Centrifugation-Assisted Single-Cell Trapping in a Truncated Cone-Shaped Microwell Array Chip for the Real-Time Observation of Cellular Apoptosis.

Microfluidic devices have been extensively used in single-cell assays. However, most of them have complicated structures (multiple layers, valves, and channels) and require the assistance of a pump or pressure-controlling system. In this paper, we present a facile centrifugation-assisted single-cell trapping (CAScT) approach based on a truncated cone-shaped microwell array (TCMA) chip for real-time observation of cellular apoptosis. Our method requires neither a pump nor a pressure-controlling system, and it greatly reduces the complexity of other cell-trapping devices. This method is so fast and efficient that single-cell occupancy could reach approximately 90% within a few seconds. Combined with modern fluorescence microscopy, CAScT makes the highly ordered and addressable TCMA a high-throughput platform (10(4)-10(5) single-cell trapping sites per cm(2)) for single-cell analysis. Cells trapped in it could be exposed to various chemicals by directly immersing it in bulk solutions without the significant loss of cells due to the truncated cone shape of the microwells. As a proof of concept, we demonstrated the ability of our chip for the real-time observation of the apoptosis of single HeLa cells induced by the common anticancer drug doxorubicin. This simple, robust, and efficient approach possesses great potential in diverse applications, such as drug screening, biosensing, and fundamental biological research.

[1]  Arum Han,et al.  A high-throughput microfluidic single-cell screening platform capable of selective cell extraction. , 2015, Lab on a chip.

[2]  Sumio Sugano,et al.  Single-cell analysis of lung adenocarcinoma cell lines reveals diverse expression patterns of individual cells invoked by a molecular target drug treatment , 2015, Genome Biology.

[3]  Huabing Yin,et al.  Raman-activated cell sorting based on dielectrophoretic single-cell trap and release. , 2015, Analytical chemistry.

[4]  Huaying Chen,et al.  High-throughput, deterministic single cell trapping and long-term clonal cell culture in microfluidic devices. , 2015, Lab on a chip.

[5]  D. Pe’er,et al.  Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands , 2015, Proceedings of the National Academy of Sciences.

[6]  I. Hsing,et al.  Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells. , 2015, ACS applied materials & interfaces.

[7]  Hongkai Wu,et al.  Recent Developments in Microfluidics for Cell Studies , 2014, Advanced materials.

[8]  Shawn M. Gillespie,et al.  Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma , 2014, Science.

[9]  Amy E. Herr,et al.  Single-cell western blotting , 2014, Nature Methods.

[10]  Qian Wang,et al.  Highly sensitive and homogeneous detection of membrane protein on a single living cell by aptamer and nicking enzyme assisted signal amplification based on microfluidic droplets. , 2014, Analytical chemistry.

[11]  Jong-In Han,et al.  In situ analysis of heterogeneity in the lipid content of single green microalgae in alginate hydrogel microcapsules. , 2013, Analytical chemistry.

[12]  Pei-Yu Chiou,et al.  Microfluidic integrated optoelectronic tweezers for single-cell preparation and analysis. , 2013, Lab on a chip.

[13]  Klaus Eyer,et al.  A microchamber array for single cell isolation and analysis of intracellular biomolecules. , 2012, Lab on a chip.

[14]  Chun-Ping Jen,et al.  Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells , 2011, Sensors.

[15]  Rong Fan,et al.  Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells , 2011, Proceedings of the National Academy of Sciences.

[16]  Catherine A. Rivet,et al.  Imaging single-cell signaling dynamics with a deterministic high-density single-cell trap array. , 2011, Analytical chemistry.

[17]  Hongkai Wu,et al.  Single-cell assays. , 2011, Biomicrofluidics.

[18]  Min Cheol Park,et al.  High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging. , 2011, Lab on a chip.

[19]  David K. Wood,et al.  Single cell trapping and DNA damage analysis using microwell arrays , 2010, Proceedings of the National Academy of Sciences.

[20]  Hongkai Wu,et al.  A prototypic microfluidic platform generating stepwise concentration gradients for real-time study of cell apoptosis. , 2010, Biomicrofluidics.

[21]  Donald Wlodkowic,et al.  Microfluidic single-cell array cytometry for the analysis of tumor apoptosis. , 2009, Analytical chemistry.

[22]  David R Walt,et al.  Multianalyte single-cell analysis with multiple cell lines using a fiber-optic array. , 2007, Analytical chemistry.

[23]  C. Greiner,et al.  SU-8: a photoresist for high-aspect-ratio and 3D submicron lithography , 2007 .

[24]  Bo Huang,et al.  Counting Low-Copy Number Proteins in a Single Cell , 2007, Science.

[25]  K. Jensen,et al.  Cells on chips , 2006, Nature.

[26]  Lionel Buchaillot,et al.  Variation of absorption coefficient and determination of critical dose of SU-8 at 365 nm , 2006 .

[27]  Y. Kakinuma,et al.  Doxorubicin induces apoptosis by activation of caspase-3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. , 2006, Journal of pharmacological sciences.

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

[29]  Da Xing,et al.  Measuring dynamics of caspase‐3 activity in living cells using FRET technique during apoptosis induced by high fluence low‐power laser irradiation , 2005, Lasers in surgery and medicine.

[30]  R. Hancock,et al.  Internal organisation of the nucleus: assembly of compartments by macromolecular crowding and the nuclear matrix model , 2004, Biology of the cell.

[31]  R. Zare,et al.  Chemical cytometry on a picoliter-scale integrated microfluidic chip. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Joy Joseph,et al.  Doxorubicin Induces Apoptosis in Normal and Tumor Cells via Distinctly Different Mechanisms , 2004, Journal of Biological Chemistry.

[33]  A. Rebbaa,et al.  Caspase inhibition switches doxorubicin-induced apoptosis to senescence , 2003, Oncogene.

[34]  Chi-Hung Lin,et al.  Critical role of mitochondrial reactive oxygen species formation in visible laser irradiation-induced apoptosis in rat brain astrocytes (RBA-1). , 2002, Journal of biomedical science.

[35]  David R Walt,et al.  Optical imaging fiber-based single live cell arrays: a high-density cell assay platform. , 2002, Analytical chemistry.

[36]  Y. Pu,et al.  Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells. , 2001, Biochemical and biophysical research communications.

[37]  A. Thor,et al.  Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis. , 2001, Cancer research.

[38]  D R Walt,et al.  Application of high-density optical microwell arrays in a live-cell biosensing system. , 2000, Analytical biochemistry.

[39]  V. Dixit,et al.  Death receptors: signaling and modulation. , 1998, Science.

[40]  S. Nagata,et al.  Apoptosis by Death Factor , 1997, Cell.