Single Cell Manipulation Research Articles

6AM2-D-4 超並列デジタル細胞操作ステーションの開発 : 電場駆動力を利用した単一細胞の同時並列操作(6AM2-D OS3 マイクロ・ナノ生体医工学)

6AM2-D-4 超並列デジタル細胞操作ステーションの開発 : 電場駆動力を利用した単一細胞の同時並列操作(6AM2-D OS3 マイクロ・ナノ生体医工学)

Optoelectronic tweezers integrated with lensfree holographic microscopy for wide-field interactive cell and particle manipulation on a chip.

We demonstrate an optoelectronic tweezer (OET) coupled to a lensfree holographic microscope for real-time interactive manipulation of cells and micro-particles over a large field-of-view (FOV). This integrated platform can record the holographic images of cells and particles over the entire active area of a CCD sensor array, perform digital image reconstruction to identify target cells, dynamically track the positions of cells and particles, and project light beams to trigger light-induced dielectrophoretic forces to pattern and sort cells on a chip. OET technology has been previously shown to be capable of performing parallel single cell manipulation over a large area. However, its throughput has been bottlenecked by the number of cells that can be imaged within the limited FOV of a conventional microscope objective lens. Integrating lensfree holographic imaging with OET solves this fundamental FOV barrier, while also creating a compact on-chip cell/particle manipulation platform. Using this unique platform, we have successfully demonstrated real-time interactive manipulation of thousands of single cells and micro-particles over an ultra-large area of e.g., 240 mm(2) (i.e. 17.96 mm × 13.52 mm).

Analysis and suppression of residual vibration in microhand for high-speed single-cell manipulation

Stable grasping of micro-objects at high speed is a desired feature for micromanipulation systems. At high speed, however, the residual vibration tends to be greater. In this paper, we analyse the residual vibration of the end effector of the microhand during the high-speed grasping motion. Based on the analysis results, we propose suppression of residual vibration in two steps. First, the stiffness of the end effector is increased by structural design. Second, feedforward control is implemented since the behaviour of the vibration can predicted for the specific conditions. Preliminary experimental results show that the settling time decreases by 74% and the amplitude of the vibration by 47%.

An opto-thermocapillary cell micromanipulator.

An opto-thermocapillary micromanipulator (OTMm) capable of single-cell manipulation and patterning is presented here. The OTMm uses a near-infrared laser focused on an ITO substrate to induce thermocapillary convection that can trap and transport living cells with forces of up to 40 pN. The OTMm complements other cell-manipulation technologies, such as optical tweezers and dielectrophoresis, as it is less dependent upon the optical and electrical properties of the working environment, and can function in many types of cell culture media. The OTMm was used to construct single-cell matrices in two popular hydrogels: PEGDA and agarose. High viability rates were observed in both hydrogels, and cells patterned in agarose spread and migrated during subsequent culturing.

Hippocampal Place Fields Emerge upon Single-Cell Manipulation of Excitability During Behavior

The origin of the spatial receptive fields of hippocampal place cells has not been established. A hippocampal CA1 pyramidal cell receives thousands of synaptic inputs, mostly from other spatially tuned neurons; however, how the postsynaptic neuron's cellular properties determine the response to these inputs during behavior is unknown. We discovered that, contrary to expectations from basic models of place cells and neuronal integration, a small, spatially uniform depolarization of the spatially untuned somatic membrane potential of a silent cell leads to the sudden and reversible emergence of a spatially tuned subthreshold response and place-field spiking. Such gating of inputs by postsynaptic neuronal excitability reveals a cellular mechanism for receptive field origin and may be critical for the formation of hippocampal memory representations.

Bubble Jet agent release cartridge for chemical single cell stimulation

We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d <30μm) on-demand micro gradients can be generated for the specific manipulation of single cells. A single channel and a double channel agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V = 15pl and V = 10pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.

On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves

Techniques that can dexterously manipulate single particles, cells, and organisms are invaluable for many applications in biology, chemistry, engineering, and physics. Here, we demonstrate standing surface acoustic wave based "acoustic tweezers" that can trap and manipulate single microparticles, cells, and entire organisms (i.e., Caenorhabditis elegans) in a single-layer microfluidic chip. Our acoustic tweezers utilize the wide resonance band of chirped interdigital transducers to achieve real-time control of a standing surface acoustic wave field, which enables flexible manipulation of most known microparticles. The power density required by our acoustic device is significantly lower than its optical counterparts (10,000,000 times less than optical tweezers and 100 times less than optoelectronic tweezers), which renders the technique more biocompatible and amenable to miniaturization. Cell-viability tests were conducted to verify the tweezers' compatibility with biological objects. With its advantages in biocompatibility, miniaturization, and versatility, the acoustic tweezers presented here will become a powerful tool for many disciplines of science and engineering.

Selection of B16 Melanoma Cells by Single Cell Manipulation and Analysis of Tyrosinase Gene Expression and Epigenetic Regulation

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High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.

Segmented magnetic nanofibers for single cell manipulation

We report a simple but straightforward approach to fabricate magnetic nanofiber segments for cell manipulation. Electrospinning was used to produce nanofibers from a magnetic nanoparticles containing polymethylglutarimide (PMGI) precursor solution. After sonication, the fabricated nanofibers were uniformly segmented. When dispersed in an aqueous solution, the orientation of the fiber segments could easily be controlled by an external magnetic field. NIH 3T3 cells were then cultured in a medium containing magnetic fibers, resulting in stable cell-nanofiber hybrids which can be conveniently manipulated with a magnet.

Sensitive and high-throughput isolation of rare cells from peripheral blood with ensemble-decision aliquot ranking.

Enrichment of cells: An approach called ensemble-decision aliquot ranking (eDAR) for isolating rare cells from peripheral blood is described. The eDAR process has a recovery of over 93 % (number of runs n=9) with a zero false positive rate (n=8) and provides direct easy access to individual isolated live cells for downstream single-cell manipulation and analysis (CTCs=circulating tumor cells).

Towards Single-Cell Analysis For Pharmacokinetics

Traditional 'macroscopic' pharmacokinetics (PK) investigates the fate of drugs or toxicants administered externally to living organisms, described by the extent and rate of absorption, distribution, metabolism and excretion. However, how a single cell affects a specific pharmaceutical after administration still remains a largely untouched area, primarily due to the technical restrictions imposed by minute amounts of chemicals involved. With the fast development of high-temporal and spatial-resolution detection techniques and single-cell handling techniques, it becomes possible to pursue single-cell PK. This review summarizes useful methodological and experimental techniques to investigate PK at the level of the single cell, including the microfluidics-based single-cell manipulation and the MS and electrochemical methods for single-cell analysis.

GABA Signaling Promotes Synapse Elimination and Axon Pruning in Developing Cortical Inhibitory Interneurons

Accumulating evidence indicates that GABA acts beyond inhibitory synaptic transmission and regulates the development of inhibitory synapses in the vertebrate brain, but the underlying cellular mechanism is not well understood. We have combined live imaging of cortical GABAergic axons across time scales from minutes to days with single-cell genetic manipulation of GABA release to examine its role in distinct steps of inhibitory synapse formation in the mouse neocortex. We have shown previously, by genetic knockdown of GABA synthesis in developing interneurons, that GABA signaling promotes the maturation of inhibitory synapses and axons. Here we found that a complete blockade of GABA release in basket interneurons resulted in an opposite effect, a cell-autonomous increase in axon and bouton density with apparently normal synapse structures. These results not only demonstrate that GABA is unnecessary for synapse formation per se but also uncover a novel facet of GABA in regulating synapse elimination and axon pruning. Live imaging revealed that developing GABAergic axons form a large number of transient boutons, but only a subset was stabilized. Release blockade led to significantly increased bouton stability and filopodia density, increased axon branch extension, and decreased branch retraction. Our results suggest that a major component of GABA function in synapse development is transmission-mediated elimination of subsets of nascent contacts. Therefore, GABA may regulate activity-dependent inhibitory synapse formation by coordinately eliminating certain nascent contacts while promoting the maturation of other nascent synapses.

Investigating the Effects of Dynamic External Stimuli on Single Cell Fitness and Gene Expression in Escherichia Coli

In this work, we study the bacterial cellular response to time-dependent external stimuli in single living cells. We developed a microfluidic platform for single cell analysis that allows for dynamic control of well-defined environmental growth conditions and culture media. Using this platform, we studied the effect of small molecule inducers on gene expression in the lac operon using fluorescent reporter proteins and cell growth rates as a proxy of cellular fitness. We applied temporally varying inducer concentrations by translating single cells between two adjacent fluid streams containing either growth medium or growth medium and inducer. We observed that single cell gene expression depends on growth rate and frequency of exposure to inducer concentrations. Single cell induction experiments are compared to control experiments with and without continuous fluid flow in microfluidic channels. For these experiments, single cell analysis is facilitated by a microfluidic-based hydrodynamic trap recently developed in our lab. The hydrodynamic trap enables confinement and manipulation of single cells in free solution using the sole action of fluid flow. Automated feedback control is integrated into the device using an “on-chip” valve, which allows for precise confinement of cells in free solution. Using this device, cells are confined at a fluid stagnation point generated in a cross-slot microfluidic geometry, thereby enabling non-perturbative trapping of cells for long time scales. Using optical microscopy, we observe single Escherichia coli cells growing and dividing both at room temperature and at 370C, and cell division rates in the microfluidic trap compare favorably to the growth rates of E. coli measured in bulk studies. We anticipate that the microfluidic-based trap is an ideal platform to study cellular regulation and gene network dynamics of single living cells in free solution.

Voltage controlled nano-injection system for single-cell surgery

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.

Targeted delivery to single cells in precisely controlled microenvironments

The heterogeneous nature of cells can be an issue for in vitro analysis of cell function due to cell type differences within a population. Observations are most often averaged and dependent on the homogeneity or lack thereof for most cell types. Patterning of features at the sub-cellular scale (< 10 μm) allows for single cell manipulation. Additionally, the ability to pattern multiple materials simultaneously with nanoscale precision enables facile fabrication of multiplexed cellular microenvironment arrays. Here we use this ability to deliver different materials to single or few cells within hundreds of microns of each other on the same substrate. Calcein AM, Calcein Red AM and quantum dots are delivered to live single or few cells. This allows for exposing limited cell numbers to many well defined conditions, thus opening the possibility of single cell based assays.

Single-Cell Biopsy using Nanopipettes

To fully understand cell functioning and stochastic gene expression patterns, it necessary to measure molecular interactions at the single cell level. Advances in microscopy as well as genomics technology have enabled detailed analysis of gene expression at the single-cell level, but the means to manipulate individual cells are still limited. Nanotechnology-based tools having high sensitivity and low invasiveness are holding great promises as new biomedical devices for single cell manipulation. The fully electrical read-out as well as the ease and low cost of fabrication are unique features that give nanopipette technology enormous potential. We developed a single-cell manipulation platform based on nanopipettes. The system uses scanning microscopy techniques to position the nanopipette with nanoscale precision, and electro-wetting to aspirate minute amount of cytoplasmic material from individual cells without comprising cell viability. It is well documented that the application of a voltage at the interface across two immiscible liquids changes their surface tension. The resulting force is sufficient to cause liquid to flow into/out of the nanopipette, with displacement volumes much smaller than a typical mammalian cell. Multiple biopsies can be taken from the same cell to study gene expression patterns. We will present preliminary RNA-seq data of material biopsied from a single cell. We envision that this technology will allow biological analysis with unprecedented level of detail, allowing a better understanding of single cell dynamics.

Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability

BackgroundDespite the availability of conventional devices for making single-cell manipulations, determining the hardness of a single cell remains difficult. Here, we consider the cell to be a linear elastic body and apply Young’s modulus (modulus of elasticity), which is defined as the ratio of the repulsive force (stress) in response to the applied strain. In this new method, a scanning probe microscope (SPM) is operated with a cantilever in the “contact-and-push” mode, and the cantilever is applied to the cell surface over a set distance (applied strain).ResultsWe determined the hardness of the following bacterial cells: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and five Bacillus spp. In log phase, these strains had a similar Young’s modulus, but Bacillus spp. spores were significantly harder than the corresponding vegetative cells. There was a positive, linear correlation between the hardness of bacterial spores and heat or ultraviolet (UV) resistance.ConclusionsUsing this technique, the hardness of a single vegetative bacterial cell or spore could be determined based on Young’s modulus. As an application of this technique, we demonstrated that the hardness of individual bacterial spores was directly proportional to heat and UV resistance, which are the conventional measures of physical durability. This technique allows the rapid and direct determination of spore durability and provides a valuable and innovative method for the evaluation of physical properties in the field of microbiology.

Microfluidic single-cell cultivation chip with controllable immobilization and selective release of yeast cells

We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.

Electromagnetically actuated micromanipulator using an acoustically oscillating bubble

A novel non-invasive micromanipulation technique has been developed where a microrobot swimming in an aqueous medium manipulates micro-objects, through electromagnetic actuation using an acoustically oscillating bubble attached to the microrobot as a grasping tool. This micromanipulation concept was experimentally verified; an investigation of electromagnetic actuation and acoustic excitation was also performed. Two-dimensional propulsion of a magnetic piece was demonstrated through electromagnetic actuation, using three pairs of electric coils surrounding the water chamber, and confirming that the propulsion speed of the magnetic piece was linearly proportional to the applied current intensity. Micro-object manipulation was separately demonstrated using an air bubble with glass beads (80 µm diameter) and a steel ball (800 µm diameter) in an aqueous medium. Upon acoustic excitation of the bubble by a piezo-actuator around its resonant frequency, the generated radiation force attracted and captured the neighboring glass beads and steel ball. The grasping force was indirectly measured by exposing the glass beads captured by the oscillating bubble to a stream generated by an auto-syringe pump in a mini-channel. By measuring the maximum speed of the streaming flow when the glass beads detached from the oscillating bubble and flowed downstream, the grasping force was calculated as 50 nN, based on Stokes' drag approximation. Finally, a fish egg was successfully manipulated with the integration of electromagnetic actuation and acoustic excitation, using a mini-robot consisting of a millimeter-sized magnetic piece with a bubble attached to its bottom. This novel micromanipulation may be an efficient tool for both micro device assembly and single-cell manipulation.