Biology subjects

Toh, Y.-C.

Publications and source records attributed to Toh, Y.-C..

11 recordsLinked to original sources

bioRxiv2026

Dissecting Immune-Epithelial Interactions in Airway Infection at Single-Cell Resolution Using a Compartmentalised Microfluidic Device

Immune-epithelial interactions govern the initiation and progression of airway diseases, yet their heterogeneity is difficult to capture using existing in vitro models. Although conventional Transwell and lung-on-chip systems reproduce airway compartmentalisation and permit epithelial-immune interactions, they lack the spatial and analytical resolution needed to visualise dynamic immune behaviour during infection. Here, we present the "Single Cell resolved Airway-Immune Recruitment" (scAIR) platform designed to interrogate immune-epithelial interactions during airway infection. The scAIR device features a modular central chamber accommodating a Transwell insert with primary airway epithelial cells (AECs) pre-differentiated under air-liquid interface (ALI), flanked by immune compartments connected through a precision-patterned microchannel array. This architecture enables real-time single-cell imaging of immune cell migration while preserving epithelial physiology. The scAIR device coupled with a machine learning analysis (MLA) pipeline enables automated tracking and quantification of individual immune cell speed, direction, and behavioural heterogeneity. Using this platform, respiratory syncytial virus (RSV) infection is modelled to generate a type 1 inflammatory airway epithelium that drives neutrophil recruitment. TNF-alpha neutralisation with adalimumab reveals distinct migratory behaviours that are obscured by population-averaged measurements. This integrated platform quantifies airway immune responses during infection and therapeutic modulation, enabling mechanistic studies, drug evaluation, and precision modelling of airway inflammation.

bioengineering↗

bioRxiv2026

From Fabrication to Flow: Impact of Print Orientation on Surface Qualities and Capillary-Driven Flow in Laser SLA-based Open Microchannels

Stereolithography (SLA) 3D printing has become increasingly popular for fabricating microfluidic devices, with applications including hydrogel patterning and tissue modeling. In open-channel systems with surface tension-driven flow, 3D-printer-induced discrepancies in channel surface texture can significantly impact fluid flow and device performance. While previous work has focused on comparing different 3D printing methods for microchannel fabrication, the effect of device orientation during SLA printing on microchannel morphology and capillary-driven flow has not been systematically evaluated. Furthermore, there is minimal research elucidating the influence of channel surface texture on the flow of biologically relevant hydrogel precursors commonly used in organ-on-a-chip applications. Herein, we investigated the impact of print orientation on channel morphology, fluid wetting behavior, and fluid flow by comparing laser SLA-based parts where the length of the channel was tilted at 0{degrees}, 15{degrees}, 45{degrees}, or 90{degrees} during printing. We demonstrated that channel floor surface texture is greatly affected by print orientation: the highest axial surface roughness was measured in 15{degrees} printed channels, and the highest axial surface tortuosity-which describes the real length along the surface-was measured in 45{degrees} printed channels. Print angles of 15{degrees} and 45{degrees} also resulted in asymmetric roughness of the channel floor, which caused asymmetric wetting of glycerol solution. Surface tension-driven flow of glycerol solution, agarose precursor solution, and collagen precursor solution was affected by print orientation, in which the 45{degrees} printed flow devices had slowest flow for all test fluids. Root mean square roughness was not a reliable predictor of slower flow; instead, surface tortuosity should be considered. Potential alternatives to better theoretically model how print angle-induced surface texture affects open-channel flow are discussed as well. These findings provide a framework of fabrication considerations for laser SLA printing of open microchannels that can also be applied to other layer-by-layer, vat photopolymerization-based 3D printing technologies.

bioengineering↗

bioRxiv2025

Suspended Tissue Engineering with Assemblable Microfluidics (STEAM)

Suspended tissue culture systems enable cellular responses to mechanical forces critical for tissue development and function. Tissues develop in complex environments containing both mechanical and chemical cues that vary spatiotemporally; thus modeling both of these physiochemistries in vitro through integration of spatial patterning with mechanical manipulation simultaneously is an important aspect in microphysiological tissue modeling which has yet to be achieved. Here, we introduce Suspended Tissue Engineering with Assemblable Microfluidics (STEAM), a modular tissue fabrication platform that allows for spatially heterogeneous suspended tissue architectures. With STEAM, we achieve tissue constructs with multiple regions through the addition of capillary pinning features to control hydrogel precursor flow. STEAM tissues can easily be moved from patterning setup to well-plate to microscope slide, which also enables stacking of separately generated layers. Mechanical manipulation post-fabrication is also possible via static stretching, where cell-embedded 3D tissues can be stretched farther apart to induce strain along an axis. To demonstrate the utility of post fabrication strain ability, we showed that myotube alignment increases when strain is applied to STEAM generated engineered muscle tissue containing mouse myoblasts. Finally, by modifying the channel geometry of the fluidic-based patterning rails, we generate complex nonplanar suspended tissues. STEAM leverages microfluidic principles to generate suspended tissues that integrate patterning precision, mechanical functionality, and experimental versatility, providing a suite of construct combinations for modeling tissue behaviors from the interplay of spatial organization and mechanical forces.

bioengineering↗

bioRxiv2025

From Home to Transcriptome: Comparing the transcriptomic profile of induced immune response via lipopolysaccharide stimulation in homeRNA and venous blood

Remote blood sampling offers multiple advantages over traditional clinic-based blood sampling studies, including greater patient inclusion, more frequent sampling, and broader geographical reach. Combining remote blood sampling with transcriptomic analysis opens potential in translational applications for capturing acute and dynamic immune responses to various exposures. In this study, we establish the feasibility of homeRNA, a capillary blood collection and RNAlater-based stabilization kit, for use in downstream bulk RNA-sequencing applications via capturing a lipopolysaccharide (LPS)-induced inflammatory response. We also compared the baseline gene expression profiles and induced inflammatory response following LPS stimulation between homeRNA-stabilized samples and venous blood stabilized with RNAlater or PAXgene. We found that homeRNA was successfully able to capture an inflammatory response to LPS, specifically targeting various cytokines (e.g., IL6, IL12B, IL1B), chemokines (e.g., CCL3, CXCL10, CCL4), and other transcriptional factors in the toll-like receptor pathway, the primary pathway activated during LPS stimulation. Importantly, we also found that homeRNA captured a LPS-induced inflammatory response comparable to that of venous blood samples stabilized with either RNAlater or PAXgene. Overall, this work demonstrates that the homeRNA platform is compatible with downstream bulk RNA-sequencing analysis and can capture transcriptomic immune responses to a known stimulus which are analogous to results in traditional stabilized venous blood samples.

bioengineering↗

bioRxiv2025

A Patient-Specific 3D Printed Carotid Artery Model Integrating Vascular Structure, Flow and Endothelium Responses

The progression of atherosclerosis is driven by the interplay between vascular anatomy, hemodynamic forces, and endothelial responses. However, existing in vitro vascular models have yet to integrate all these elements into a cohesive, patient-specific system. Here, we present the first instance of direct 3D printing of a miniaturized, patient-specific carotid artery model that recapitulates anatomical-dependent hemodynamic changes and vascular cell remodeling. Phase-contrast MRI scans from a healthy donor were used to generate miniaturized 3D carotid artery models, which were analyzed via computational fluid dynamics (CFD) and particle imaging velocimetry (PIV) to validate the preservation of physiological hemodynamic properties. Using digital light processing (DLP) 3D printing, we fabricated the miniaturized carotid artery vessel using GelMA, containing embedded human aortic smooth muscle cells and an endothelialized lumen. Perfusion culture replicated physiological arterial shear stress of up to 10 dynes cm-{superscript 2}, resulting in differential endothelial cell alignment and inflammatory monocyte adhesion corresponding to laminar and turbulent flow regions within the carotid artery. This model serves as a powerful platform to investigate how anatomical variation influences susceptibility to atherosclerosis through its impact on local flow dynamics.

bioengineering↗

bioRxiv2024

Simulating big mechanically-active culture systems (BigMACS) using paired biomechanics-histology FEA modelling to derive mechanobiology design relationships.

Big mechanically-active culture systems (BigMACS) are promising to stimulate, control, and pattern cell and tissue behaviours with less soluble factor requirements, however, it remains challenging to predict if and how distributed mechanical forces impact single-cell behaviours to pattern tissue. In this study, we introduce a centimetre, tissue-scale, finite element analysis (FEA) framework able to correlate sub-cellular quantitative histology with centimetre-scale biomechanics. Our framework is relevant to diverse bigMACS; media perfusion, tensile-stress, magnetic, and pneumatic tissue culture platforms. We apply our framework to understand how the design and operation of a multi-axial soft robotic bioreactor can spatially control mesenchymal stem cell (MSC) proliferation, orientation, differentiation to smooth muscle, and extracellular vascular matrix deposition. We find MSC proliferation and matrix deposition correlate positively with mechanical stimulation but cannot be locally patterned by soft robot mechanical stimulation within a centimetre scale tissue. In contrast, local stress distribution was able to locally pattern MSC orientation and differentiation to smooth muscle phenotypes, where MSCs aligned perpendicular to principal stress direction and expressed increased -SMA with increasing 3D Von Mises Stresses from 0 to 15 kPa. Altogether, our new biomechanical-histological simulation framework is a promising technique to derive the future mechanical design equations to control cell behaviours and engineer patterned tissue generation.

bioengineering↗

bioRxiv2023

Organ-on-a-Chip Fabrication using Dynamic Photomask

Organ-on-a-chip (OoC) technology is a powerful tool for creating physiologically relevant microscale models applicable to biomedical studies. Despite the advances in OoC technology, its fabrication method still primarily relies on soft lithography, which (1) lacks the adaptability to accommodate dynamic cell culture (e.g., spheroids and organoid culture) and (2) has a long design-to-prototype cycle that lowers its manufacturability. To overcome these challenges, we developed a system to fabricate OoC (consisting of microchannels and multiple cell types in a well-defined spatial arrangement) dynamically using a digital photomask aligned with a microchamber. Our approach used a pre-defined microfluidic chamber customized by xurography and cell-laden microfluidic channels photopatterned by a digital photomask; the entire design-to-prototype cycle was achieved within two hours. The versatility of our approach offered previously unattainable crucial features in the fabrication of OoC, including a gradual change in the height of the microchannels, and real-time modification of channel designs to trap live tissues (e.g., spheroids). In summary, this work highlights a versatile system to fabricate OoC that can accommodate various design requirements of microenvironments of specific organ tissues. We envision the effectiveness of our system for the rapid fabrication of OoC to contribute to the wide adoption of the technology for therapeutic screening and elucidation of disease mechanisms in both academic and industrial settings.

bioengineering↗

bioRxiv2023

Localized oxygen control in a microfluidic osteochondral interface model recapitulates bone-cartilage crosstalk during osteoarthritis

Osteoarthritis (OA) is characterized by the dysregulation of the osteochondral interface between bone and cartilage. In vitro models that accurately mimic this interface hold great potential for understanding OA pathophysiology and screening therapeutic agents. Presently, research efforts have focused on emulating heterogeneity in structural and mechanical attributes of the extracellular matrix (ECM) at the osteochondral interface. However, the precise simulation of differential oxygen gradients experienced by chondrocytes and osteoblasts in vivo remains a substantial obstacle for modeling osteo-chondral interactions effectively. To overcome this limitation, we show that micropatterned granular hydrogels, which are small microgel particles swelled in liquid culture media to create a shear-yielding jammed-packed solid, can be used to control the delivery of oxygen scavenging agents in a simple and scalable manner. Hypoxic granular hydrogels formulated with Oxyrase could maintain <1% oxygen concentration in a conventional cell culture incubator. Primary human chondrocytes maintained in the hypoxic hydrogels expressed a more anabolic phenotype similar to those cultured in a hypoxic incubator. The granular hydrogels can be readily patterned in a microfluidic device to generate a localized hypoxic environment, mimicking the differential oxygen levels at the osteochondral tissue interface (i.e. osteoblast at 20% and chondrocyte at 2% oxygen). Using this microfluidic coculture model, we paired healthy human chondrocytes with osteoblasts isolated from non-sclerotic and sclerotic subchondral bone to investigate how oxygen environment modulates osteoblast-chondrocyte crosstalk during OA. In a differential oxygen environment, the osteoblast-chondrocyte co-culture model showed sclerotic osteoblasts inducing chondrocyte collagen expression changes through increased MMP13 and ADAM15 production, unlike in a uniform normoxic oxygen environment, where the change was driven by altered collagen gene expression favoring Type I over Type II collagen. Furthermore, differential oxygen conditions enabled the identification of extensive transcriptional alterations induced by sclerotic osteoblasts, which involved inflammatory NF-{kappa}{beta}, TGF-{beta}/BMP, and IGF signaling pathways, that was otherwise not detectable in a uniform normoxic co-culture. The microfluidic model with localized oxygen variations effectively mimics physiologically relevant osteoblast-chondrocyte crosstalk, providing valuable insights into OA pathophysiology.

bioengineering↗

bioRxiv2022

FEZ1 participates in human embryonic brain development by modulating neuronal progenitor subpopulation specification and migration

Abnormal neuronal networks arising from perturbations during early brain development contribute to neurodevelopmental disorders. Mutations and deletions of human Fasciculation and Elongation Protein Zeta 1 (FEZ1) are found in schizophrenia and Jacobsen syndrome patients. However, its roles in human brain development and manifestation of clinical pathological symptoms remain unknown. Here, using human cerebral organoids (hCOs), we observed that FEZ1 expression is turned on early during brain development and is detectable in both neuroprogenitor subtypes and immature neurons. Deletion of FEZ1 disrupts expression of genes involved in neuronal and synaptic development. Using single-cell RNA sequencing, we further uncovered an abnormal expansion of homeodomain-only protein homeobox (HOPX)- outer radial glia (oRG) in FEZ1-null hCOs, occurring at the expense of HOPX+ oRG. HOPX- oRGs show higher cell mobility as compared to HOPX+ oRGs, which is accompanied by the ectopic localization of the neuroprogenitors to the outer layer of FEZ1-null hCOs. Moreover, abnormal encroachment of TBR2+ intermediate progenitors into CTIP2+ deep layer neurons indicated that cortical layer formation is disrupted in FEZ1-null hCOs. Collectively, our findings highlight the involvement of FEZ1 in early cortical brain development and how it contributes to neurodevelopmental disorders.

neuroscience↗

bioRxiv2021

Biomimetic Vasculatures by 3D-Printed Porous Molds

Anatomically and biologically relevant vascular models are critical to progress our understanding of cardiovascular diseases (CVDs) that can lead to effective therapies. Despite advances in 3D bioprinting, recapitulating complex architectures (i.e., freestanding, branching, multilayered, perfusable) of a cell-laden vascular construct remains technically challenging, and the development of new techniques that can recapitulate both anatomical and biological features of blood vessels is of paramount importance. In this work, we introduce a unique, microfluidics-enabled molding technique that allows us to fabricate anatomically-relevant, cell-laden hydrogel vascular models. Our approach employed 3D-printed porous molds of poly(ethylene glycol) diacrylate (PEGDA) as templates to cast alginate-containing bioinks. Due to the porous and aqueous nature of the PEGDA mold, the calcium ion (Ca2+) was diffusively released to crosslink the bioinks to create hollow structures. Applying this technique, multiscale, multilayered vascular constructs that were freestanding and perfusable were readily fabricated using cell-compatible bioinks (i.e., alginate and gelatin methacryloyl (GelMA)). The bioinks were also readily customizable to either improve the compatibility with specific vascular cells or tune the mechanical modulus to mimic native blood vessels. Importantly, we successfully integrated smooth muscle cells and endothelial cells in a biomimetic organization within our vessel constructs and demonstrated a significant increase in monocyte adhesion upon stimulation with an inflammatory cytokine, tumor necrosis factor-alpha (TNF-). We also demonstrated that the fabricated vessels were amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiologically-relevant mechanical states, such as vessel stretching and bending. Overall, we introduce a versatile fabrication technique with multi-faceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of CVD progression and the development of therapeutic interventions.

bioengineering↗

bioRxiv2019

Enhancement of endothelialization by topographical features is mediated by PTP1B-dependent endothelial adherens junctions remodeling

RationaleFailure of small synthetic vascular grafts is largely due to late endothelialization and has been an ongoing challenge in the treatment of cardiovascular diseases.\n\nObjectivePrevious strategies developed to promote graft endothelialization include surface topographical modulation and biochemical modifications. However, these have been met with limited success. Importantly, although the integrity of Endothelial Cell (EC) monolayer is crucial for endothelialization, the crosstalk between surface topography and cell-cell connectivity is still not well understood. Here we explored a combined strategy that utilizes both topographical features and pharmacological perturbations.\n\nMethods and resultWe characterized EC behaviors in response to micron-scale grating topography in conjunction with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We studied the EA.hy 926 cell-cell junctions and monolayer integrity using the junctional markers upon the inhibitory effect of EAJ regulator on both planar and grating topographies substrates.\n\nWe identified a protein tyrosine phosphatase, PTP1B, as a potent regulator of EAJ stability. Next, we studied the physiologically relevant behaviors of EC using primary human coronary arterial endothelial cells (HCAEC). Our results showed that PTP1B inhibition synergized with grating topographies to modulate EAJ rearrangement, thereby controlling global EC monolayer sheet orientation, connectivity and collective cell migration to promote endothelialization.\n\nOur results showed that PTP1B inhibition synergized with grating topographies to modulate EAJ rearrangement, thereby controlling global EC monolayer sheet orientation, connectivity and collective cell migration and proliferation.\n\nConclusionThe synergistic effect of PTP1B inhibition and grating topographies could be useful for the promotion of endothelialization by enhancing EC migration and proliferation.

cell biology↗