We fabricated piezoelectric energy harvesters composed of
c-axis-oriented Pb(Zr,Ti)O
3 (PZT) films transferred onto stainless steel cantilevers for improved energy conversion efficiency and ...toughness. PZT films were epitaxially grown on Pt/MgO substrates using rf-magnetron sputtering and transferred to 50-μm-thick stainless steel cantilevers. The transferred PZT films had a low dielectric constant (
ɛ
r
=
166) and high transverse piezoelectric coefficient (
e
31*
=
d
31/
s
11
=
−4.5
C/m
2); these values are almost the same as those of the as-grown epitaxial PZT films on MgO substrates. Because of the small thickness of the cantilever, the first resonant frequency could be as low as 126
Hz without a seismic mass at the tip of the cantilever. At an acceleration of 5
m/s
2, the energy harvester generated an average output power of 5.3
μW at the optimal load resistance of 50
kΩ. The output power increased monotonically with the acceleration, reaching 244
μW under an acceleration of 50
m/s
2 without damage to the cantilever.
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.
We fabricated piezoelectric energy harvesters with lead-free (K,Na)NbO3 (KNN) thin films and compared their power-generation performance with that of Pb(Zr,Ti)O3 (PZT) thin film energy harvesters. ...The KNN and PZT thin films were deposited on Pt/Ti/Si substrates by rf-magnetron sputtering. The transverse piezoelectric properties of the KNN and PZT films were essentially the same, e31,f∼−11C/m2. The peak average output power of the unimorph cantilevers of KNN/Si and PZT/Si were 1.1 and 1.0μW, respectively. Thus, the performance of lead-free KNN thin films as a piezoelectric energy harvester is comparable to that of PZT films.
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Cell surface reactions such as material surface characteristics and cell proliferation/adhesion rates are correlated and this correlation can be used as early determination criteria ...for shortening the development period and reducing costs of selecting materials used for bio-experiments, and of bio-experimental instruments. When fabricating Cells-on-Chips, a microfluidic device fabricated using MEMS technology, the influence of the scaffold material should be considered. Since a wide variety of materials are used as constituent materials of Cells-on-Chips, highly versatile early determination criteria corresponding to a specific environment and various materials are needed. In this research, we propose the early determination criteria of scaffolding material composing Cells-on-Chips by using surface free energy, which is calculated from contact angle that can be measured easily and inexpensively. Six materials were selected, regardless of inorganic/organic and photosensitive/non-photosensitive properties. When HeLa cells were cultured on each material, a significant correlation was observed between surface free energy and cell proliferation/adhesion rates. Therefore, the surface free energy of the material is appropriate as the early determination criteria for selecting the constituent material of Cells-on-Chips. Compared with the conventional criteria, the proposed criteria have high versatility regardless of the kind of material and can be used for evaluation of various materials.
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.
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/SiO
) substrate with spacings of 100 nm to 500 nm. The thiol-polyethylene glycol (PEG)-biotin self-assembled monolayer (SAM) and silane-PEG-CH
SAM were then selectively formed on the pillars and SiO
surface, respectively. This allowed for both immobilization of kinesin molecules on Au nano-pillars in a precise manner and repulsion of kinesins from the SiO
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.
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.
High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs ...morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.
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•hiPSC-CMs are seeded on aligned nanofibers to obtain 3D cardiac tissue-like constructs•Drug assessment using CTLCs can be more robust than conventional cultures•CTLCs can be used to model in vitro cardiac tissue engraftment•CTLCs improve the function of rat hearts with myocardial infarction
Although high-purity cardiomyocytes can be achieved, they are randomly distributed and resemble immature rather than adult CMs. In this article, Liu Li and her colleagues show that 3D and aligned cardiac tissue-like constructs (CTLCs) can be obtained using hPSC-CMs and aligned nanofiber. They also demonstrate the robust drug responses and repair capability of CTLCs in a myocardial infarction model.
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-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.
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