Scalable pattern formation of skeletal myotubes by synergizing microtopographic cues and chiral nematics of cells.

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotubes deviating from the groove boundaries is commonly observed but has long been unattended. In this study, we report a method to guide the formation of skeletal myotubes into scalable and controlled patterns. By inducing C2C12 myoblasts onto grooved patterns with different widths (from 0.4 to 200µm), we observed an enhanced chiral orientation of cells developing on wide grooves (50 and 100µm width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of the cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, these chiral myotubes were formed with enhanced length, diameter, and contractility on wide grooves. Treatment of latrunculin A (Lat A) suppressed the chiral rotation and migration of cells as well as the myotube formation, suggesting the essence of chiral nematics of cells for myogenesis. Finally, by arranging wide grooved/striped patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes were formed, providing a strategy for engineering skeletal muscle tissue formation.

Microfluidic immunoassay for detection of serological antibodies: A potential tool for rapid evaluation of immunity against SARS-CoV-2.

In December 2019, coronavirus disease 2019 became a pandemic affecting more than 200 countries and territories. Millions of lives are still affected because of mandatory quarantines, which hamstring economies and induce panic. Immunology plays a major role in the modern field of medicine, especially against virulent infectious diseases. In this field, neutralizing antibodies are heavily studied because they reflect the level of infection and individuals' immune status, which are essential when considering resumption of work, flight travel, and border entry control. More importantly, it also allows evaluating the antiviral vaccine efficacy as vaccines are still known for being the ultimate intervention method to inhibit the rapid spread of virulent infectious diseases. In this Review, we first introduce the host immune response after the infection of SARS-CoV-2 and discuss the latest results using conventional immunoassays. Next, as an enabling platform for detection with sufficient sensitivity while saving analysis time and sample size, the progress of microfluidic-based immunoassays is discussed and compared based on surface modification, microfluidic kinetics, signal output, signal amplification, sample matrix, and the detection of anti-SARS-CoV-2 antibodies. Based on the overall comparison, this Review concludes by proposing the future integration of visual quantitative signals on microfluidic devices as a more suitable approach for general use and large-scale surveillance.

Early Committed Clockwise Cell Chirality Upregulates Adipogenic Differentiation of Mesenchymal Stem Cells.

Cell chirality is observed with diverse forms and coordinates various left-right (LR) asymmetry in tissue morphogenesis. To give rise to such diversity, cell chirality may be coupled with cell differentiation. Here, using micropatterned human mesenchymal stem cells (hMSCs), an early committed clockwise (CW) cell chirality that can itself upregulate the adipogenic differentiation is reported. hMSC chirality enables a positively tilted chiral orientation on micropatterned stripes. When cultured as single cells on circular micropatterns, an anticlockwise (ACW)-biased nucleus rotation and swirling pattern of actin filament are observed. Interestingly, with adipogenic induction for 3-6 days, such chirality is reversed to negative chiral orientation and CW-biased rotation, which is earlier than the maturation of other differentiation markers, and consistently expressed in terminally differentiated adipocytes. Using latrunculin A (LatA), cytochalasin D (CD), and nocodazole (Noco) that forces a CW-biased actin filament and nucleus rotation resembling the early differentiated chirality upon adipogenic induction, an upregulation of adipogenic differentiation is found. The result demonstrates that the early differentiated chirality may serve as a mechanical precursor to engage the lineage commitment, suggesting a feedback mechanism of chiral actin in regulating cell differentiation and LR morphogenesis.

Light field display with ellipsoidal mirror array and single projector.

We present a method to create light field display using a single projector and an array of plane mirrors. Mirrors can reproduce densely arranged virtual projectors regardless of the physical size of the real projector, thus producing a light field display of competitive ray density. We propose an ellipsoidal geometric framework and a design pipeline, and use parametric modelling technique to automatically generate the display configurations satisfying target design parameters. Three units of mirror array light field display systems have been implemented to evaluate the proposed methodologies. More importantly, we have experimentally verified that the high-density light field produced by our method can naturally evoke accommodation of the eyes, thereby reducing the vergence-accommodation conflict. The mirror array approach allows flexible trading between the spatial and angular resolutions for accommodating different applications, thus providing a practical solution to realize projection-based light field display.

Remnant Effects of Culture Density on Cell Chirality After Reseeding.

Proper muscle function requires specific orientation of myotubes. Cell chirality, a mechanical behavior of cells, may participate in myogenesis and give rise to left-right (LR) orientation of muscle tissue. Thus, it is essential to understand the factors effecting the cell chirality. Here, using C2C12 cells as a model system, we report that prior culture condition with high/low density can create remnant effects on cell chirality after reseeding. C2C12 myoblasts were first conditioned by a series of subcultures with plating density at 2200 cells/cm2 (low density) or 22 000 cells/cm2 (high density). After reseeding on micropatterned stripes fabricated on glass or polydimethylsiloxane (PDMS) substrates, we found that the cells after low-density cultures exhibited a reduced cell aspect ratio and intercellular alignment, leading to an attenuated chiral orientation only appearing on glass substrate. In contrast, chiral orientation was observed in cells after high-density culture on both substrates. By comparing it to the original cells without being subcultured with high/low density, we found that the series of low-density cultures disorganized the formation of actin rings in single cells, which is an essential structure for cell chirality. Moreover, by using high-density culture supplemented with inhibitors of actin polymerization, the effect of low-density cultures was recaptured, suggesting that the series of subcultures with high/low density may be an in vitro aging process that modifies the actin cytoskeleton, causing a remnant attenuation of cell chirality even after trypsin digestion and reseeding. Together, our result suggests a mechanistic insight of how cytoskeletal structures "memorize" the previous experience through modification of the actin filament, opening up new possibilities for morphogenesis and mechanobiology.

Outline-etching image segmentation reveals enhanced cell chirality through intercellular alignment.

Cells cultured on micropatterns exhibit a chiral orientation, which may underlie the development of left-right asymmetry in tissue microarchitectures. To investigate this phenomenon, fluorescence staining of nuclei has been used to reveal such orientation. However, for images with high cell density, analysis is difficult because of the overlapping nuclei. Here, we report an image processing method that can acquire cell orientations within dense cell populations. After initial separation based on Boolean addition of binarized images using global and adaptive thresholds, the overlapping nucleus contours in the binarized images were segmented by iteratively etching the outlines of nuclei, which allowed the orientations of each cell to be extracted from densely packed cell clusters. In applying this technique to cultured C2C12 myoblasts in micropatterned stripes on different substrates, we found an enhanced chiral orientation on glass substrate. More important, this enhanced chirality was consistently observed with increased intercellular alignment and independent of cell-cell distance or cell density, suggesting that intercellular alignment plays a role in determining the chiral orientation. By segmenting single cells with intact orientation, this technique offers an automated method for quantitative analysis with improved accuracy, providing an essential tool for studying left-right asymmetry and other morphogenic dynamics in tissue formation.

Discrete Element Model for Suppression of Coffee-Ring Effect.

When a sessile droplet evaporates, coffee-ring effect drives the suspended particulate matters to the droplet edge, eventually forming a ring-shaped deposition. Because it causes a non-uniform distribution of solid contents, which is undesired in many applications, attempts have been made to eliminate the coffee-ring effect. Recent reports indicated that the coffee-ring effect can be suppressed by a mixture of spherical and non-spherical particles with enhanced particle-particle interaction at air-water interface. However, a model to comprehend the inter-particulate activities has been lacking. Here, we report a discrete element model (particle system) to investigate the phenomenon. The modeled dynamics included particle traveling following the capillary flow with Brownian motion, and its resultant 3D hexagonal close packing of particles along the contact line. For particles being adsorbed by air-water interface, we modeled cluster growth, cluster deformation, and cluster combination. We found that the suppression of coffee-ring effect does not require a circulatory flow driven by an inward Marangoni flow at air-water interface. Instead, the number of new cluster formation, which can be enhanced by increasing the ratio of non-spherical particles and the overall number of microspheres, is more dominant in the suppression process. Together, this model provides a useful platform elucidating insights for suppressing coffee-ring effect for practical applications in the future.

Substrate Stiffness Regulates the Development of Left-Right Asymmetry in Cell Orientation.

Left-right (LR) asymmetry of tissue/organ structure is a morphological feature essential for many tissue functions. The ability to incorporate the LR formation in constructing tissue/organ replacement is important for recapturing the inherent tissue structure and functions. However, how LR asymmetry is formed remains largely underdetermined, which creates significant hurdles to reproduce and regulate the formation of LR asymmetry in an engineering context. Here, we report substrate rigidity functioning as an effective switch that turns on the development of LR asymmetry. Using micropatterned cell-adherent stripes on rigid substrates, we found that cells collectively oriented at a LR-biased angle relative to the stripe boundary. This LR asymmetry was initiated by a LR-biased migration of cells at stripe boundary, which later generated a velocity gradient propagating from stripe boundary to the center. After a series of cell translocations and rotations, ultimately, an LR-biased cell orientation within the micropatterned stripe was formed. Importantly, this initiation and propagation of LR asymmetry was observed only on rigid but not on soft substrates, suggesting that the LR asymmetry was regulated by rigid substrate probably through the organization of actin cytoskeleton. Together, we demonstrated substrate rigidity as a determinant factor that mediates the self-organizing LR asymmetry being unfolded from single cells to multicellular organization. More broadly, we anticipate that our findings would pave the way for rebuilding artificial tissue constructs with inherent LR asymmetry in the future.

Nanowire Magnetoscope Reveals a Cellular Torque with Left-Right Bias.

Cellular force regulates many types of cell mechanics and the associated physiological behaviors. Recent evidence suggested that cell motion with left-right (LR) bias may be the origin of LR asymmetry in tissue architecture. As actomyosin activity was found essential in the process, it predicts a type of cellular force that coordinates the development of LR asymmetry in tissue formation. However, due to the lack of appropriate platform, cellular force with LR bias has not yet been found. Here we report a nanowire magnetoscope that reveals a rotating force-torque-exerted by cells. Ferromagnetic nanowires were deposited and internalized by micropatterned cells. Within a uniform, horizontal magnetic field, the nanowires that initially aligned with the magnetic field were subsequently rotated due to the cellular torque. We found that the torque is LR-biased depending on cell types. While NIH 3T3 fibroblasts and human vascular endothelial cells exhibited counterclockwise torque, C2C12 myoblasts showed torque with slight clockwise bias. Moreover, an actin ring composed of transverse arcs and radial fibers was identified as a major factor determining the LR bias of cellular torque, since the disruption of actin ring by biochemical inhibitors or elongated cell shape abrogated the counterclockwise bias of NIH 3T3 fibroblasts. Our finding reveals a LR-biased torque of single cells and a fundamental origin of cytoskeletal chirality. More broadly, we anticipate that our method will provide a different perspective on mechanics-related cell physiology and force transmission necessary for LR propagation in tissue formation.

Optimization of Combinatory Nicking Endonucleases for Accurate Identification of Nucleic Acids in Low Abundance.

Nucleic acid biomarkers embody inherent importance for differentiating disease-causing organisms or environmental pathogens. Identifying unknown nucleic acids in low abundance remains extremely challenging. Previously, we reported a method to identify complementary DNA (cDNA) molecules based on sequence-specific topographical labels measured by atomic force microscopy (AFM). However, the accuracy is limited because only one type of nicking endonuclease was used as the labeling agent. Here we investigate how accuracy is improved using multiple types of nicking endonucleases in combinations. The numerical experiments created cDNA molecules incorporating measurement error or labeling defects, which were later compared with the 29,563 human messenger RNA (mRNA) transcript database with ideal labels. After comparison, the unknown cDNA molecule was identified as the transcript with the highest matching score. Thus, the accuracy was determined by the rate of true positives. We found that the accuracy is positively proportional to the label number. Compared with cases using single nicking endonuclease, which has an average accuracy of 51.2% ± 34.4%, the average accuracy was improved to 97.1% ± 5.6% using an optimized combination of NtBsmAI + NtBstNBI + NtAlwI. This improved accuracy is applicable to more than 85% of human mRNA transcripts. Together, our study suggests an optimization strategy for identifying nucleic acids in low abundance using the AFM-based method, with implications for diseases diagnosis, pathogen identification, and forensics at the single molecule level.

Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope.

We discuss a novel atomic force microscope-based method for identifying individual short DNA molecules (<5000 bp) within a complex mixture by measuring the intra-molecular spacing of a few sequence-specific topographical labels in each molecule. Using this method, we accurately determined the relative abundance of individual DNA species in a 15-species mixture, with fewer than 100 copies per species sampled. To assess the scalability of our approach, we conducted a computer simulation, with realistic parameters, of the hypothetical problem of detecting abundance changes in individual gene transcripts between two single-cell human messenger RNA samples, each containing roughly 9000 species. We found that this approach can distinguish transcript species abundance changes accurately in most cases, including transcript isoforms which would be challenging to quantitate with traditional methods. Given its sensitivity and procedural simplicity, our approach could be used to identify transcript-derived complementary DNAs, where it would have substantial technical and practical advantages versus established techniques in situations where sample material is scarce.

An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study.

Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.