Reconstitution of tissue morphology with inherent left–right (LR) asymmetry is essential for tissue/organ functions. For skeletal muscle, the largest tissue in mammalian organisms, successful myogenesis requires the regulation of the LR asymmetry to form the appropriate muscle alignment. However, the key factor for reproducing the LR asymmetry of skeletal tissues in a controllable, engineering context remains largely unknown. Recent reports indicate that cell chirality may underlie the LR development in tissue morphogenesis. Here, we report that a rigid substrate is required for the chirality of skeletal muscle cells. By using alternating micropatterned cell-adherent and cell-repellent stripes on a rigid substrate, we found that C2C12 skeletal muscle myoblasts exhibited a unidirectional tilted orientation with respect to the stripe boundary. Importantly, such chiral orientation was reduced when soft substrates were used instead. In addition, we demonstrated the key role of actin stress fibers in the formation of the chiral orientation. This study reveals that a rigid substrate is required for the chiral pattern of myoblasts, paving the way for reconstructing damaged muscle tissue with inherent LR asymmetry in the future.

determine the LR asymmetry in morphogenesis. [2] For example, chirality has been found in Xenopus egg cortex before fertilization, [1] and can be passed down to more differentiated cells that establish LR body axis of animal body plan, [7] organ distribution, [8-10] and epithelial movement that leads to axial torsion and overall handedness of hindgut. [11,12] For specialized adult cells derived from somatic tissue, footprints of cell chirality can still be seen by their ability of generating cellular torque, [6,13] migration with LR bias, [14-16] or forming specific alignment in the multicellular level. [15,17,18] Through cell-cell communication, the chiral behavior causes LR-biased cell assembly of multicellular structure [15] and regulates permeability of intercellular junctions. [19] Clearly, cell chirality can be manifested in diverse forms and coordinate different morphogenic dynamics, resulting in distinct forms of tissue and organ architecture. Actin cytoskeleton plays an important role in cell chirality. When cultured on micropatterned substrate, the accumulation of actomyosin stress fibers at micropattern boundary is essential to activate the LR bias in cell migration and orientation. [15,17] Molecular studies suggest helical motion of actin filament as the underlying mechanism for the chirality at cellular level. [20,21] Functioning as a built-in machinery, actomyosin cytoskeleton allows chiral nucleus rotation of single cell [21] and generation of cellular torque with rotational bias. [13] Through a series of amplification process, the actin chirality ultimately determines the symmetry breaking in early embryonic development, [1,7,22] cardiac looping, [4,8,23] and organ laterality [9] in vivo. To give rise to such diverse forms of cell chirality, cell differentiation should play a role. [13,15,17] Cell differentiation is a process coupling with chemical [24] and physical factors. [25-27] Based on variation of key proteins in cytoskeleton, [28] cytoskeleton can be changed at early stage of cell differentiation, as shown by upregulation of cytoskeletal contractility [29] and cell morphological features, which can even forecast the cell lineage fate. [28] It suggests that cytoskeletal components, particularly actin, may early respond to the induction of cell differentiation and then actively participate the signal cascades to engage cell fate. Evidences can be found by regulation of cell differentiation via changed cell shape and cell spreading by physical cues [30-38]

Various COVID-19 vaccines are currently deployed, but their immunization varies and decays with time. Antibody level is a potent correlate to immune protection, but its quantitation relies on intensive laboratory techniques. Here, we report a decentralized, instrument-free microfluidic device that directly visualizes SARS-CoV-2 antibody levels. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) can bind to SARS-CoV-2 antibodies simultaneously. In a microfluidic chip, this binding reduces the incidence of free PMPs escaping from magnetic separation and shortens PMP accumulation length at a particle dam. This visual quantitative result enables use in either sensitive mode [limit of detection (LOD): 13.3 ng/ml; sample-to-answer time: 70 min] or rapid mode (LOD: 57.8 ng/ml; sample-to-answer time: 20 min) and closely agrees with the gold standard enzyme-linked immunosorbent assay. Trials on 91 vaccinees revealed higher antibody levels in mRNA vaccinees than in inactivated vaccinees and their decay in 45 days, demonstrating the need for point-of-care devices to monitor immune protection.

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.

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotube 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 on the groove patterns with different widths (from 0.4 to 200 μm), we observed an enhanced chiral orientation of cells developed on wide grooves (50 and 100 μm of width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, those 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 numtatics of cells for myogenesis. Finally, by arranging wide groove/stripe patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes are formed, providing a strategy for engineering skeletal muscle tissue formation.