Precise Manipulation Of Cells Research Articles

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Polymeric DNA network is a kind of material that can realize functional integration while preserving the specific properties of DNA. DNA networks enable precise manipulation of cells and can be used as a universal tool for understanding the fundamental laws of life and disease diagnosis and treatment, such as cell isolation, cell delivery and cell interference. More details are discussed in the article by Yang et al. on page 1875—1887.image

A 50 µm acoustic resonator microchannel enables focusing 100 nm polystyrene beads and sub-micron bioparticles

The manipulation and focusing of submicron/nanometre-scale objects are becoming increasingly important in the fields of biology, chemistry, and materials science. Acoustofluidics provides for the high throughput and precise manipulation of cells and particles with simple equipment. However, application to submicron/nanoparticles is challenging due to the particle volume dependence of the acoustic radiation force and the relatively larger effect of the viscous drag force on small objects. Here, we present an acoustofluidic device that can focus submicron/nanoparticles, bacterial cells, and extracellular vesicles (EVs) using a high-frequency two-dimensional acoustic standing wave. We fabricate 50 µm × 50 µm square cross-section channels as acoustic resonators and generate a two-dimensional acoustic standing wave at an actuation frequency of 14.9 MHz. This configuration enables a strong acoustic radiation force towards the centre of the microchannel and a cross-section-centred single-vortex stream that does not counteract the acoustic radiation force. Using this device, polystyrene beads ranging in diameter from 50 to 500 nm were focused toward the channel centre, where a focused bead stream width of less than 5 µm was achieved at a flow rate of 3 µL min−1 for 100 nm beads and 9 µL min−1 for 200 nm beads. Furthermore, bacterial cells were successfully focused at a flow rate of 40 µL min−1, and the focusing of EVs was also demonstrated. Our acoustofluidic device can become a promising tool for a wide range of biological analyses targeting submicron cells, viruses, and EVs.

Magnetophoretic Micro-Distributor for Controlled Clustering of Cells.

Cell clustering techniques are important to produce artificial cell clusters for in vitro models of intercellular mechanisms at the single‐cell level. The analyses considering physical variables such as the shape and size of cells have been very limited. In addition, the precise manipulation of cells and control of the physical variables are still challenging. In this paper, a magnetophoretic device consisting of a trampoline micromagnet and active elements that enable the control of individual selective jumping motion and positioning of a micro‐object is proposed. Based on a numerical simulation under various conditions, automatic separation or selective clustering of micro‐objects according to their sizes is performed by parallel control and programmable manipulation. This method provides efficient control of the physical variables of cells and grouping of cells with the desired size and number, which can be a milestone for a better understanding of the intercellular dynamics between clustered cells at the single‐cell level for future cell‐on‐chip applications.

A DNA Molecular Robot that Autonomously Walks on the Cell Membrane to Drive Cell Motility

AbstractSynthetic molecular robots can execute sophisticated molecular tasks at nanometer resolution. However, a molecular robot capable of controlling cellular behavior remains unexplored. Herein, we report a self‐propelled DNA robot operating on the cell membrane to control the migration of a cell. Driven by DNAzyme catalytic activity, the DNA robot could autonomously and stepwise move on the membrane‐floating cell‐surface receptors in a stochastic manner and simultaneously trigger the receptor‐dimerization to activate downstream signaling for cell motility. The cell membrane‐associated continuous motion and operation of a DNA robot allowed for the ultrasensitive regulation of MET/AKT signaling and cytoskeleton remodeling to enhance cell migration. Finally, we designed distinct conditional DNA robots to orthogonally manipulate the cell migration in a coculture of mixed cell populations. We have developed a novel strategy to engineer a cell‐driving molecular robot, representing a promising avenue for precise cell manipulation with nanoscale resolution.

A DNA Molecular Robot that Autonomously Walks on the Cell Membrane to Drive Cell Motility.

Synthetic molecular robots can execute sophisticated molecular tasks at nanometer resolution. However, a molecular robot capable of controlling cellular behavior remains unexplored. Herein, we report a self-propelled DNA robot operating on the cell membrane to control the migration of a cell. Driven by DNAzyme catalytic activity, the DNA robot could autonomously and stepwise move on the membrane-floating cell-surface receptors in a stochastic manner and simultaneously trigger the receptor-dimerization to activate downstream signaling for cell motility. The cell membrane-associated continuous motion and operation of a DNA robot allowed for the ultrasensitive regulation of MET/AKT signaling and cytoskeleton remodeling to enhance cell migration. Finally, we designed distinct conditional DNA robots to orthogonally manipulate the cell migration in a coculture of mixed cell populations. We have developed a novel strategy to engineer a cell-driving molecular robot, representing a promising avenue for precise cell manipulation with nanoscale resolution.

Swimming nanorobots for opening a cell membrane mechanically

AbstractSwimming nanorobots performing efficient self‐propulsion in various biofluids have drawn tremendous attention over 15 years due to their application including active drug delivery, precise cell manipulation, noninvasive surgery, rapid biosensor, and mobile in vivo imaging in the field of biomedicine. However, there are still many challenges in using swimming nanorobots in practice for active drug delivery and therapy, such as biocompatibility of chemical fuels or externally physical fields, biodegradation of synthetic materials, biofouling in bodily fluids, in vivo imaging of swimming nanorobots, and self‐navigation for active targeting. In this review, we highlight the mechanical drilling of cell membranes by swimming nanorobots and discuss the issues, because the cell membranes are one of the key barriers for intracellular drug delivery and drug delivery efficiency of swimming nanorobots. We will summarize the recent advances in swimming nanorobots with different propulsion mechanism and introduce the fundamental issues of interaction between swimming nanorobot and cell membrane. Then, the process, mechanism, and optimization of mechanically opening a cell membrane by swimming nanorobots are discussed and the perspective on the challenge and solution is also included. Such swimming nanorobots capable of mechanically opening a cell membrane could help to better understand the biophysical property of cells and pave the development of precision medicine.

Grafting of 3D Bioprinting to In Vitro Drug Screening: A Review.

The inadequacy of conventional cell-monolayer planar cultures and animal experiments in predicting the toxicity and clinical efficacy of drug candidates has led to an imminent need for in vitro methods with the ability to better represent in vivo conditions and facilitate the systematic investigation of drug candidates. Recent advances in 3D bioprinting have prompted the precise manipulation of cells and biomaterials, rendering it a promising technology for the construction of in vitro tissue/organ models and drug screening devices. This review presents state-of-the-art in vitro methods used for preclinical drug screening and discusses the limitations of these methods. In particular, the significance of constructing 3D in vitro tissue/organ models and microfluidic analysis devices for drug screening is emphasized, and a focus is placed on the grafting process of 3D bioprinting technology to the construction of such models and devices. The in vitro methods for drug screening are generalized into three types: mini-tissue, organ-on-a-chip, and tissue/organ construct. The revolutionary process of the in vitro methods is demonstrated in detail, and relevant studies are listed as examples. Specifically, the tumor model is adopted as a precedent to illustrate the possible grafting of 3D bioprinting to antitumor drug screening.

Capturing magnetic bead-based arrays using perpendicular magnetic anisotropy

Designing and implementing means of locally trapping magnetic beads and understanding the factors underlying the bead capture force are important steps toward advancing the capture-release process of magnetic particles for biological applications. In particular, capturing magnetically labeled cells using magnetic microstructures with perpendicular magnetic anisotropy (PMA) will enable an approach to cell manipulation for emerging lab-on-a-chip devices. Here, a Co (0.2 nm)/Ni (0.4 nm) multilayered structure was designed to exhibit strong PMA and large saturation magnetization (M s ). Finite element simulations were performed to assess the dependence of the capture force on the value of M s . The simulated force profile indicated the largest force at the perimeter of the disks. Arrays of Co/Ni disk structures of (4-7) μm diameter were fabricated and tested in a microchannel with suspended fluorescent magnetic beads. The magnetic beads were captured and localized to the edge of the disks as predicted by the simulations. This approach has been demonstrated to enable uniform assembly of magnetic beads without external fields and may provide a pathway toward precise cell manipulation methods.

Automated Indirect Transportation of Biological Cells with Optical Tweezers and a 3D Printed Microtool

Optical tweezers are widely used for noninvasive and precise micromanipulation of living cells to understand biological processes. By focusing laser beams on cells, direct cell manipulation with optical tweezers can achieve high precision and flexibility. However, direct exposure to the laser beam can lead to negative effects on the cells. These phenomena are also known as photobleaching and photodamage. In this study, we proposed a new indirect cell micromanipulation approach combined with a robot-aided holographic optical tweezer system and 3D nano-printed microtool. The microtool was designed with a V-shaped head and an optical handle part. The V-shaped head can push and trap different sizes of cells as the microtool moves forward by optical trapping of the handle part. In this way, cell exposure to the laser beam can be effectively reduced. The microtool was fabricated with a laser direct writing system by two-photon photopolymerization. A control strategy combined with an imaging processing algorithm was introduced for automated manipulation of the microtool and cells. Experiments were performed to verify the effectiveness of our approach. First, automated microtool transportation and rotation were demonstrated with high precision. Second, indirect optical transportations of cells, with and without an obstacle, were performed to demonstrate the effectiveness of the proposed approach. Third, experiments of fluorescent cell manipulation were performed to confirm that, indicated by the photobleaching effect, indirect manipulation with the microtool could induce less laser exposure compared with direct optical manipulation. The proposed method could be useful in complex biomedical applications where precise cell manipulation and less laser exposure are required.

Translational and rotational manipulation of filamentous cells using optically driven microrobots.

Optical cell manipulation has become increasingly valuable in cell-based assays. In this paper, we demonstrate the translational and rotational manipulation of filamentous cells using multiple cooperative microrobots automatically driven by holographic optical tweezers. The photodamage of the cells due to direct irradiation of the laser beam can be effectively avoided. The proposed method will enable fruitful biomedical applications where precise cell manipulation and less photodamage are required.

Whole genome amplification of single epithelial cells dissociated from snap-frozen tissue samples in microfluidic platform.

Single cell sequencing is a technology capable of analyzing the genome of a single cell within a population. This technology is mostly integrated with microfluidics for precise cell manipulation and fluid handling. So far, most of the microfluidic-based single cell genomic studies have been focused on lab-cultured species or cell lines that are relatively easy to handle following standard microfluidic-based protocols without additional adjustments. The major challenges for performing single cell sequencing on clinical samples is the complex nature of the samples which requires additional sample processing steps to obtain intact single cells of interest without using amplification-inhibitive agents. Fluorescent-activated cell sorting is a common option to obtain single cells from clinical samples for single cell applications but requires >100 000 viable cells in suspension and the need for specialized laboratory and personnel. In this work, we present a protocol that can be used to obtain intact epithelial cells from snap-frozen postsurgical human endometrial tissues for single cell whole genome amplification. Our protocol includes sample thawing, cell dissociation, and labeling for genome amplification of targeted cells. Between 80% and 100% of single cell replicates lead to >25 ng of DNA after amplification with no measurable contamination, sufficient for downstream sequencing.

(Invited) Hybrid Microfluidic Cell Sorting for Rare Cell Isolation

Selective isolation of specific cell subpopulations from highly heterogeneous biological samples is an essential process in various biological studies and clinical applications. The ability to isolate rare cell populations, for example circulating tumor cells, provides unprecedented capabilities for the study of disease progression and better disease assessment. Currently, sorting and isolation of rare cell populations is typically performed using fluorescence-activated cell sorting (FACS) system, a technology developed nearly 60 years ago. These FACS systems are however complex, bulky, and expensive, require highly trained personnel for operation, and may produce bio-hazardous aerosols in open environments. Microfluidics studies the unique behavior and interactions of fluids and particles at the micrometer length scale, typically in confined microscale channels. Microfluidic technology capable of precise cell manipulation thus has great potential to reinvent the next-generation cell sorting technology. We are currently developing two microfluidic cell sorting technologies including high-throughput hydrodynamic cell sorting and high-accuracy acoustic cell sorting. Hydrodynamic cell sorting is using an interesting inertial particle focusing behavior in an intermediate Reynolds number regime (~100 > Re > ~1). We have designed and fabricated a new type of hydrodynamic sorting device with a series of reverse wavy channel structures. These repeated wavy channels produce periodically reversed Dean secondary flow perpendicular to the main flow direction. The hydrodynamic inertial lift force and the Dean drag force scale with the particle size very distinctively, which leads to distinct equilibrium positions of differently sized particles. We have demonstrated the use of this new hydrodynamic sorting device to isolate cancer cells from blood cells without the use of sheath flows. Acoustic cell sorting is developed based on the study of interactions between high-frequency sound waves and microscale particles. In particular, I will discuss a unique design of a focused interdigital transducer (FIDT) structure, which is able to generate a highly localized acoustic field on the order of 25 µm wide. This highly focused acoustic beam has an effective manipulation area size that is comparable to individual micron-sized particles. We have demonstrated the use of this highly localized acoustic field for fluorescence-activated single cell level sorting with sub-millisecond pulses. By combining the two microfluidic cell sorting technologies, we have demonstrated the capability of enriching target cells by 1,000 to 10,000 times, providing a new solution of rare cell isolation.

Editorial for Special Issue: Advances in Microfluidic Devices for Cell Handling and Analysis

Microfluidics is a technology that is expanding rapidly in many areas of research, especially in the biological areas of cell handling and analysis.[...]

Gravity-Based Precise Cell Manipulation System Enhanced by In-Phase Mechanism.

This paper proposes a gravity-based system capable of generating high-resolution pressure for precise cell manipulation or evaluation in a microfluidic channel. While the pressure resolution of conventional pumps for microfluidic applications is usually about hundreds of pascals as the resolution of their feedback sensors, precise cell manipulation at the pascal level cannot be done. The proposed system successfully achieves a resolution of 100 millipascals using water head pressure with an in-phase noise cancelation mechanism. The in-phase mechanism aims to suppress the noises from ambient vibrations to the system. The proposed pressure system is tested with a microfluidic platform for pressure validation. The experimental results show that the in-phase mechanism effectively reduces the pressure turbulence, and the pressure-driven cell movement matches the theoretical simulations. Preliminary experiments on deformability evaluation with red blood cells under incremental pressures of one pascal are successfully performed. Different deformation patterns are observed from cell to cell under precise pressure control.

Patterned Thermoresponsive Microgel Coatings for Noninvasive Processing of Adherent Cells.

Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments.

Acoustic devices for particle and cell manipulation and sensing.

An emerging demand for the precise manipulation of cells and particles for applications in cell biology and analytical chemistry has driven rapid development of ultrasonic manipulation technology. Compared to the other manipulation technologies, such as magnetic tweezing, dielectrophoresis and optical tweezing, ultrasonic manipulation has shown potential in a variety of applications, with its advantages of versatile, inexpensive and easy integration into microfluidic systems, maintenance of cell viability, and generation of sufficient forces to handle particles, cells and their agglomerates. This article briefly reviews current practice and reports our development of various ultrasonic standing wave manipulation devices, including simple devices integrated with high frequency (>20 MHz) ultrasonic transducers for the investigation of biological cells and complex ultrasonic transducer array systems to explore the feasibility of electronically controlled 2-D and 3-D manipulation. Piezoelectric and passive materials, fabrication techniques, characterization methods and possible applications are discussed. The behavior and performance of the devices have been investigated and predicted with computer simulations, and verified experimentally. Issues met during development are highlighted and discussed. To assist long term practical adoption, approaches to low-cost, wafer level batch-production and commercialization potential are also addressed.

Design and Performance of a High-Efficiency Cell-Electrofusion Device

A microfluidic cell-electrofusion device was developed for high fusion efficiency and automatic manipulation. It included an experimental platform, an electric signal generator and a microchip. Microstructure and controlling signal were elaborately chosen in order to realize precise cell manipulation, alignment and electrofusion. Compared with the traditional cell-electrofusion device, this device has some outstanding advantages. For example, short distance between two counter-microelectrodes will reduce the required voltage. A large number of microelectrodes can be fabricated on one small chip for high-throughput fusion. Transparent chip architecture benefited for microscopic observation and real-time recording. The integration of the experimental platform, signal generator and microchip is helpful for developing a portable cell-fusion system. On-chip electrofusion experiments of cucumber mesophyll protoplasts have been carried out and up to 40% fusion efficiency was obtained.

Embryonic stem cell application in drug discovery

Embryonic stem (ES) cells and their differentiated progeny offer tremendous potential for regenerative medicine, even in the field of drug discovery. There is an urgent need for clinically relevant assays that make use of ES cells because of their rich biological utility. Attention has been focused on small molecules that allow the precise manipulation of cells in vitro, which could allow researchers to obtain homogeneous cell types for cell-based therapies and discover drugs for stimulating the regeneration of endogenous cells. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, and homing. In the present paper, we reviewed the use of ES cell models for high-throughput/content drug screening and toxicity assessment. In addition, we examined the role of stem cells in large pharmaceutical companies' R&D and discussed a novel subject, nicheology, in stem cell-related research fields.

Electroporation of micro‐droplet encapsulated HeLa cells in oil phase

Electroporation (EP) is a method widely used to introduce foreign genes, drugs or dyes into cells by permeabilizing the plasma membrane with an external electric field. A variety of microfluidic EP devices have been reported so far. However, further integration of prior and posterior EP processes turns out to be very complicated, mainly due to the difficulty of developing an efficient method for precise manipulation of cells in microfluidics. In this study, by means of a T-junction structure within a delicate microfluidic device, we encapsulated HeLa cells in micro-droplet of poration medium in oil phase before EP, which has two advantages: (i) precise control of cell-encapsulating droplets in oil phase is much easier than the control of cell populations or individuals in aqueous buffers; (ii) this can minimize the electrochemical reactions on the electrodes. Finally, we successfully introduced fluorescent dyes into the micro-droplet encapsulated HeLa cells in oil phase. Our results reflected a novel way to realize the integrated biomicrofluidic system for EP.

A chemical approach to stem-cell biology and regenerative medicine

An improved understanding of stem-cell and regenerative biology, as well as a better control of stem-cell fate, is likely to produce treatments for many devastating diseases and injuries. Chemical approaches are starting to have an increasingly important role in this young field. Attention has focused on chemical approaches that allow the precise manipulation of cells in vitro to obtain homogeneous cell types for cell-based therapies. Another promising approach is the development of conventional chemical and biological therapeutics to stimulate endogenous cells to regenerate. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, reprogramming and homing.