Single Cell Manipulation Research Articles

Hippo pathway regulation by cell morphology and stress fibers

The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.

Generation and Staining of MARCM Clones in Drosophila: Figure 1.

INTRODUCTIONDendrite morphological diversity helps to define the properties of neural circuits by influencing circuit organization and information processing. Dendrite development has been shown to be driven by a combination of cell-intrinsic and -extrinsic factors. However, much remains to be discovered about the cellular and molecular mechanisms that give rise to unique yet highly stereotyped dendrite arbors of diverse neuronal types. Work in the last decade has established the Drosophila system as an excellent model for studies of dendrite morphogenesis. Mosaic analysis with a repressible cell marker (MARCM) permits resolution of dendrites at a single-cell level and genetic manipulation of individual neurons to assess gene function during neuronal morphogenesis. Mosaic systems for examining mutant phenotypes are advantageous for studies of larval stages because of the ability to discern cell-autonomous phenotypes at very high resolution. The protocol described here can be used to generate and label MARCM clones for the analysis of dendritic patterning and branching control in Drosophila.

Characterization of a fully human monoclonal antibody against extracellular domain of matrix protein 2 of influenza A virus

The extra-cellular domain of the influenza virus matrix protein 2 (M2e) is highly conserved between influenza A virus strains compared to hemagglutinin and neuraminidase, and has long been viewed as a potential and universal vaccine target. M2e induces no or only weak and transient immune responses following infection, making it difficult to detect M2e-specific antibodies producing B-cells in human peripheral blood lymphocytes. Recently, using a single-cell manipulation method, immunospot array assay on a chip (ISAAC), we obtained an M2e-specific human antibody (Ab1-10) from the peripheral blood of a healthy volunteer. In this report, we have demonstrate that Ab1-10 reacted not only to seasonal influenza A viruses, but also to pandemic (H1N1) 2009 virus (2009 H1N1) and highly pathogenic avian influenza A virus, and that the antibody-bound M2e of 2009 H1N1 inactivated the virus with high affinity (∼10 −10 M). More importantly, it inhibited 2009 H1N1 viral propagation in vitro. These results suggest that Ab1-10 might be a potential candidate for antibody therapeutics for a wide range of influenza A viruses.

Drop-on-Demand Single Cell Isolation and Total RNA Analysis

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods.

High-throughput single-cell manipulation system for a large number of target cells

A sequential and high-throughput single-cell manipulation system for a large volume of cells was developed and the successive manipulation for single cell involving single-cell isolation, individual labeling, and individual rupture was realized in a microhydrodynamic flow channel fabricated by using two-dimensional simple flow channels. This microfluidic system consisted of the successive single-cell handlings of single-cell isolation from a large number of cells in cell suspension, labeling each isolated single cell and the lysate extraction from each labeled single cell. This microfluidic system was composed of main channels, cell-trapping pockets, drain channels, and single-cell content collection channels which were fabricated by polydimethylsiloxane. We demonstrated two kinds of prototypes for sequential single-cell manipulations, one was equipped with 16 single-cell isolation pockets in microchannel and the other was constructed of 512 single-cell isolation pockets. In this study, we demonstrated high-throughput and high-volume single-cell isolation with 512 pocket type device. The total number of isolated single cells in each isolation pocket from the cell suspension at a time was 426 for the cell line of African green monkey kidney, COS-1, and 360 for the rat primary brown preadipocytes, BAT. All isolated cells were stained with fluorescence dye injected into the same microchannel successfully. In addition, the extraction and collection of the cell contents was demonstrated using isolated stained COS-1 cells. The cell contents extracted from each captured cell were individually collected within each collection channel by local hydrodynamic flow. The sequential trapping, labeling, and content extraction with 512 pocket type devices realized high-throughput single-cell manipulations for innovative single-cell handling, feasible staining, and accurate cell rupture.

Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics

Single-cell genetic manipulation is expected to substantially advance the field of systems neuroscience. However, existing gene delivery techniques do not allow researchers to electrophysiologically characterize cells and to thereby establish an experimental link between physiology and genetics for understanding neuronal function. In the mouse brain in vivo, we found that neurons remained intact after 'blind' whole-cell recording, that DNA vectors could be delivered through the patch-pipette during such recordings and that these vectors drove protein expression in recorded cells for at least 7 d. To illustrate the utility of this approach, we recorded visually evoked synaptic responses in primary visual cortical cells while delivering DNA plasmids that allowed retrograde, monosynaptic tracing of each neuron's presynaptic inputs. By providing a biophysical profile of a cell before its specific genetic perturbation, this combinatorial method captures the synaptic and anatomical receptive field of a neuron.

Single Cell Sensing and Manipulation by Scanning Nanopore Microscopy

Signal Transduction by Ion Nano-Gating (STING) technology is a label-free biosensor based on quartz nanopipettes capable of detecting DNA and proteins. Using conical quartz nanopipettes a Scanning Ion Conductance Microscope (SICM) was developed and used for single cell imaging, sensing and manipulation. The height of the nanopore above a surface can be accurately controlled by measuring the ionic current through the pore as it approaches a surface. By maintaining a predefined distance from a surface and raster scanning the pore, a topographical image can be acquired. Additionally, material can be ejected through nanopipette using electrophoretic and electroosmotic properties. The cost of fabrication and ease of use of nanopipettes provides an ideal system for developing cellular systems for both cell detection and control. We demonstrate the capability of this sensor to image living cells, deposit material for controlled single cell growth on a substrate, and a platform for single cell injection and detection using an integrated microfluidic chip. The combination of STING technology with traditional SICM technology can be leveraged to acquire both topographical data and functional data, such as local ion concentration. This form of complementary microscopy is henceforth termed functional Scanning Ion Conductance Microscopy (fSICM). Preliminary fSICM results will be presented. This work illustrates the applicability of the STING sensor to a variety of applications, from single cell detection to definition of cellular circuits.

Single Sickle Cell Occlusion and Manipulation in Microchannels

Sickle cell disease arises because a point mutation permits the hemoglobin in erythrocytes to form stiff polymers and obstruct the microcirculation. We have developed a microfluidic system to model vaso-occlusion in the smallest vessels to fill a critical gap in understanding the pathophysiology of sickle cell disease. Microfluidic methods heretofore have only studied far larger vessels, and could not observe events of speed comparable to physiologic transit times. We use microchannels of approximately rectangular cross section (3 μm x 1.5 μm). Because the cells fill the channel top-to-bottom, absorbance can determine intracellular concentration. Cells can be flowed, viewed statically, or stopped and restarted. By laser photolysis of COHbS, any desaturation of the cells can be created in milliseconds. Channels are constructed of PDMS, and cells are driven by hydrostatic pressure which can be controlled to stop the cell within the channel. The cell can then be photolyzed, and studied rheologically. Polymerized cells stick in the channels, but can be pushed through, with pressure that rises with the mass of the polymers formed. Cells sickled at the entry into such channels (see Figure) are found to maintain occlusion against much higher pressures.

Emerging fabrication techniques for 3D nano-structuring in plasmonics and single molecule studies

The application of new methods and techniques to fields such as biology and medicine is becoming more and more demanding since the request of detailed information down to single molecules is a scientific necessity and a technical realistic possibility. In this effort a key role is played by emerging fabrication techniques. One of the hardest challenges is to incorporate the third dimension in the design and fabrication of novel devices. Significantly, this means that conventional nano-fabrication methods, intrinsically useful for planar structuring, have to be substituted or complemented with new approaches. In this paper we show how emerging techniques can be used for 3D structuring of noble metals down to nanoscale. In particular, the paper deals with electroless deposition of silver, ion and electron beam induced deposition, focused ion beam milling, and two-photon lithography. We exploited these techniques to fabricate different plasmonics nanolenses, nanoprobes and novel beads for optical tweezers. In the future these devices will be used for the manipulation and chemical investigation of single cells with sensitivity down to a few molecules in label free conditions and native environment. Although this paper is only devoted to nanofabrication, we foresee that the fields of biology and medicine will directly gain substantial advantages from this approach.

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells.

P-1-8 単一細胞操作デバイスにおける流路幅の影響に関する研究(マイクロナノメカトロニクス,ポスター要旨講演)

In this study, we investigated influence of the microchannel width on a microdevice for single-cell manipulation. The proposed device was fabricated by the Single-Mask inclined exposure method which can fabricated a lot of micro-holes to fix cells and lower microchannel to load reagent. We estimated the restriction of the fabrication process due to the width of the lower microchannel. Moreover, we proposed a reduction method of the warp of the fabricated device made of the thick permanent film resist to achieve a wide lower microchannel.

Quantitative modeling of forces in electromagnetic tweezers.

This paper discusses numerical simulations of the magnetic field produced by an electromagnet for generation of forces on superparamagnetic microspheres used in manipulation of single molecules or cells. Single molecule force spectroscopy based on magnetic tweezers can be used in applications that require parallel readout of biopolymer stretching or biomolecular binding. The magnetic tweezers exert forces on the surface-immobilized macromolecule by pulling a magnetic bead attached to the free end of the molecule in the direction of the field gradient. In a typical force spectroscopy experiment, the pulling forces can range between subpiconewton to tens of piconewtons. In order to effectively provide such forces, an understanding of the source of the magnetic field is required as the first step in the design of force spectroscopy systems. In this study, we use a numerical technique, the method of auxiliary sources, to investigate the influence of electromagnet geometry and material parameters of the magnetic core on the magnetic forces pulling the target beads in the area of interest. The close proximity of the area of interest to the magnet body results in deviations from intuitive relations between magnet size and pulling force, as well as in the force decay with distance. We discuss the benefits and drawbacks of various geometric modifications affecting the magnitude and spatial distribution of forces achievable with an electromagnet.

Single Cell Deposition and Patterning with a Robotic System

Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.

SmashCell: a software framework for the analysis of single-cell amplified genome sequences

Summary: Recent advances in single-cell manipulation technology, whole genome amplification and high-throughput sequencing have now made it possible to sequence the genome of an individual cell. The bioinformatic analysis of these genomes, however, is far more complicated than the analysis of those generated using traditional, culture-based methods. In order to simplify this analysis, we have developed SmashCell (Simple Metagenomics Analysis SHell-for sequences from single Cells). It is designed to automate the main steps in microbial genome analysis—assembly, gene prediction, functional annotation—in a way that allows parameter and algorithm exploration at each step in the process. It also manages the data created by these analyses and provides visualization methods for rapid analysis of the results.Availability: The SmashCell source code and a comprehensive manual are available at http://asiago.stanford.edu/SmashCellContact: eoghanh@stanford.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Nanoliters Discharge/Suction by Thermoresponsive Polymer Actuated Probe and Applied for Single Cell Manipulation

We propose the Thermoresponsive Polymer Actuated (TPA) probe which uses thermoresponsive polymer poly (N-isopropylacrylamide) (PNIPAAm) volume change as an actuator. The proposed probe is applicable to single cell analysis, especially single cell manipulation. The TPA probe can discharge and suck solution in several nanoliters (nl) using the volume change. Normally, it is difficult to realize solution discharge and suction less than several dozen nl by the conventional air- or oil-pressure-actuated probe. We designed the TPA probe for low-cost fabrication and disposable use. The probe also takes in and ejects on a nl order by simply switching a heater on and off. PNIPAAm solution volume change was evaluated in this paper. The manipulation of single microbead and the suction of target cell were also demonstrated by the TPA probe in the semi-closed microchip. It is considered that the TPA probe can contribute to the manipulation of single cell.

Rapid and Direct Cell-to-Cell Adherence Using Avidin-Biotin Binding System: Large Aggregate Formation in Suspension Culture and Small Tissue Element Formation Having a Precise Microstructure Using Optical Tweezers

Effectively organizing isolated cells to tissue elements having an appropriate microstructure is a fundamental issue in future tissue engineering, but biological cell-to-cell adhesion is too weak to assemble single cells directly. In order to overcome the difficulty, we applied an Avidin-Biotin Binding System (ABBS) to cell surfaces, and avidinylated and biotinylated cells could mutually bind in the short time they were mixed together. Unlike conventional intact cells, ABBS helped make larger spheroids. Interestingly, avidinylated and biotinylated cell adherence occurred within 1 sec using laser trapping, enabling single cell manipulation. We showed precise, direct single-cell-based tissue assembly using ABBS and optical tweezers, followed by damage-free tissue culture. The combination of ABBS and single cell manipulation has considerable potential for use in application such as tissue engineering, regenerative medicine, and drug screening system.

Single-cell manipulation on microfluidic chip by dielectrophoretic actuation and impedance detection

This paper presents the design, fabrication, and characterization of a microfluidic biochip with integrated actuation electrodes used to manipulate a cell and a microbead by dielectrophoresis and sensing electrodes to detect the trapping by using the impedance detection method. Combining deflective dielectrophoretic barriers with controlled pressure-driven liquid flows allows the accurate control of a cell/microbead in suspensions. The threshold voltage for microbead trapping was experimentally verified at various flow rates. The impedance change caused by the blockage of the electrical conducting path between sensing electrodes with the trapping of an MCF7 cell and a polystyrene microbead was measured. The impedance before the trapping of an MCF7 cell was 10.9 MΩ at 1 kHz and increased to 12 MΩ when the cell was placed between sensing electrodes.

Driving method of microtool by horizontally arranged permanent magnets for single cell manipulation

This paper presents an innovative driving method for a magnetically driven microtool to achieve precise positioning control while maintaining a high power output derived from commercialized permanent magnets. An effective driving methodology using permanent magnets, whose axes are parallel to driving direction, is applied to reduce friction force on the microtool. The positioning accuracy improves by five times and the response speed becomes ten times faster against the driving stage than in the conventional method. Furthermore, this method has been extended to two-degree-of-freedom movements, and the performance of the magnetically driven microtools is experimentally validated by oocyte manipulation.