Effects of Confined Microenvironments with Protein Coating, Nanotopography, and TGF-ß Inhibitor on Nasopharyngeal Carcinoma Cell Migration through Channels.

Distant metastasis is the primary cause of unsuccessful treatment in nasopharyngeal carcinoma (NPC), suggesting the crucial need to comprehend this process. A tumor related to NPC does not have flat surfaces, but consists of confined microenvironments, proteins, and surface topography. To mimic the complex microenvironment, three-dimensional platforms with microwells and connecting channels were designed and developed with a fibronectin (FN) coating or nanohole topography. The potential of the transforming growth factor-ß (TGF-ß) inhibitor (galunisertib) for treating NPC was also investigated using the proposed platform. Our results demonstrated an increased traversing probability of NPC43 cells through channels with an FN coating, which correlated with enhanced cell motility and dispersion. Conversely, the presence of nanohole topography patterned on the platform bottom and the TGF-ß inhibitor led to a reduced cell traversing probability and decreased cell motility, likely due to the decrease in the F-actin concentration in NPC43 cells. This study highlights the significant impact of confinement levels, surface proteins, nanotopography, and the TGF-ß inhibitor on the metastatic probability of cancer cells, providing valuable insights for the development of novel treatment therapies for NPC. The developed platforms proved to be useful tools for evaluating the metastatic potential of cells and are applicable for drug screening.

Detecting Nanotopography Induced Changes in Cell Migration Directions Using Oxygen Sensors.

This study investigates the oxygen (O2) consumption of single cells during changes in their migration direction. This is the first integration of nanotopographies with an O2 biosensor in a platform, allowing the real-time monitoring of O2 consumption in cells and the ability to distinguish cells migrating in the same direction from those migrating in the opposite direction. Advanced nanofabrication technologies were used to pattern nanoholes or nanopillars on grating ridges, and their effects were evaluated using fluorescence microscopy, cell migration assays, and O2 consumption analysis. The results revealed that cells on the nanopillars over grating ridges exhibited an enhanced migration motility and more frequent directional changes. Additionally, these cells showed an increased number of protrusions and filopodia with denser F-actin areas and an increased number of dotted F-actin structures around the nanopillars. Dynamic metabolic responses were also evident, as indicated by the fluorescence intensity peaks of platinum octaethylporphyrin ketone dye, reflecting an increased O2 consumption and higher mitochondria activities, due to the higher energy required in response to directional changes. The study emphasizes the complex interplay between O2 consumption and cell migration directional changes, providing insights into biomaterial science and regenerative medicine. It suggests innovative designs for biomaterials that guide cell migration and metabolism, advocating nanoengineered platforms to harness the intricate relationships between cells and their microenvironments for therapeutic applications.

Biointerfaces with ultrathin patterns for directional control of cell migration.

In the context of wound healing and tissue regeneration, precise control of cell migration direction is deemed crucial. To address this challenge, polydimethylsiloxane (PDMS) platforms with patterned 10 nm thick TiOx in arrowhead shape were designed and fabricated. Remarkably, without tall sidewall constraints, MC3T3-E1 cells seeded on these platforms were constrained to migrate along the tips of the arrowheads, as the cells were guided by the asymmetrical arrowhead tips which provided large contact areas. To the best of our knowledge, this is the first study demonstrating the use of thin TiOx arrowhead pattern in combination with a cell-repellent PDMS surface to provide guided cell migration unidirectionally without tall sidewall constraints. Additionally, high-resolution fluorescence imaging revealed that the asymmetrical distribution of focal adhesions, triggered by the patterned TiOx arrowheads with arm lengths of 10, 20, and 35 µm, promoted cell adhesion and protrusion along the arrowhead tip direction, resulting in unidirectional cell migration. These findings have important implications for the design of biointerfaces with ultrathin patterns to precisely control cell migration. Furthermore, microelectrodes were integrated with the patterned TiOx arrowheads to enable dynamic monitoring of cell migration using impedance measurement. This microfluidic device integrated with thin layer of guiding pattern and microelectrodes allows simultaneous control of directional cell migration and characterization of the cell movement of individual MC3T3-E1 cells, offering great potential for the development of biosensors for single-cell monitoring.

PEGylated Paclitaxel Nanomedicine Meets 3D Confinement: Cytotoxicity and Cell Behaviors.

Investigating the effect of nanomedicines on cancer cell behavior in three-dimensional (3D) platforms is beneficial for evaluating and developing novel antitumor nanomedicines in vitro. While the cytotoxicity of nanomedicines on cancer cells has been widely studied on two-dimensional flat surfaces, there is little work using 3D confinement to assess their effects. This study aims to address this gap by applying PEGylated paclitaxel nanoparticles (PEG-PTX NPs) for the first time to treat nasopharyngeal carcinoma (NPC43) cells in 3D confinement consisting of microwells with different sizes and a glass cover. The cytotoxicity of the small molecule drug paclitaxel (PTX) and PEG-PTX NPs was studied in microwells with sizes of 50 × 50, 100 × 100, and 150 × 150 µm2 both with and without a concealed top cover. The impact of microwell confinement with varying sizes and concealment on the cytotoxicity of PTX and PEG-PTX NPs was analyzed by assessing NPC43 cell viability, migration speed, and cell morphology following treatment. Overall, microwell isolation was found to suppress drug cytotoxicity, and differences were observed in the time-dependent effects of PTX and PEG-PTX NPs on NPC43 cells in isolated and concealed microenvironments. These results not only demonstrate the effect of 3D confinement on nanomedicine cytotoxicity and cell behaviors but also provide a novel method to screen anticancer drugs and evaluate cell behaviors in vitro.

Effects of nanopillars and surface coating on dynamic traction force.

The extracellular matrix serves as structural support for cells and provides biophysical and biochemical cues for cell migration. Topography, material, and surface energy can regulate cell migration behaviors. Here, the responses of MC3T3-E1 cells, including migration speed, morphology, and spreading on various platform surfaces, were investigated. Polydimethylsiloxane (PDMS) micropost sensing platforms with nanopillars, silicon oxide, and titanium oxide on top of the microposts were fabricated, and the dynamic cell traction force during migration was monitored. The relationships between various platform surfaces, migration behaviors, and cell traction forces were studied. Compared with the flat PDMS surface, cells on silicon oxide and titanium oxide surfaces showed reduced mobility and less elongation. On the other hand, cells on the nanopillar surface showed more elongation and a higher migration speed than cells on silicon oxide and titanium oxide surfaces. MC3T3-E1 cells on microposts with nanopillars exerted a larger traction force than those on flat PDMS microposts and had more filopodia and long protrusions. Understanding the relationships between platform surface condition, migration behavior, and cell traction force can potentially lead to better control of cell migration in biomaterials capable of promoting tissue repair and regeneration.

Enhanced motility and interaction of nasopharyngeal carcinoma with epithelial cells in confined microwells.

The three-dimensional (3D) structure of the extracellular matrix and cell-cell contacts are two important cues to altering cell migration behavior and the tumor formation process. In this work, we designed and fabricated microwell arrays with a grating-patterned bottom in polydimethylsiloxane platforms to systematically study the effects of confinement, changes in topography, and cell-cell contacts on the migration behavior of nasopharyngeal carcinoma (NPC43) and immortalized nasopharyngeal epithelial (NP460) cells by time-lapse imaging. When two types of cells were co-cultured in microwells, the migration speed and spreading area of NPC43 cells were significantly increased, which might be attributed to the heterotypic cell-cell contacts with NP460 cells. On a flat surface, NPC43 cells could not form clusters due to the frequent interruptions by the active movements of NP460 cells. However, in 3D microwell arrays, clusters of NPC43 cells formed on the bottom surface while the majority of NP460 cells migrated onto the sidewalls. These cell clusters could be further processed to form spheroids for drug screening. These results also revealed that the 3D microenvironments and cell-cell contacts could have significant implications for NPC cell migration and initiation of tumor formation, which will provide insight for NPC progression and dissemination.

A Microfluidic Platform Revealing Interactions between Leukocytes and Cancer Cells on Topographic Micropatterns.

Immunoassay for detailed analysis of immune-cancer intercellular interactions can achieve more promising diagnosis and treatment strategies for cancers including nasopharyngeal cancer (NPC). In this study, we report a microfluidic live-cell immunoassay integrated with a microtopographic environment to meet the rising demand for monitoring intercellular interactions in different tumor microenvironments. The developed assay allows: (1) coculture of immune cells and cancer cells on tunable (flat or micrograting) substrates, (2) simultaneous detection of different cytokines in a wide working range of 5-5000 pg/mL, and (3) investigation of migration behaviors of mono- and co-cultured cells on flat/grating platforms for revealing the topography-induced intercellular and cytokine responses. Cytokine monitoring was achieved on-chip by implementing a sensitive and selective microbead-based sandwich assay with an antibody on microbeads, target cytokines, and the matching fluorescent-conjugated detection antibody in an array of active peristaltic mixer-assisted cytokine detection microchambers. Moreover, this immunoassay requires a low sample volume down to 0.5 µL and short assay time (30 min) for on-chip cytokine quantifications. We validated the biocompatibility of the co-culture strategy between immune cells and NPC cells and compared the different immunological states of undifferentiated THP-1 monocytic cells or PMA-differentiated THP-1 macrophages co-culturing with NP460 and NPC43 on topographical and planar substrates, respectively. Hence, the integrated microfluidic platform provides an efficient, broad-range and precise on-chip cytokine detection approach, eliminates the manual sampling procedures and allows on-chip continuous cytokine monitoring without perturbing intercellular microenvironments on different topographical ECM substrates, which has the potential of providing clinical significance in early immune diagnosis, personalized immunotherapy, and precision medicine.

A three-dimensional spheroid-specific role for Wnt-ß-catenin and Eph-ephrin signaling in nasopharyngeal carcinoma cells.

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-ß-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.

Directing osteoblastic cell migration on arrays of nanopillars and nanoholes with different aspect ratios.

To realize highly directional guidance for cell migration, both micro- and nano-scale topographies were studied to better understand and mimic the complex extracellular matrix environment. Polydimethylsiloxane-based platforms with micro- and nano-topographies were developed to systematically study their guidance effects on cell migration behaviors. Compared to microtopography such as flat surface or grating, nanotopographies such as nanoholes and nanopillars could promote the generation of filopodia and extension of long protrusions with increased migration speed for MC3T3-E1 cells. Although cells on the grating structures showed lower migration speed, more directional cell migration was achieved due to their anisotropic topography compared to nanohole or nanopillar arrays with isotropic structures. To further enhance the cell migration directionality, the nanotopographies were patterned in grating arrangements and the results showed that both nanoholes and nanopillars in grating arrangements introduced more directional cell migration compared to gratings. The effects of physical dimensions of the nanotopographies on cell migration were studied and the results showed that there was less cell elongation and less directional migration of the nanoholes in grating arrangements with increasing depth of nanoholes. However, the nanopillars in grating arrangements showed more cell elongation, more directional migration, and higher migration speed with increasing height of the nanopillars. Platforms with nanopillars in grating arrangements and large height could be used to control cell migration speed and directionality, which could potentially lead to cell sorting and screening.

Spreading and Migration of Nasopharyngeal Normal and Cancer Cells on Microgratings.

Cell spreading and migration play a pivotal role in many diseases such as tumor metastasis. In particular, nasopharyngeal tumor cells have known of their tendency of migration to pterygoid muscles and further distant metastasis. Although existing studies revealed key characteristics of the nasopharyngeal tumor cells, their migration preference is yet to be thoroughly understood, especially in the physical aspects including the microtopographical factors. Researchers have developed techniques in recent years to study microtopography-related cell behaviors but they are not yet applied in investigating the nasopharyngeal tumor cells. In this work, we elaborate the spreading and migration characteristics of normal and cancerous nasopharyngeal cells on micrograting substrates mimicking the microtopography of myotubes of the pterygoid muscles. We further apply interference reflection microscopy (IRM) to visualize the cell-substrate adhesion dynamics. We are interested in examining the microtopography-related cell spreading and migration behaviors and their correlations, providing insights for deeper understanding and more promising prediction on the nasopharyngeal tumor metastasis.

Migration of immortalized nasopharyngeal epithelia and carcinoma cells through porous membrane in 3D platforms.

In the present study, 3D biomimetic platforms were fabricated with guiding grating to mimic extracellular matrix topography, porous membrane to resemble the epithelial porous interface and trenches below to represent blood vessels as an in vitro tissue microenvironment. Fabrication technologies were developed to integrate the transparent biocompatible polydimethylsiloxane platforms with preciously controlled dimensions. Cell migration behaviors of an immortalized nasopharyngeal epithelial cell line (NP460) and a nasopharyngeal carcinoma cell line (NPC43) were studied on the 2D and 3D platforms. The NP460 and NPC43 cells traversing through the porous membrane and migrating in the trenches below were studied by time-lapse imaging. Before traversing through the pores, NP460 and NPC43 cells migrated around the pores but NPC43 cells had a lower migration speed with less lamellipodia spreading. After traversing to trenches below, NPC43 cells moved faster with an alternated elongated morphology (mesenchymal migration mode) and round morphology (amoeboid migration mode) compared with only mesenchymal migration mode for NP460 cells. The cell traversing probability through porous membrane on platforms with 30 µm wide trenches below was found to be the highest when the guiding grating was perpendicular to the trenches below and the lowest when the guiding grating was parallel to the trenches below. The present study shows important information on cell migration in complex 3D microenvironment with various dimensions and could provide insight for pathology and treatment of nasopharyngeal carcinoma.

3D nanoplasmonic biosensor for detection of filopodia in cells.

Filopodia are thin finger-like protrusions from cells and they are hard to detect using electrical, mechanical, or optical sensors because of their nanometer scale features. Besides, the signals from filopodia and the cell membrane are often mixed together which makes the detection of filopodia challenging. Here, a 3D nanoplasmonic biosensor with microposts is proposed to overcome these limitations. By using suitable chemical coating and physical dimensions, the signals from filopodia and the cell membrane were separated by having the microposts keep the cell membrane from making contact with the nanoplasmonic biosensor. The filopodia were detected by the 3D asymmetrical nanopillars with sharp Fano resonance. The sensitivity and figure of merit of the nanoplasmonic biosensor were 650 nm per refractive index unit and 28.3, respectively. A large peak shift of 6 nm was observed for the detection of MC3T3 osteoblastic cell filopodia at a concentration of 1300 cells per mm2. To the best of our knowledge, this is the first demonstration of filopodia detection using nanoplasmonic biosensors, where microposts were used to separate the cell membrane from filopodia and the 3D nanoplasmonic biosensors were used to monitor filopodia on the nanometer scale. These combined 3D micro- and nano-structures allow filopodia to be detected using different sensors without interference from the cell membrane.

Deconstructing, Replicating, and Engineering Tissue Microenvironment for Stem Cell Differentiation.

One of the most crucial components of regenerative medicine is the controlled differentiation of embryonic or adult stem cells into the desired cell lineage. Although most of the reported protocols of stem cell differentiation involve the use of soluble growth factors, it is increasingly evident that stem cells also undergo differentiation when cultured in the appropriate microenvironment. When cultured in decellularized tissues, for instance, stem cells can recapitulate the morphogenesis and functional specialization of differentiated cell types with speed and efficiency that often surpass the traditional growth factor-driven protocols. This suggests that the tissue microenvironment (TME) provides stem cells with a holistic "instructive niche" that harbors signals for cellular reprogramming. The translation of this into medical applications requires the decoding of these signals, but this has been hampered by the complexity of TME. This problem is often addressed by a reductionist approach, in which cells are exposed to substrates decorated with simple, empirically designed geometries, textures, and chemical compositions ("bottom-up" approach). Although these studies are invaluable in revealing the basic principles of mechanotransduction mechanisms, their physiological relevance is often uncertain. This review examines the recent progress of an alternative, "top-down" approach, in which the TME is treated as a holistic biological entity. This approach is made possible by recent advances in systems biology and fabrication technologies that enable the isolation, characterization, and reconstitution of TME. It is hoped that these new techniques will elucidate the nature of niche signals so that they can be extracted, replicated, and controlled. This review summarizes these emerging techniques and how the data they generated are changing our view on TME. Impact statement This review summarizes the current state of art of the understanding of instructive niche in the field of tissue microenvironment. Not only did we survey the use of different biochemical preparations as stimuli of stem cell differentiation and summarize the recent effort in dissecting the biochemical composition of these preparations, through the application of extracellular matrix (ECM) arrays and proteomics, but we also introduce the use of open-source, high-content immunohistochemistry projects in contributing to the understanding of tissue-specific composition of ECM. We believe this review would be highly useful for our peer researching in the same field. "Mr. Tulkinghorn is always the same… so oddly out of place and yet so perfectly at home." -Charles Dickens, Bleak House.

Capturing instructive cues of tissue microenvironment by silica bioreplication.

Cells in tissues are enveloped by an instructive niche made of the extracellular matrix. These instructive niches contain three general types of information: topographical, biochemical and mechanical. While the combined effects of these three factors are widely studied, the functions of each individual one has not been systematically characterised, because it is impossible to alter a single factor in a tissue microenvironment without simultaneously affecting the other two. Silica BioReplication (SBR) is a process that converts biological samples into silica, faithfully preserving the original topography at the nano-scale. We explored the use of this technique to generate inorganic replicas of intact mammalian tissues, including tendon, cartilage, skeletal muscle and spinal cord. Scanning electron and atomic force microscopy showed that the resulting replicas accurately preserved the three-dimensional ultrastructure of each tissue, while all biochemical components were eradicated by calcination. Such properties allowed the uncoupling the topographical information of a tissue microenvironment from its biochemical and mechanical components. Here, we showed that human mesenchymal stem cells (MSC) cultured on the replicas of different tissues displayed vastly different morphology and focal adhesions, suggesting that the topography of the tissue microenvironment captured by SBR could profoundly affect MSC biology. MSC cultured on tendon replica elongated and expressed tenocytes marker, while MSC on the spinal cord replica developed into spheroids that resembled neurospheres, in morphology and in the expression of neurosphere markers, and could be further differentiated into neuron-like cells. This study reveals the significance of topographical cues in a cell niche, as tissue-specific topography was sufficient in initiating and directing differentiation of MSC, despite the absence of any biochemical signals. SBR is a convenient and versatile method for capturing this topographical information, facilitating the functional characterisation of cell niches. STATEMENT OF SIGNIFICANCE: Various studies have shown that three major factors, topographical, biochemical and mechanical, in a tissue microenvironment (TME) are essential for cellular homeostasis and functions. Current experimental models are too simplistic to represent the complexity of the TME, hindering the detailed understanding of its functions. In particular, the importance each factor in a tissue microenvironment have not been individually characterised, because it is challenging to alter one of these factors without simultaneously affecting the other two. Silica bioreplication (SBR) is a process that converts biological samples into silica replicas with high structural fidelity. SBR is a convenient and versatile method for capturing this topographical information on to a biologically inert material, allowing the functional characterisation of the architecture of a TME.

Microenvironmental topographic cues influence migration dynamics of nasopharyngeal carcinoma cells from tumour spheroids.

Tumour metastasis is a complex process that strongly influences the prognosis and treatment of cancer. Apart from intracellular factors, recent studies have indicated that metastasis also depends on microenvironmental factors such as the biochemical, mechanical and topographical properties of the surrounding extracellular matrix (ECM) of tumours. In this study, as a proof of concept, we conducted tumour spheroid dissemination assay on engineered surfaces with micrograting patterns. Nasopharyngeal spheroids were generated by the 3D culture of nasopharyngeal carcinoma (NPC43) cells, a newly established cell line that maintains a high level of Epstein-Barr virus, a hallmark of NPC. Three types of collagen I-coated polydimethylsiloxane (PDMS) substrates were used, with 15 µm deep "trenches" that grated the surfaces: (a) 40/10 µm ridges (R)/trenches (T), (b) 18/18 µm (R/T) and (c) 50/50 µm (R/T). The dimensions of these patterns were designed to test how various topographical cues, different with respect to the size of tumour spheroids and individual NPC43 cells, might affect dissemination behaviours. Spreading efficiencies on all three patterned surfaces, especially 18/18 µm (R/T), were lower than that on flat PDMS surface. The outspreading cell sheets on flat and 40/10 µm (R/T) surfaces were relatively symmetrical but appeared ellipsoid and aligned with the main axes of the 18/18 µm (R/T) and 50/50 µm (R/T) grating platforms. Focal adhesions (FAs) were found to preferentially formed on the ridges of all patterns. The number of FAs per spheroid was strongly influenced by the grating pattern, with the least FAs on the 40/10 µm (R/T) and the most on the 50/50 µm (R/T) substrate. Taken together, these data indicate a previously unknown effect of surface topography on the efficiency and directionality of cancer cell spreading from tumour spheroids, suggesting that topography, like ECM biochemistry and stiffness, can influence the migration dynamics in 3D cell culture models.

Natural killer cell migration control in microchannels by perturbations and topography.

Natural killer (NK) cells are lymphocytes which play an important role in the immune system by recognizing and killing potentially malignant cells without antigen sensitization, and could be utilized in cancer therapy. NK cell migration is an essential process to find and kill target cells, which is well known to be driven by the chemotaxis effect. NK cells also experience a topographical effect induced by the extracellular matrix (ECM) during their migration. However, topographical effects on NK cell locomotion in three dimensional (3D) environments are not well studied yet. In this work, polydimethylsiloxane based platforms containing microchannels with different types of perturbations and decorated with various surface patterns were fabricated to systematically study the topographical effect on NK cell migration with and without the chemotaxis effect. The results showed that perturbation sites in channels induced pauses and reversals in chemotaxis driven NK cell migration. Surface topography such as gratings in confined environments could introduce directional preference to NK cell movement even without chemoattractants. These findings showed that NK cell migration could be controlled by contact guidance, which provides future possibility to manipulate NK cell migration in controlled in vitro bioengineering systems. Results in this study showed that the complex topography of 3D microenvironments in the ECM could have significant effects on NK cell migration in different tissues and organs, and provided insight for explaining the dynamics of NK cell activities in clinical experiments.

Cell migration on microposts with surface coating and confinement.

Understanding cell migration in a 3D microenvironment is essential as most cells encounter complex 3D extracellular matrix (ECM) in vivo Although interactions between cells and ECM have been studied previously on 2D surfaces, cell migration studies in 3D environment are still limited. To investigate cell migration under various degrees of confinements and coating conditions, 3D platforms with micropost arrays and controlled fibronectin (FN) protein coating were developed. MC3T3-E1 cells spread and contacted the top surface of microposts if FN was coated on top. When FN was coated all over the microposts, cells were trapped between microposts with 3 µm spacing and barely moved. As the spacing between microposts increased from 3 to 5 µm, cells became elongated with limited cell movement of 0.18 µm/min, slower than the cell migration speed of 0.40 µm/min when cells moved on top. When cells were trapped in between the microposts, cell nuclei were distorted and actin filaments formed along the sidewalls of microposts. With the addition of a top cover to introduce cell confinement, the cell migration speed was 0.23 and 0.84 µm/min when the channel height was reduced from 20 to 10 µm, respectively. Cell traction force was monitored at on the top and bottom microposts with 10 µm channel height. These results show that the MC3T3-E1 cell morphology, migration speed, and movement position were affected by surface coating and physical confinement, which will provide significant insights for in vivo cell migration within a 3D ECM.

Cell traction force in a confined microenvironment with double-sided micropost arrays.

Three-dimensional (3D) cell migrations are regulated by force interactions between cells and a 3D extracellular matrix (ECM). Mapping the 3D traction force generated by cells on the surrounding ECM with controlled confinement and contact area will be useful in understanding cell migration. In this study, double-sided micropost arrays were fabricated. The cell traction force was mapped by microposts on the top and bottom of opposing surfaces with a controlled separating distance to create different confinements. The density of micropost arrays was modified to investigate the effect of cell contact area on 3D traction force development. Using MC3T3-E1 osteoblastic cells, the leading traction force was found to increase with additional contact surface on the top. Summing force vectors on both surfaces, a large force imbalance was found from the leading to trailing regions for fast migrating cells. With 10 µm separation and densely arranged microposts, the traction force on the top surface was the largest at 28.6 ± 2.5 nN with the highest migration speed of 0.61 ± 0.07 µm min-1. Decreasing the density of the top micropost arrays resulted in a reduced traction force on the top and lower migration speed. With 15 µm separation, the cell traction force on the top and migration speed further decreased simultaneously. These results revealed traction force development on 3D ECM with varied degrees of confinement and contact area, which is important in regulating 3D cell migration.

Highly sensitive detection of exosomes by 3D plasmonic photonic crystal biosensor.

In this study, two-dimensional (2D), quasi-three-dimensional (3D), and 3D plasmonic photonic crystal (PPC) nanostructures with point-defect cavities were developed and fabricated using direct and reversal nanoimprint lithography. As a result of the hybrid coupling of localized surface plasmon resonance and Fabry-Perot cavity modes, the quasi-3D plasmonic nanoholes showed higher electromagnetic field intensity and sensitivity than the 2D plasmonic nanoholes. Specifically, the sensitivity of the quasi-3D plasmonic nanoholes was 483 nm per refractive index unit (RIU), whereas that of the 2D plasmonic nanoholes was 276 nm RIU-1. In addition, by enhancing electromagnetic field intensity around corners and generating an additional subradiant dark mode, the symmetrical breakage of the quasi-3D plasmonic nanoholes further increased the sensitivity to 946 nm RIU-1. Among all the nanostructures developed in the study, the 3D PPC nanostructures with point-defect cavities showed the highest sensitivity up to 1376 nm RIU-1 and highest figure of merit of 11.6 as the result of the hybrid coupling of plasmonics and photonic crystal modes with multilayered plasmonic nanostructures. The spacing between the 3D PPC nanostructures was comparable with the average size of exosomes derived from fibroblast L cells, which allowed the exosomes to spread around the 3D PPC nanostructures with increased sensing area. This effect further enhanced the detection sensitivity with a large peak shift of 9 nm when using the 3D PPC biosensor to detect exosomes at the concentration of 1 × 104 particles per ml, and the peak shift increased to 102 nm as exosome concentration increased to 1 × 1011 particles per ml.

Microfluidic implementation of functional cytometric microbeads for improved multiplexed cytokine quantification.

Functional microbeads have been widely applied in molecular identification and other biochemical applications in the past decade, owing to the compatibility with flow cytometry and the commercially available microbeads for a wide range of molecular identification. Nevertheless, there is still a technical hurdle caused by the significant sample volume required (∼50 µl), limited molecular detection limit (∼20 pg/ml), complicated liquid/microbead handling procedures, and the long reaction time (>2 h). In this work, we optimize the operation of an automated microbead-based microfluidic device for the reagent mixing and the dynamic cytokine detection. In particular, we adopt fluorescence microscopy for quantification of multiple microbeads in each microchamber instead of flow cytometry for a lower detection limit. The operation parameters are then configured for improved measurement performance. As demonstrated, we consider the cytokine secretion of human macrophage-differentiating lymphocytes stimulated by lipopolysaccharides. We examine requirements on the mixing duration, minimal sample volume, and the image analysis scheme for the smaller biosample volume (<5 µl), the lower cytokine detection limit (∼5 pg/ml), and shorter process time (∼30 min). Importantly, this microfluidic strategy can be further extended in the molecular profiling using other functional microbeads for a broad range of biomedical applications.