Distinct Kinetics in Electrophoretic Extraction of Cytoplasmic RNA from Single Cells.

The physical fractionation of cytoplasmic versus nuclear components of cells is a key step for studying the subcellular localization of molecules. The application of an electric field is an emerging method for subcellular fractionation of proteins and nucleic acids from single cells. However, the multibiophysical process that involves electrical lysis of cytoplasmic membranes, electrophoresis, and diffusion of charged molecules remains unclear. Here we study RNA dynamics in single cells during the electrophoretic extraction via a microfluidic system that enables stringent fractionation of the subcellular components leveraging a focused electric field. We identified two distinct kinetics in the extraction of RNA molecules, which were respectively associated with soluble RNA and mitochondrial RNA. We show that the extraction kinetics of soluble RNA is dominated by electrophoresis over diffusion and has a time constant of 0.15 s. Interestingly, the extraction of mitochondrial RNA showed unexpected heterogeneity in the extraction with slower kinetics (3.8 s), while reproducibly resulting in the extraction of 98.9% ± 2% after 40 s. Together, we uncover that the microfluidic system uniquely offers length bias-free fractionation of RNA molecules for quantitative analysis of correlations among subcellular compartments by exploiting the homogeneous electrophoretic properties of RNA.

Spatial Patterning of Kinesin-1 and Dynein Motor Proteins in an In Vitro Assay using Aqueous Two-Phase Systems (ATPS).

Cooperativity of motor proteins is essential for intracellular transport. Although their motion is unidirectional, they often cause bidirectional movement by different types of motors as seen in organelles. However, in vitro assessments of such cellular functions are still inadequate owing to the experimental limitations in precisely patterning multiple motors. Here, we present an approach to immobilize two motor proteins, kinesin-1 and dynein, using the aqueous two-phase system (ATPS) made of poly(ethylene glycol) and dextran polymers. The negligible influence of polymer solutions on the attachment and velocity of motor proteins ensures the compatibility of using ATPS as the patterning technique. The selective fixation of kinesin and dynein was assessed using polarity-marked microtubules (PMMTs). Our experimental results show that on a patterned kinesin surface, 72% of PMMTs display minus-end leading motility, while on a dynein surface, 79% of PMMTs display plus-end leading motility. This work offers a universal and biocompatible method to pattern motor proteins of different classes at the nanoscale, providing a new route to study different cellular functions performed by molecular motors such as the formation of mitotic spindles.

Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate).

We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.

Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells.

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregates were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality.

On-chip separation and analysis of RNA and DNA from single cells.

The simultaneous analysis of RNA and DNA of single cells remains a challenge as these species have very similar physical and biochemical properties and can cross-contaminate each other. Presented is an on-chip system that enables selective lysing of single living cells, extraction, focusing, and absolute quantification of cytoplasmic RNA mass and its physical separation from DNA in the nucleus using electrical lysing and isotachophoresis (ITP). This absolute quantitation is performed without enzymatic amplification in less than 5 min. The nucleus is preserved, and its DNA fluorescence signal can be measured independently. We demonstrate the technique using single mouse lymphocyte cells, for which we extracted an average of 14.1 pg of total RNA per cell. We also demonstrate correlation analysis between the absolute amount of RNA and relative amount of DNA, showing heterogeneity associated with cell cycles. The technique is compatible with fractionation of DNA and RNA and with downstream assays of each.

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

A perfusable microfluidic device with on-chip total internal reflection fluorescence microscopy (TIRFM) for in situ and real-time monitoring of live cells.

A microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis was developed. This integrated device enables in situ Total Internal Reflection Fluorescence Microscopy (TIRFM) on adherent cells cultured under continuous medium perfusion. This TIR-based chip, allows TIRFM to be easily performed on cells without the assembly of complicated optical components and cell culture chambers. The integrated device was evaluated by tracking the movement of fluorescent beads and monitoring the location of insulin granules in mouse pancreatic ß-cells. This system offers higher signal-to-noise (S/N) ratio than epi-fluorescence microscopy (EPIFM), and comparable image quality to commercial TIRFM systems when imaging insulin granules. We also detected repetitive changes in intracellular Ca(2+) concentration in MIN6-m9 cells stimulated with KCl, which demonstrates quick perfusion for cell analysis while maintaining high S/N ratio.

Dielectrophoresis-assisted massively parallel cell pairing and fusion based on field constriction created by a micro-orifice array sheet.

In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two-dimensional micro-orifice array. The sheet is sandwiched by a pair of micro-chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size-independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two-dimensional orifice arrangement at the pitch of 50 µm realizes simultaneous fusion of 6 × 10³ cells on a 4 mm diameter chip, and the fusion yield of 78-90% is achieved for various sizes and types of cells.

Cell Culture on Low-Fluorescence and High-Resolution Photoresist.

2D and 3D topographic cues made of photoresist, a polymer, are used for cell culture and cell analysis. Photoresists used for cell analysis provide the surface conditions necessary for proper cell growth, along with patterning properties of a wide range and high precision, and low auto-fluorescence that does not affect fluorescence imaging. In this study, we developed a thick negative photoresist SJI-001 possessing the aforementioned properties. We evaluated the surface conditions of SJI-001 affecting cell culture. First, we studied the wettability of SJI-001, which was changed by plasma treatment, conducted as a pretreatment on a plastic substrate before cell seeding. SJI-001 was more chemically stable than SU-8 used for fabricating the micro-electromechanical systems (MEMS). Furthermore, the doubling time and adhesion rate of adherent HeLa cells cultured on untreated SJI-001 were 25.2 h and 74%, respectively, thus indicating its suitability for cell culture over SU-8. In addition, we fabricated a cell culture plate with a 3D lattice structure, three micrometers in size, using SJI-001. HeLa cells seeded on this plate remained attached over five days. Therefore, SJI-001 exhibits surface conditions suitable for cell culture and has several bioapplications including microstructures and cell chips for cell culture and cell analysis.

Different motilities of microtubules driven by kinesin-1 and kinesin-14 motors patterned on nanopillars.

Kinesin is a motor protein that plays important roles in a variety of cellular functions. In vivo, multiple kinesin molecules are bound to cargo and work as a team to produce larger forces or higher speeds than a single kinesin. However, the coordination of kinesins remains poorly understood because of the experimental difficulty in controlling the number and arrangement of kinesins, which are considered to affect their coordination. Here, we report that both the number and spacing significantly influence the velocity of microtubules driven by nonprocessive kinesin-14 (Ncd), whereas neither the number nor the spacing changes the velocity in the case of highly processive kinesin-1. This result was realized by the optimum nanopatterning method of kinesins that enables immobilization of a single kinesin on a nanopillar. Our proposed method enables us to study the individual effects of the number and spacing of motors on the collective dynamics of multiple motors.

Surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopy for sensitive detection of tumor markers.

Surface plasmon resonance (SPR), which provides real-time, in situ analysis of dynamic surface events, is a valuable tool for studying interactions between biomolecules. In the clinical diagnosis of tumor markers in human blood, SPR is applied to detect the formation of a sandwich-type immune complex composed of a primary antibody immobilized on a sensor surface, the tumor marker, and a secondary antibody. However, the SPR signal is quite low due to the minute amounts (ng-pg/mL) of most tumor markers in blood. We have shown that the SPR signal can be amplified by applying an antibody against the secondary antibody or streptavidin-conjugated nanobeads that specifically accumulate on the secondary antibody. Another method employed for highly sensitive detection is the surface plasmon field-enhanced fluorescence spectroscopy-based immunoassay, which utilizes the enhanced electric field intensity at a metal/water interface to excite a fluorophore. Fluorescence intensity attributed to binding of a fluorophore-labeled secondary antibody is increased due to the enhanced field in the SPR condition and can be monitored in real time.

Transport of microtubules according to the number and spacing of kinesin motors on gold nano-pillars.

Motor proteins function in in vivo ensembles to achieve cargo transport, flagellum motion, and mitotic cell division. Although the cooperativity of multiple motors is indispensable for physiological function, reconstituting the arrangement of motors in vitro is challenging, so detailed analysis of the functions of motor ensembles has not yet been achieved. Here, we developed an assay platform to study the motility of microtubules driven by a defined number of kinesin motors spaced in a definite manner. Gold (Au) nano-pillar arrays were fabricated on a silicon/silicon dioxide (Si/SiO2) substrate with spacings of 100 nm to 500 nm. The thiol-polyethylene glycol (PEG)-biotin self-assembled monolayer (SAM) and silane-PEG-CH3 SAM were then selectively formed on the pillars and SiO2 surface, respectively. This allowed for both immobilization of kinesin molecules on Au nano-pillars in a precise manner and repulsion of kinesins from the SiO2 surface. Using arrayed kinesin motors, we report that motor number and spacing do not influence the motility of microtubules driven by kinesin-1 motors. This assay platform is applicable to all kinds of biotinylated motors, allows the study of the effects of motor number and spacing, and is expected to reveal novel behaviors of motor proteins.

Clonal Isolation of Human Pluripotent Stem Cells on Nanofibrous Substrates Reveals an Advanced Subclone for Cardiomyocyte Differentiation.

Human pluripotent stem cells (hPSCs) have been widely used for various applications including disease modeling and regenerative medicine, among others. Recently, an increasing number of studies has focused on heterogeneity among hPSCs, which could affect cell quality and subsequent applications. In this study, a nanofibrous platform is developed for single human induced pluripotent stem cell isolation and culture. One type of single cell-derived subclone is established and found to have a distinct morphology compared to other subclones. When used for differentiation toward cardiomyocytes, this type of subclone demonstrates higher differentiation efficiency, increased maturation, and stronger beating compared to those derived from the other subclones. The findings provide a convenient method for single-cell isolation and culture, and demonstrate that variations in differentiation tendencies exist among subclones from the same cell line. This substrate adhesion-based selection process could be used to obtain cell lines with improved differentiation efficiency toward cardiomyocytes and other cell types, which would be advantageous for studies in various fields.

Gene transfer device utilizing micron-spiked electrodes produced by the self-organization phenomenon of Fe-alloy.

In the diffusional phase transformation of two-phase alloys, the new phase precipitates form the matrix phase at specific temperatures, followed by the formation of a mixed microstructure comprising the precipitate and the matrix. It has been found that by specific chemical-etching treatment, the precipitate in Fe-25Cr-6Ni alloy projects substantially and clusters at the surface. The configuration of the precipitate has an extremely high aspect ratio: it is several microns in width and several tens of microns in length (known as micron-spiked). This study targets the development of a gene transfer device with a micro-spike produced based on the self-organization phenomenon of the Fe-25Cr-6Ni alloy. With this spike-projected device, we tried to efficiently transfer plasmid DNA into adherent cells by electric pulse-triggered gene transfer using a plasmid-loaded electrode (electroporation-based reverse transfection). The spiked structure was applied to a substrate of the device to allow efficient gene transfer into adherent cells, although the general substrate was flat and had a smooth surface. The results suggest that this unique spike-projected device has potential applications in gene transfer devices for the analysis of the human genome in the post-genome period.

Engineering of vascularized 3D cell constructs to model cellular interactions through a vascular network.

Current in vitro 3D culture models lack a vascular system to transport oxygen and nutrients, as well as cells, which is essential to maintain cellular viability and functions. Here, we describe a microfluidic method to generate a perfusable vascular network that can form inside 3D multicellular spheroids and functionally connect to microchannels. Multicellular spheroids containing endothelial cells and lung fibroblasts were embedded within a hydrogel inside a microchannel, and then, endothelial cells were seeded into both sides of the hydrogel so that angiogenic sprouts from the cell spheroids and the microchannels were anastomosed to form a 3D vascular network. Solution containing cells and reagents can be perfused inside the cell spheroids through the vascular network by injecting it into a microchannel. This method can be used to study cancer cell migration towards 3D co-culture spheroids through a vascular network. We recapitulated a bone-like microenvironment by culturing multicellular spheroids containing osteo-differentiated mesenchymal stem cells (MSCs), as well as endothelial cells, and fibroblasts in the device. After the formation of vascularized spheroids, breast cancer cells were injected into a microchannel connected to a vascular network and cultured for 7 days on-chip to monitor cellular migration. We demonstrated that migration rates of the breast cancer cells towards multicellular spheroids via blood vessels were significantly higher in the bone-like microenvironment compared with the microenvironment formed by undifferentiated MSCs. These findings demonstrate the potential value of the 3D vascularized spheroids-on-a-chip for modeling in vivo-like cellular microenvironments, drug delivery through blood vessels, and cellular interactions through a vascular network.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 µM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

SINC-seq: correlation of transient gene expressions between nucleus and cytoplasm reflects single-cell physiology.

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level.

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells.

We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Separation of long DNA chains using a nonuniform electric field: a numerical study.

In the present study, we investigate the migration of DNA molecules through a microchannel using a series of electric traps controlled by an ac electric field. We describe the motion of DNA based on Brownian dynamics simulations of a bead-spring chain. The DNA chain captured by an electric field escapes due to thermal fluctuation. The mobility of the DNA chain was determined to depend on the chain length, the mobility of which sharply increases when the length of the chain exceeds a critical value that is strongly affected by the amplitude of the applied ac field. Thus we can optimize the separation selectivity of the channel for DNA molecules that is to be separated, without changing the structure of the channel. In addition, we present a phenomenological description for the relationship between the critical chain length and the strength of binding electric field.