13-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity , 2001, Nature Medicine.

[31]  Timothy K Lee,et al.  Single-cell NF-κB dynamics reveal digital activation and analogue information processing , 2010, Nature.

[32]  Pengyu Hong,et al.  Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening , 2010, Neuroinformatics.

[33]  Christopher S. Chen,et al.  Cells lying on a bed of microneedles: An approach to isolate mechanical force , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Quake,et al.  Monolithic microfabricated valves and pumps by multilayer soft lithography. , 2000, Science.

[35]  Vincent Studer,et al.  A nanoliter-scale nucleic acid processor with parallel architecture , 2004, Nature Biotechnology.

[36]  Hong Wu,et al.  A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens. , 2010, Cancer research.

[37]  P. Mitchell.  Microfluidics—downsizing large-scale biology , 2001, Nature Biotechnology.

[38]  Sukdeb Pal,et al.  High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device. , 2011, Lab on a chip.

[39]  B. Lin,et al.  Characterization of the interaction between fibroblasts and tumor cells on a microfluidic co‐culture device , 2010, Electrophoresis.

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

[41]  Francis E H Tay,et al.  A quantitative observation and imaging of single tumor cell migration and deformation using a multi-gap microfluidic device representing the blood vessel. , 2006, Microvascular research.

[42]  Andre Levchenko,et al.  High Content Cell Screening in a Microfluidic Device*S , 2009, Molecular & Cellular Proteomics.

[43]  G. Whitesides.  The origins and the future of microfluidics , 2006, Nature.

[44]  Ravi A. Desai,et al.  Mechanical regulation of cell function with geometrically modulated elastomeric substrates , 2010, Nature Methods.

[45]  Arul Jayaraman,et al.  Rapid Fabrication of Bio‐inspired 3D Microfluidic Vascular Networks , 2009 .

[46]  I. Vermes,et al.  Microfluidic Technology in Vascular Research , 2009, Journal of biomedicine & biotechnology.