Raymond H. W. Lam

Nanotopography Influences Adhesion, Spreading, and Self-Renewal of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.

Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.

Surface‐Micromachined Microfiltration Membranes for Efficient Isolation and Functional Immunophenotyping of Subpopulations of Immune Cells

An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this bulk assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined poly-dimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay (AlphaLISA), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples.

Multiparametric Biomechanical and Biochemical Phenotypic Profiling of Single Cancer Cells Using an Elasticity Microcytometer

Deep phenotyping of single cancer cells is of critical importance in the era of precision medicine to advance understanding of relationships between gene mutation and cell phenotype and to elucidate the biological nature of tumor heterogeneity. Existing microfluidic single-cell phenotyping tools, however, are limited to phenotypic measurements of 1-2 selected morphological and physiological features of single cells. Herein a microfluidic elasticity microcytometer is reported for multiparametric biomechanical and biochemical phenotypic profiling of free-floating, live single cancer cells for quantitative, simultaneous characterizations of cell size, cell deformability/stiffness, and surface receptors. The elasticity microcytometer is implemented for measurements and comparisons of four human cell lines with distinct metastatic potentials and derived from different human tissues. An analytical model is developed from first principles for the first time to convert cell deformation and adhesion information of single cancer cells encapsulated inside the elasticity microcytometer to cell deformability/stiffness and surface protein expression. Together, the elasticity microcytometer holds great promise for comprehensive molecular, cellular, and biomechanical phenotypic profiling of live cancer cells at the single cell level, critical for studying intratumor cellular and molecular heterogeneity using low-abundance, clinically relevant human cancer cells.

Effects of 4-methylbenzylidene camphor (4-MBC) on neuronal and muscular development in zebrafish (Danio rerio) embryos

The negative effects of overexposure to ultraviolet (UV) radiation in humans, including sunburn and light-induced cellular injury, are of increasing public concern. 4-Methylbenzylidene camphor (4-MBC), an organic chemical UV filter, is an active ingredient in sunscreen products. To date, little information is available about its neurotoxicity during early vertebrate development. Zebrafish embryos were exposed to various concentrations of 4-MBC in embryo medium for 3xa0days. In this study, a high concentration of 4-MBC, which is not being expected at the current environmental concentrations in the environment, was used for the purpose of phenotypic screening. Embryos exposed to 15xa0μM of 4-MBC displayed abnormal axial curvature and exhibited impaired motility. Exposure effects were found to be greatest during the segmentation period, when somite formation and innervation occur. Immunostaining of the muscle and axon markers F59, znp1, and zn5 revealed that 4-MBC exposure leads to a disorganized pattern of slow muscle fibers and axon pathfinding errors during the innervation of both primary and secondary motor neurons. Our results also showed reduction in AChE activity upon 4-MBC exposure both in vivo in the embryos (15xa0μM) and in vitro in mammalian Neuro-2A cells (0.1xa0μM), providing a possible mechanism for 4-MBC-induced muscular and neuronal defects. Taken together, our results have shown that 4-MBC is a teratogen and influences muscular and neuronal development, which may result in developmental defects.

Dynamics of microvalve operations in integrated microfluidics

Pneumatic microvalves are widely used key components for automating liquid manipulation and flow control in microfluidics for more than one decade. Due to their robust operations and the ease of fabrication, tremendous microfluidic systems have been developed with the multiple microvalves for higher throughput and extended functionalities. Therefore, operation performance of the microvalves in the integrated microfluidic devices is crucial to the related applications, in fields such as micro-flows, cell analyses, drug discovery, and physical/chemical detections. It has been reported that operation performance of the microvalves are highly sensitive to the device configuration and pressurization scheme. This implies the further development of integrated microfluidics with a larger number of the valves may suffer the problems of undetermined microvalve behaviors during operations, which can become an unavoidable hurdle in the device design and optimization processes. Herein, we characterize responses of the individual microvalves for different operation configurations, e.g., membrane thicknesses and driving pressures. We investigate also the effects in microfluidics integrated with the more valves, through experiments, modeling and simulations. We show that dynamics of the microvalves is indeed influenced by the configurations, levels of design complexity and positions in the devices. Overall, taken dynamics of the microvalve responses into considerations, this work provides insights and guidelines for better designs of integrated microfluidics for the future applications requiring higher throughput and improved operation performance.

Hacking macrophage-associated immunosuppression for regulating glioblastoma angiogenesis

Glioblastoma (GBM) is the most lethal primary adult brain tumor and its pathology is hallmarked by distorted neovascularization, diffuse tumor-associated macrophage infiltration, and potent immunosuppression. Reconstituting organotypic tumor angiogenesis models with biomimetic cell heterogeneity and interactions, pro-/anti-inflammatory milieu and extracellular matrix (ECM) mechanics is critical for preclinical anti-angiogenic therapeutic screening. However, current inxa0vitro systems do not accurately mirror inxa0vivo human brain tumor microenvironment. Here, we engineered a three-dimensional (3D), microfluidic angiogenesis model with controllable and biomimetic immunosuppressive conditions, immune-vascular and cell-matrix interactions. We demonstrate inxa0vitro, GL261 and CT-2A GBM-like tumors steer macrophage polarization towards a M2-like phenotype for fostering an immunosuppressive and proangiogenic niche, which is consistent with human brain tumors. We distinguished that GBM and M2-like immunosuppressive macrophages promote angiogenesis, while M1-like pro-inflammatory macrophages suppress angiogenesis, which we coin inflammation-driven angiogenesis. We observed soluble immunosuppressive cytokines, predominantly TGF-β1, and surface integrin (αvβ3) endothelial-macrophage interactions are required in inflammation-driven angiogenesis. We demonstrated tuning cell-adhesion receptors using an integrin (αvβ3)-specific collagen hydrogel regulated inflammation-driven angiogenesis through Src-PI3K-YAP signaling, highlighting the importance of altered cell-ECM interactions in inflammation. To validate the preclinical applications of our 3D organoid model and mechanistic findings of inflammation-driven angiogenesis, we screened a novel dual integrin (αvβ3) and cytokine receptor (TGFβ-R1) blockade that suppresses GBM tumor neovascularization by simultaneously targeting macrophage-associated immunosuppression, endothelial-macrophage interactions, and altered ECM. Hence, we provide an interactive and controllable GBM tumor microenvironment and highlight the importance of macrophage-associated immunosuppression in GBM angiogenesis, paving a new direction of screening novel anti-angiogenic therapies.

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.

Microfluidic long-term differential oxygenation for bacterial growth characteristics analyses

Dissolved oxygen is a critical micro-environmental factor to determine the growth characteristics of bacteria, such as cell viability, migration, aggregation and metabolic processes. Here, we report a microfluidic long-term oxygenator which can support high-throughput parallel cell cultures under multiple defined oxygenation conditions (0–42 ppm). An array of oxygen–nitrogen gas micro-mixers is developed and fabricated in the device to generate stable oxygen concentrations for the oxygenation process. A water-jacket layer located between the gas layer and the cell culture chamber is applied to block evaporation and maintain the conditions of the culture media in the chamber. Furthermore, we perform simulations and experiments to investigate the functions of the gas mixers and the water jackets. We also conduct culture experiments to study the long-term growth (up to one week) and aggregation of three selected dental bacteria (Streptococcus mutans, Actinomyces viscosus and Fusobacterium nucleatum) under ranges of oxygenation conditions. These particular results can provide important insights into their roles in dental biofilm formation. Overall, this work demonstrates that the long-term microfluidic oxygenation approach can effectively regulate defined dissolved-oxygen levels in cell microenvironments. Importantly, this oxygenation approach can be further applied to general long-term analyses of cells for their behavioral, metabolic and genetic responses, and their biofilm formation processes.

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.