Biology subjects

Ong, H. T.

Publications and source records attributed to Ong, H. T..

10 recordsLinked to original sources

bioRxiv2026

Microdomes: Micro-engineered Microdome arrays enable standardised and shear-free 3D biology with full-spectrum optical imaging compatibility

Three-dimensional cellular models such as organoids and spheroids hold major promise for developmental biology, disease modelling and precision medicine, yet their large-scale production and analysis remain constrained by handling-induced shear stress, sample fragility, positional instability and limited compatibility with advanced imaging workflows. Here, we introduce Microdomes, dense arrays of open-top, dome-shaped microcavities. Each cavity holds a single specimen in its own miniature aquarium, accessed through a narrow apical opening that admits cells, medium, matrix and staining reagents while shielding it from the shear generated during routine pipetting. Each Microdomes chip accommodates more than 100 spheroids or organoids, supporting diverse cell types, co-culture formats and both matrix-free and matrix-embedded culture. All specimens are retained through prolonged culture, repeated medium exchange, fixation and immunostaining. Because every specimen occupies a fixed, addressable position against a thin transparent film, the same spheroid can be relocated and re-imaged over weeks of culture and across microscopes, from array-wide overviews to subcellular details, without transfer, embedding or other perturbation. Microdomes also support AI-based automated segmentation, from whole-spheroid outlines to individual nuclei in 3D, using common image analysis software. Microdomes thereby turn each array into a self-contained quality control unit, providing specimen-level traceability that organoid production pipelines currently lack.

cell biology↗

bioRxiv2025

Ordering from the edge: Chiral multicellular pattern formation directed by actin self-organisation in cells

The mechanisms underlying both the establishment of mirror symmetry and deviations from it in development of bilateral multicellular organisms remain insufficiently understood. Actin cytoskeletons of individual cells exhibit intrinsic chirality, and a strong correlation exists between single-cell actin fibres chiral organisation and the collective alignment of cells confined to rectangular adhesive islands (2D-microtissues). Here, we demonstrate how multicellular chiral patterns can be inferred from the chiral behaviour of actin fibres in individual cells. By analysing chiral actin systems in cells with elliptical and semi-circular shapes, representing inner and boundary positions within 2D-microtissues, we defined the rules of chiral motile behaviour and formulated two models of cell alignment: (i) chiral rotation of inner cells and (ii) chiral tilting of boundary cells relative to island edges. In both models, neighbouring cells are also mutually aligned. Systematic variation of island area and aspect ratio, combined with dynamic observations, revealed the primary role of boundary cells. Chiral order first emerged at tissue boundaries and then propagated inward. This outside-in mechanism also explains why mirror-symmetric cell groups are difficult to achieve with uniformly chiral cells, but can be obtained either by reversing chirality in one subgroup or by enlarging the microtissue to minimise boundary influence.

biophysics↗

bioRxiv2025

Scaffolded hiPSC liver organoids recapitulating bile duct tubulogenesis and periportal architecture.

Recapitulating the liver periportal area in vitro remains a major challenge due to its complex cellular composition and the coordinated development of both ductal and endothelial networks. Most existing bile duct organoid models fail to reproduce tubular extension and multicellular organization. We present a 3D ECM micro-rods system biofunctionalized with grafted growth factors mimicking fetal liver paracrine signaling to initiate and to coordinate the development of bile ducts, parenchymal cells, and vascular structures. Our system recapitulates key developmental stages, from the formation of the ductal plate to the emergence of elongated tubular ducts. The ECM rod alone provides the biophysical cues initiating the elongation of the bile ducts and the spatial structuration of the cellular organization, while the biofunctionalization with growth factors enhance the organization and maturation of the biliary and vascular systems. We further demonstrate that this approach is scalable and amenable to quantitative analysis through label-free Optical Coherence Tomography combined with AI-driven 3D segmentation.

cell biology↗

bioRxiv2023

E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes.

Actomyosin tension has been shown to be a ubiquitous driver of tissue morphogenesis1, 2. The Rho pathway, a prominent regulatory network influencing cortical tension, plays a central role in both tissue reorganisation and cell migration3-6. While viscous dissipation in the actin network is commonly regarded as a constant passive parameter in cell migration in both 2D and 3D contexts, there is limited knowledge concerning the regulation of dissipative forces arising from viscous drag between cells during collective rearrangement. Here, we found that the phosphorylation of Epithelial Growth Factor Receptor (EGFR) downstream of de novo E-cadherin adhesion7, 8 orchestrates a feedback loop, thereby governing intercellular viscosity via the Rac pathway regulating actin dynamics. Our findings highlight how the E-cadherin-dependent EGFR activity controls the migration mode of collective cell movements independently of intercellular tension. Combining molecular cell biology, micropatterning, and in silico simulation, our work suggests the existence of a regulatory loop by which cells can tune junctional actin viscosity, with implications for the phenomenology of morphogenetic movements.

cell biology↗

bioRxiv2023

Digitalized organoids: integrated pipeline for 3D high-speed analysis of organoid structures using multilevel segmentation and cellular topology

Analysing the tissue morphogenesis and function is crucial for unravelling the underlying mechanisms of tissue development and disease. Organoids, 3D in vitro models that mimic the architecture and function of human tissues, offer a unique opportunity to study effects of external perturbators that are difficult to replicate in vivo. However, large-scale screening procedures for studying the effects of different stress on cellular morphology and topology of these 3D tissue-like system face significant challenges, including limitations in high-resolution 3D imaging, and accessible 3D analysis platforms. These limitations impede the scale and throughput necessary to accurately quantify the effects of mechanical and chemical cues. Here, we present a novel, fine-tuned pipeline for screening morphology and topology modifications in 3D cell culture using multilevel segmentations and cellular topology, based on confocal microscopy and validated across different image qualities. Our pipeline incorporates advanced image analysis algorithms and artificial intelligence (AI) for multiscale 3D segmentation, enabling quantification of morphology changes at both the nuclear and cytoplasmic levels, as well as at the organoid scale. Additionally, we investigate cell relative position and employ neighbouring topology analysis to identify tissue patterning and their correlations with organoid microniches. Eventually, we have organized all the extracted features, 3D segmented masks and raw images into a single database to allow statistical and data mining approaches to facilitate data analysis, in a biologist-friendly way. We validate our approach through proof-of-concept experiments, including well-characterized conditions and poorly explored mechanical stressors such as microgravity, showcasing the versatility of our pipeline. By providing a powerful tool for discovery-like assays in screening 3D organoid models, our pipeline has wide-ranging interests from biomedical applications in development and aging-related pathologies to tissue engineering and regenerative medicine.

cell biology↗

bioRxiv2021

Actin polymerization and crosslinking drive left-right asymmetry in single cell and cell collectives

Deviations from mirror symmetry in the development of bilateral organisms are highly stereotypic and genetically predetermined, but their mechanisms are not sufficiently understood. At the cellular level, self-organization of the actin cytoskeleton results in chiral actin swirling, and cells in groups confined to micropatterns demonstrate chiral cell alignment. The relationship between individual and collective cell chirality is unclear, and molecular players involved remain essentially unidentified. Here, by screening major actin-associated proteins and deep-learning-based morphometric analysis of actin patterns, we found that knockdowns of specific actin polymerization regulators, such as mDia1, ARPC2, and cofilins 1&2, abolished chiral actin swirling, while depletion of profilin 1 and CapZ{beta}, reversed its direction in an actin crosslinker -actinin1-dependent manner. Analysis of these and other knockdowns and pharmacological treatments revealed a robust correlation between their effects on the chirality of individual cells and confined cell groups. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry. One Sentence SummaryCell chirality determined by specific regulators of actin polymerization drives left-right asymmetry emergence in cell groups

cell biology↗

bioRxiv2021

High content 3D imaging method for quantitative characterization of organoid development and phenotype.

Quantitative analysis on a large number of organoids can provide meaningful information from the morphological variability observed in 3D organotypic cultures, called organoids. Yet, gathering statistics of growing organoids is currently limited by existing imaging methods and subsequent image analysis workflows that are either restricted to 2D, limited in resolution, or with a low throughput. Here, we present an automated high content imaging platform synergizing high density organoid cultures with 3D live light-sheet imaging. The platform is an add-on to a standard inverted microscope. We demonstrate our capacity to collect libraries of 3D images at a rate of 300 organoids per hour, enabling training of artificial intelligence-based algorithms to quantify the organoid morphogenetic organization at multiple scales with subcellular resolution. We validate our approach on different organotypic cell cultures (stem, primary, and cancer), and quantify the development of hundreds of neuroectoderm organoids (from human Embryonic Stem Cells) at cellular, multicellular and whole organoid scales.

biophysics↗

bioRxiv2019

Mechanical Roles of Vinculin/β-catenin interaction in Adherens Junction

Mechanical force transmission through the adherens junctions (AJs) are highly regulated processes essential for multicellular organization of tissues. AJ proteins such as E-cadherin, -catenin, and vinculin have been shown to be sensing or bearing mechanical forces being transmitted between the actin cytoskeleton and the intercellular contacts. However, the molecular organization and connectivity of these components remains not well understood. Using a super-resolution microscopy approach, we report that vinculin, once activated, could form a direct structural connection with {beta}-catenin, which can bypass -catenin, one of the main mechanotransducers in AJs. Direct vinculin/{beta}-catenin interaction is capable of supporting mechanical tension and contributes to the stabilization of the cadherin-catenin complexes. These findings suggest a multi-step model for the force-dependent reinforcement of AJs whereby -catenin may serve as the initial catalytic activator of vinculin, followed by vinculin translocation to form a direct link between E-cadherin-bound {beta}-catenin and the actin cytoskeleton.

cell biology↗

bioRxiv2019

Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity

Eph receptors, a family of receptor tyrosine kinases, play a crutial role in the assembly and maintenance of healthy tissues. Dysfunction in Eph signaling are causally and correlatively associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been largely linked to alterations in receptor clustering abilities. In the present study, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptor in carcinoma cells of different origin. Using this assay, we found that cancer cells retained EphA clustering phenotype upon cell division for several generations and degree of clustering reported for population as well as single-cell migration potential. Finally, using patient-derived cancer cell lines, we probed the evolution of EphA signalling in cancer cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our simple and scalable approach provides a reliable quantitation of EphA associated gene expression and phenotypes in multiple carcinomas and can assay the heterogeneity of cancer cell populations in a cost- and time-effective manner.

cancer biology↗

bioRxiv2017

Force dependence of filopodia adhesion: involvement of myosin II and formins

Filopodia are dynamic membrane protrusions driven by polymerization of an actin filament core, mediated by formin molecules at the filopodia tips. Filopodia can adhere to the extracellular matrix and experience both external and cell generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we induced sustained growth of filopodia by applying pulling force to their tips via attached fibronectin-coated beads trapped by optical tweezers. Strikingly, pharmacological inhibition or knockdown of myosin IIA, which localized to the base of filopodia, resulted in weakening of filopodia adherence strength. Inhibition of formins, which caused detachment of actin filaments from formin molecules, produced similar effect. Thus, myosin IIA-generated centripetal force transmitted to the filopodia tips through interactions between formins and actin filaments are required for filopodia adhesion. Force-dependent adhesion led to preferential attachment of filopodia to rigid versus fluid substrates, which may underlie cell orientation and polarization.

cell biology↗