Carbon nanotubes (CNTs) are receiving much attention in medicine, electronics, consumer products, and next-generation nanocomposites because of their unique nanoscale properties. However, little is known about the toxicity and oxidative stress related anomalies of CNTs on complex multicellular behavior. This includes cell chirality, a newly discovered cellular property important for embryonic morphogenesis and demonstrated by directional migration and biased alignment on micropatterned surfaces. In this study, we report the influence of single-walled carbon nanotubes (SWCNTs) on multicellular chirality. The incubation of human umbilical vein endothelial cells (hUVECs) and mouse myoblasts (C2C12) with CNTs at different doses and time points stimulates reactive oxygen species (ROS) production and intra- and extracellular oxidative stress (OS). The OS-mediated noxious microenvironment influences vital subcellular organelles (e.g., mitochondria and centrosomes), cytoskeletal elements (microtubules), and vinculin rich focal adhesions. The disorientated nuclear-centrosome (NC) axis and centriole disintegration lead to a decreased migration rate and loss of directional alignment on micropatterned surfaces. These findings suggest that CNT-mediated OS leads to loss of multicellular chirality. Furthermore, the in vitro microscale system presented here to measure cell chirality can be extended as a prototype for testing toxicity of other nanomaterials.

The vertebrate body plan is overall symmetrical but left–right (LR) asymmetric in the shape and positioning of internal organs. Although several theories have been proposed, the biophysical mechanisms underlying LR asymmetry are still unclear, especially the role of cell chirality, the LR asymmetry at the cellular level, on organ asymmetry. Here with developing chicken embryos, we examine whether intrinsic cell chirality or handedness regulates cardiac C looping. Using a recently established biomaterial-based 3D culture platform, we demonstrate that chick cardiac cells before and during C looping are intrinsically chiral and exhibit dominant clockwise rotation in vitro. We further show that cells in the developing myocardium are chiral as evident by a rightward bias of cell alignment and a rightward polarization of the Golgi complex, correlating with the direction of cardiac tube rotation. In addition, there is an LR polarized distribution of N-cadherin and myosin II in the myocardium before the onset of cardiac looping. More interestingly, the reversal of cell chirality via activation of the protein kinase C signaling pathway reverses the directionality of cardiac looping, accompanied by a reversal in cellular biases on the cardiac tube. Our results suggest that myocardial cell chirality regulates cellular LR symmetry breaking in the heart tube and the resultant directionality of cardiac looping. Our study provides evidence of an intrinsic cellular chiral bias leading to LR symmetry breaking during directional tissue rotation in vertebrate development.

Our understanding of the left-right (LR) asymmetry of embryonic development, in particular the contribution of intrinsic handedness of the cell or cell chirality, is limited due to the confounding systematic and environmental factors during morphogenesis and a ack of physiologically relevant in vitro 3D platforms. Here we report an efficient two-layered biomaterial platform for determining the chirality of individual cells, cell aggregates, and self-organized hollow epithelial spheroids. This bioengineered niche provides a uniform defined axis allowing for cells to rotate spontaneously with a directional bias toward either clockwise or counterclockwise directions. Mechanistic studies reveal an actin-dependent, cell-intrinsic property of 3D chirality that can be mediated by actin cross-linking via α-actinin-1. Our findings suggest that the gradient of extracellular matrix is an important biophysicochemical cue influencing cell polarity and chirality. Engineered biomaterial systems can serve as an effective platform for studying developmental asymmetry and screening for environmental factors causing birth defects.cell chirality | left-right asymmetry | cell polarity | tissue morphogenesis | biomaterial A lmost all vertebrates have an asymmetric body plan, a deviation from which often leads to severe malformations (1, 2). In recent years, increasing evidence has suggested that embryonic and organ-specific left-right (LR) asymmetries, such as hindgut and genitalia rotation in Drosophila and symmetry breaking in pond snails (3-6), can arise from the LR bias at a cellular level, also termed cell chirality (7,8). In addition, this cellular asymmetry has been demonstrated in various models, including early asymmetry in Caenorhabditis elegans (9, 10), the chiral properties of Xenopus egg cortex (11, 12), asymmetric distribution of chirality related proteins at the early developmental stages of different animals (13), and migratory biases of cultured cells in vitro (12, 14-17). However, cell chirality is poorly understood in developing embryos, despite its scientific and clinical significance, due to complexities in imaging and molecular assays when dealing with animal models and confounding systematic and environmental factors that influence data explanation and hinder mechanistic findings. Therefore, it is of great importance to establish a biomimetic system for LR symmetry breaking that truly recapitulates 3D multicellular chiral morphogenesis.Cell chirality is a fundamental property of the cell, and the universality was not widely regarded until the recent use of microfabricated 2D in vitro systems (16,(18)(19)(20), including the 2D microcontact printing developed by us. In these systems, the cells were confined in a narrow area that allows the cells to exhibit their chiral biases in various formats, including cytoskeleton dynamics, cell migration, and multicellular biased alignment. With these new tools, cell chirality was found to be phenotype-dependent and related to the cross-linking of formin-associated actin bund...

Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left-right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

The development of the vertebrate body plan with left-right (LR) asymmetry (also known as handedness and chirality) requires the emerging chiral morphogenesis of epithelial cells at specific embryonic stages. In this process, cell-cell adhesions coordinate cellular organization and collective cell migration, and are critical for the directional looping of developing embryonic organs. However, the underlying biophysical mechanism is not yet well understood. Here we modeled normal and delayed epithelial LR symmetry breaking with patterned epithelial chiral morphogenesis on microscale lines with various widths. The patterned cells exhibited biased migration wherein those on opposing boundaries migrated in different directions. Disrupting adherens junctions with ethylene glycol tetraacetic acid (EGTA) resulted in a decrease in velocity difference in opposing boundaries as well as the associated biased cell alignment, along with an increase in the overall random motion. Altering the distance between the opposing boundaries did not significantly alter alignment, but significantly disturbed the velocity profile of the cell migration field. Further examination of cell polarity indicated that disruption of adherens junctions did not affect cell polarization on the boundaries, but decreased the transmission of chiral bias into the interior region of the epithelial cell sheet. Overall, our results demonstrated the dependence of the scale of collective cell migration on the strength of cell-cell adhesion, and its effects on the chirality of a multicellular structure through mediating cell polarity in the vicinity of geometric boundaries. This study demonstrated that our 2D microscale system provides a simple yet effective tool for studying the influence of collective cell migration on LR symmetry breaking, and possibly for fetal drug screening to prevent birth defects related to alteration in cell-cell adhesion.