Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Chuo University

This paper describes the utilization of giant unilamellar vesicles (GUVs) as a platform for handling chemical and biochemical reagents. GUVs with diameters of 5 to 10 µm and containing chemical/biochemical reagents together with inert polymers were fused with electric pulses (electrofusion). After reagent mixing, the fused GUVs spontaneously deformed to a budding shape, separating the mixed solution into sub-volumes. We utilized a microfluidic channel and optical tweezers to select GUVs of interest, bring them into contact, and fuse them together to mix and aliquot the reaction product. We also show that, by lowering the ambient temperature close to the phase transition temperature Tm of the lipid used, daughter GUVs completely detached (fission). This process performs all the liquid-handing features used in bench-top biochemistry using the GUV, which could be advantageous for the membrane-related biochemical assays. 

Liposome-based liquid handling platform featuring addition, mixing, and aliquoting of femtoliter volumes.

The gene expression in a clonal cell population fluctuates significantly, and its relevance to various cellular functions is under intensive debate. A fundamental question is whether the fluctuation is a consequence of the complexity and redundancy in living cells or an inevitable attribute of the minute microreactor nature of cells. To answer this question, we constructed an artificial cell, which consists of only necessary components for the gene expression (in vitro transcription and translation system) and its boundary as a microreactor (cell-sized lipid vesicle), and investigated the gene expression noise. The variation in the expression of two fluorescent proteins was decomposed into the components that were correlated and uncorrelated between the two proteins using a method similar to the one used by Elowitz and co-workers to analyze the expression noise in E. coli. The observed fluctuation was compared with a theoretical model that expresses the amplitude of noise as a function of the average number of intermediate molecules and products. With the assumption that the transcripts are partly active, the theoretical model was able to well describe the noise in the artificial system. Furthermore, the same measurement for E. coli cells harboring an identical plasmid revealed that the E. coli exhibited a similar level of expression noise. Our results demonstrated that the level of fluctuation found in bacterial cells is mostly an intrinsic property that arises even in a primitive form of the cell. 

Stochasticity in gene expression in a cell-sized compartment.

We present a method to detect the transporter activity of intact adherent cells using a microchamber device. When adherent cells are seeded onto the poly-di-methyl siloxane substrate having microchambers with openings smaller than the size of a cell, the cells form a confluent layer that covers the microchambers, creating minute, confined spaces. As substances exported across the cell membrane accumulate, transporter activity can be detected by observing the fluorescence intensity increase in the microchamber. We tested the microchamber device with HeLa cells over-expressing MDR1, an ATP-binding cassette transporter, and succeeded in detecting the transport of fluorescence-conjugated paclitaxel, the anti-cancer drug, at the single-cell level. 

Microchamber device for detection of transporter activity of adherent cells.

Herein we examined the extent of replicability of the PDMS microchamber device transferred from the master mold with complex 3D structures fabricated via micro stereolithography. Due to the elastomeric properties of PDMS, the reversely tapered micromold, with the diameter ratio of ∼5 from the largest to the narrowest part, was precisely transferred without breaking. We obtained the mathematical model to estimate the stress exerted on the mold during the demolding process. Finally, we tested the applicability of this unusual microchamber for single-cell trapping and an enzyme assay.

One-step micromolding of complex 3D microchambers for single-cell analysis.

The shapes of giant unilamellar vesicles (GUVs) enclosing polymer molecules at relatively high concentration, used as a model cytoplasm, significantly differ from those containing only small molecules. Here, we investigated the effects of the molecular weights and concentrations of polymers such as polyethylene glycol (PEG), bovine serum albumin (BSA), and DNA on the morphology of GUVs deflated by osmotic pressure. Although small PEG (MW < 1000) does not alter the mode of shape transformation even at >10% (w/w), PEG with MW > 6000 induces budding and pearling transformation at above 1% (w/w). Larger PEG frequently induced small buddings and tubulation from the membrane of mother GUVs. A similar trend was observed with BSA, indicating that the effect is irrelevant to the chemical nature of polymers. More surprisingly, long strands of DNA (>10 bp) enclosed in GUVs induced budding transformation at concentrations as low as 0.01-0.1% (w/w). We expect that this molecular size dependency arises mainly from the depletion volume effect. Our results showed that curving, budding, and tubulation of lipid membranes, which are ubiquitous in living cells, can result from simple cell-mimics consisting of the membrane and cytosolic macromolecules, but without specific shape-determining proteins.

Deformation Modes of Giant Unilamellar Vesicles Encapsulating Biopolymers.

Microelectromechanical systems (MEMS)-based high-performance accelerometers are ubiquitously used in various electronic devices. However, there is an existing need to detect physical impacts using low-cost devices with no electronic circuits or a battery. We designed and fabricated an impact sensor prototype using a commercial stereolithography apparatus that only consists of a plastic housing and working fluids. The sensor device responds to the instantaneous acceleration (impact) by deformation and pinch off of a water droplet that is suspended in oil in a sensor cavity. We tested the various geometrical and physical parameters of the impact sensor to identify their relations to threshold acceleration values. We show that the state diagram that is plotted against the dimensionless Archimedes and Bond numbers adequately describes the response of the proposed sensor.

A fluidics-based impact sensor.

We assessed the applicability of giant unilamellar vesicles (GUVs) for RNA detection using in vesicle reverse transcription polymerase chain reaction (RT-PCR). We prepared GUVs that encapsulated one-pot RT-PCR reaction mixture including template RNA, primers, and Taqman probe, using water-in-oil emulsion transfer method. After thermal cycling, we analysed the GUVs that exhibited intense fluorescence signals, which represented the cDNA amplification. The detailed analysis of flow cytometry data demonstrated that rRNA and mRNA in the total RNA can be amplified from 10-100 copies in the GUVs with 5-10 μm diameter, although the fraction of reactable GUV was approximately 60% at most. Moreover, we report that the target RNA, which was directly transferred into the GUV reactors via membrane fusion, can be amplified and detected using in vesicle RT-PCR. These results suggest that the GUVs can be used as biomimetic reactors capable of performing PCR and RT-PCR, which are important in analytical and diagnostic applications with additional functions.

Reverse Transcription Polymerase Chain Reaction in Giant Unilamellar Vesicles.

The steady streaming (SS) phenomenon is gaining increased attention in the microfluidics community, because it can generate net mass flow from zero-mean vibration. We developed numerical simulation and experimental measurement tools to analyze this vibration-induced flow, which has been challenging due to its unsteady nature. The validity of these analysis methods is confirmed by comparing the three-dimensional (3D) flow field and the resulting particle trajectories induced around a cylindrical micro-pillar under circular vibration. In the numerical modeling, we directly solved the flow in the Lagrangian frame so that the substrate with a micro-pillar becomes stationary, and the results were converted to a stationary Eulerian frame to compare with the experimental results. The present approach enables us to avoid the introduction of a moving boundary or infinitesimal perturbation approximation. The flow field obtained by the micron-resolution particle image velocimetry (micro-PIV) measurement supported the three-dimensionality observed in the numerical results, which could be important for controlling the mass transport and manipulating particulate objects in microfluidic systems.

Numerical and Experimental Analyses of Three- Dimensional Unsteady Flow around a Micro-Pillar Subjected to Rotational Vibration.

In this paper, we demonstrate that 20 μm microbeads can be preferentially assembled into substrate trenches of similar width by employing a polymer (depletant) that induces the depletion volume effect (depletion attraction). In previous works, we proved that this strategy is useful to assemble mesoscale parts in a site-specific manner. Here, we show that it is also applicable to assemble functional parts, such as fluorescent particles, into trenches engraved on the surface of two- and three-dimensional template components. A convenient advantage of this strategy is that it is independent of material properties for assembling mesoscale functional components into desired patterns.

Assembly of Microparticles to Patterned Trenches Using the Depletion Volume Effect.

Electroporation or electropermealization is a technique to open pores in the lipid bilayer membrane of cells and vesicles transiently to increase its permeability to otherwise impermeable molecules. However, the upper size limit of the materials permeable through this operation has not been studied in the past. Here, we investigate the size of the material that can be released (ejected) from giant unilamellar vesicles (GUVs) upon electrical pulsation. We confirm that the volume of GUV shrinks in a stepwise manner upon periodical pulsation, in accordance with previous studies. When the same operation is applied to GUVs that encapsulate microbeads, we find that beads as large as 20 μm can be ejected across the membrane without rupturing the whole GUV structure. We also demonstrate that functional bioactive particulate materials, such as gel balls, vesicles, and cells can be encapsulated in and ejected from GUVs. We foresee that this phenomenon can be applied to precisely regulate the time and location of release of these particulate materials in the microenvironment.

Ejection of Large Particulate Materials from Giant Unilamellar Vesicles Induced by Electropulsation.

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.

A simple microfluidic device for live-imaging of the vertical section of epithelial cells.

Deformation of liposomes, or lipid vesicles, has been investigated extensively in terms of the thermodynamic equilibrium of the bending energy of the lipid bilayer membrane. However, the range of such deformation in previous literature has been limited within the moderate surface-to-volume ratio of the vesicles, in which axisymmetric shapes are dominant. Here, we show that neuron-like morphology, in which many lipid tubes extend radially from the mother vesicle, becomes dominant upon the slow osmotic shrinkage of giant unilamellar vesicles (GUVs) initially larger than several tens of micrometers. We show that, in the time-lapse confocal imaging, the emergence of lipid tubes is initiated from the instability that appeared along the annular rim of the flat stomatocyte shape. Since these deformation dynamics into the neuron-like morphology resemble that of the milk-crown formation in liquid splashing, we discuss that the Rayleigh-Plateau capillary instability drives this transformation into a nonaxisymmetric shape.

Deformation Dynamics of Giant Unilamellar Vesicles in the Large Surface-to-Volume Ratio Regime: The Emergence of Neuron-like Morphology.

The conservation throughout evolution of membrane-bound structures that encapsulate genomic material indicates the existence of a simple, physical mechanism that facilitates the enclosing of long-stranded DNA by lipid bilayers. This study aimed to elucidate such a mechanism by investigating how molecular crowding promotes the spontaneous enveloping of model DNA into lipid bilayer membranes. Using fluorescence microscopy and giant unilamellar vesicles (GUVs) we showed that a 166 kb DNA molecule coencapsulated with a model crowder attaches to the inner membrane of the GUVs as they osmotically deflate and after the DNA-membrane complex buds out. The set of results is consistent with the hypothesis that the depletion volume effect is responsible for the spontaneous encapsulation of DNA in the GUVs. This phenomenon may offer novel insights into the basic mechanisms governing membrane encapsulation of long-stranded nucleic acids found in celluar sytems that are independent of genetic control.

Elucidating the Membrane Dynamics and Encapsulation Mechanism of Large DNA Molecules Under Molecular Crowding Conditions Using Giant Unilamellar Vesicles.

Hiroaki Suzuki

Department of Precision Mechanics,
Faculty of Science and Engineering,
Department of Precision Mechanics, Faculty of Science and Engineering

Chuo University

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Hiroaki Suzuki

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Precision Mechanics,Faculty of Science and Engineering,Department of Precision Mechanics, Faculty of Science and Engineering

Chuo University

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Department of Precision Mechanics,
Faculty of Science and Engineering,
Department of Precision Mechanics, Faculty of Science and Engineering