Mechanical Constraints in Tumor Guide Emergent Spatial Patterns of Glioblastoma Cancer Stem Cells.

The mechanical constraints in the overcrowding glioblastoma (GBM) microenvironment have been implicated in the regulation of tumor heterogeneity and disease progression. Especially, such mechanical cues can alter cellular DNA transcription and give rise to a subpopulation of tumor cells called cancer stem cells (CSCs). These CSCs with stem-like properties are critical drivers of tumorigenesis, metastasis, and treatment resistance. Yet, the biophysical and molecular machinery underlying the emergence of CSCs in tumor remained unexplored. This work employed a two-dimensional micropatterned multicellular model to examine the impact of mechanical constraints arisen from geometric confinement on the emergence and spatial patterning of CSCs in GBM tumor. Our study identified distinct spatial distributions of GBM CSCs in different geometric patterns, where CSCs mostly emerged in the peripheral regions. The spatial pattern of CSCs was found to correspond to the gradients of mechanical stresses resulted from the interplay between the cell-ECM and cell-cell interactions within the confined environment. Further mechanistic study highlighted a Piezo1-RhoA-focal adhesion signaling axis in regulating GBM cell mechanosensing and the subsequent CSC phenotypic transformation. These findings provide new insights into the biophysical origin of the unique spatial pattern of CSCs in GBM tumor and offer potential avenues for targeted therapeutic interventions.

Recent progress of organ-on-a-chip towards cardiovascular diseases: advanced design, fabrication, and applications.

Cardiovascular diseases (CVDs) are a major cause of death worldwide, leading to increased medical care costs. To turn the scale, it is essential to acquire a more in-depth and comprehensive understanding of CVDs and thus formulate more efficient and reliable treatments. Over the last decade, tremendous effort has been made to develop microfluidic systems to recapitulate native cardiovascular environments because of their unique advantages over conventional 2D culture systems and animal models such as high reproductivity, physiological relevance, and good controllability. These novel microfluidic systems could be extensively adopted for natural organ simulation, disease modeling, drug screening, disease diagnosis and therapy. Here, a brief review of the innovative designs of microfluidic devices for CVDs research is presented, with specific discussions on material selection, critical physiological and physical considerations. In addition, we elaborate on various biomedical applications of these microfluidic systems such as blood-vessel-on-a-chip and heart-on-a-chip, which are conducive to the investigation of the underlying mechanisms of CVDs. This review also provides systematic guidance on the construction of next-generation microfluidic systems for the diagnosis and treatment of CVDs. Finally, the challenges and future directions in this field are highlighted and discussed.

Charge-Powered Electrotaxis for Versatile Droplet Manipulation.

Taxis is an instinctive behavior of living organisms to external dangers or benefits. Here, we report a taxis-like behavior associated with liquid droplets on charged substrates in response to the external stimuli, referred to as droplet electrotaxis. Such droplet electrotaxis enables us to use either solid or liquid (such as water) matter, even a human finger, as stimuli to spatiotemporal precisely manipulate the liquid droplets of various physicochemical properties, including water, ethanol with low surface tension, viscous oil, and so on. Droplet electrotaxis also features a flexible configuration that even can manifest in the presence of an additional layer, such as the ceramic with a thickness of ∼10 mm. More importantly, superior to existing electricity-based strategies, droplet electrotaxis can harness the charges generated from diverse manners, including pyroelectricity, triboelectricity, piezoelectricity, and so on. These properties dramatically increase the application scenarios of droplet electrotaxis, such as cell labeling and droplet information recording.

Monitor Tumor pHe and Response Longitudinally during Treatment Using CEST MRI-Detectable Alginate Microbeads.

Imaging pHe of the tumor microenvironment has paramount importance for characterizing aggressive, invasive tumors, as well as therapeutic responses. Here, a robust approach to image pH changes in the tumor microenvironment longitudinally and during sodium bicarbonate treatment was reported. The pH-sensing microbeads were designed and prepared based on materials approved for clinical use, i.e., alginate microbead-containing computed tomography (CT) contrast-agent (iopamidol)-loaded liposomes (Iop-lipobeads). This Iop-lipobead prepared using a customized microfluidic device generated a CEST contrast of 10.6% at 4.2 ppm at pH 7.0, which was stable for 20 days in vitro. The CEST contrast decreased by 11.8% when the pH decreased from 7.0 to 6.5 in vitro. Optimized Iop-lipobeads next to tumors showed a significant increase of 19.7 ± 6.1% (p < 0.01) in CEST contrast at 4.2 ppm during the first 3 days of treatment and decreased to 15.2 ± 4.8% when treatment stopped. Notably, percentage changes in Iop-lipobeads were higher than that of amide CEST (11.7% and 9.1%) in tumors during and after treatment. These findings demonstrated that the Iop-lipobead could provide an independent and sensitive assessment of the pHe changes for a noninvasive and longitudinal monitoring of the treatment effects using multiple CEST contrast.

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.

Selective single-bacteria extraction based on capture and release of microemulsion droplets.

Human host-associated microbial communities in body sites can reflect health status based on the population distribution and specific microbial properties in the heterogeneous community. Bacteria identification at the single-cell level provides a reliable biomarker and pathological information for clinical diagnosis. Nevertheless, biosamples obtained from some body sites cannot offer sufficient sample volume and number of target cells as required by most of the existing single-cell isolation methods such as flow cytometry. Herein we report a novel integrated microfluidic system, which consists of a microemulsion module for single-bacteria encapsulation and a sequential microdroplet capture and release module for selectively extracting only the single-bacteria encapsulated in microdroplets. We optimize the system for a success rate of the single-cell extraction to be > 38%. We further verify applicability of the system with prepared cell mixtures (Methylorubrum extorquens AM1 and Methylomicrobium album BG8) and biosamples collected from human skin, to quantify the population distribution of multiple key species in a heterogeneous microbial community. Results indicate perfect viability of the single-cell extracts and compatibility with downstream analyses such as PCR. Together, this research demonstrates that the reported single-bacteria extraction system can be applied in microbiome and pathology research and clinical diagnosis as a clinical or point-of-care device.

PAM-flexible dual base editor-mediated random mutagenesis and self-activation strategies to improve CRISPRa potency.

VP64 is the smallest transactivation domain that can be packaged together with the sgRNA into a single adeno-associated virus (AAV) vector. However, VP64-based CRISPRa often exerts modest activation to the target gene when only one sgRNA is used. Herein, we used PAM-flexible dual base editor-mediated mutagenesis and self-activation strategies to derive VP64 variants with gain-of-function mutations. First, we generated an HEK293FT transgenic clone to stably expressing pTK-CRISPRa-GFP. The sgRNA of CRISPRa was designed to target the TK promoter, thereby allowing self-activation of CRISPRa-GFP. Base editors were then used to randomly mutagenesis VP64 in this transgenic cell. VP64 with enhanced potency would translate into increment of GFP fluorescence intensity, thereby allowing positive selection of the desired VP64 mutants. This strategy has enabled us to identify several VP64 variants that are more potent than the wild-type VP64. ΔCRISPRa derived from these VP64 variants also efficiently activated the endogenous promoter of anti-aging and longevity genes (KLOTHO, SIRT6, and NFE2L2) in human cells. Since the overall size of these ΔCRISPRa transgenes is not increased, it remains feasible for all-in-one AAV applications. The strategies described here can facilitate high-throughput screening of the desired protein variants and adapted to evolve any other effector domains.

A Narrow Straight Microchannel Array for Analysis of Transiting Speed of Floating Cancer Cells.

Investigating floating cells along a narrow microchannel (e.g., a blood vessel) for their transiting speeds and the corresponding roles of cell physical properties can deepen our understanding of circulating tumor cells (CTCs) metastasis via blood vessels. Many existing studies focus on the cell transiting process in blood vessel-like microchannels; further analytical studies are desired to summarize behaviors of the floating cell movement under different conditions. In this work, we perform a theoretical analysis to establish a relation between the transiting speed and key cell physical properties. We also conduct computational fluid dynamics simulation and microfluidic experiments to verify the theoretical model. This work reveals key cell physical properties and the channel configurations determining the transiting speed. The reported model can be applied to other works with various dimensions of microchannels as a more general way to evaluate the cancer cell metastasis ability with microfluidics.

Correction: Antibody-coated microstructures for selective isolation of immune cells in blood.

Correction for 'Antibody-coated microstructures for selective isolation of immune cells in blood' by Jiyu Li et al., Lab Chip, 2020, 20, 1072-1082, DOI: 10.1039/D0LC00078G.

Adhesion Strengthening Mechanism of Carbon Nanotube-Embedded Epoxy Composites: A Fracture-Based Approach.

Interfacial bonding integrity between different materials is critical to maintain the functionality of the entire physical system in any scale, ranging from building structures down to semiconductor transistors. For example, micro-patterned polymers embedded with conductive nanoparticles [e.g., carbon nanotubes (CNTs)] bonded with integrated circuits have been applied as many emerging chemical/biological microelectronic sensors. Nonetheless, it is challenging to measure and ensure the interfacial bonding integrity between materials for consistent and sustainable operations. Herein, we apply multiple interface characterization methods based on micro-engineering and microscopy as an integrative approach to reveal the mechanism of interfacial reinforcement by adding CNTs in a matrix material. An epoxy/CNT micro-beam is fabricated onto a silicon substrate, sandwiching a gold layer as an interfacial precrack. Superlayers of chromium are then repeatedly deposited onto the microstructure, inducing stepwise increasing stress over the materials and the corresponding micro-beam bending after detachment from the bonded interface. Accordingly, we can quantify key interfacial fracture parameters such as crack length, steady-state energy release rate, and fracture toughness. By further examining the formation and distribution of the micro-/nanostructures along the debonded interface using bright-field microscopy, 3D fluorescence imaging, and scanning electron microscopy, we can identify the underlying dominant interfacial strengthening and fracture toughening mechanisms. We further compare experimental results and theoretical predictions to quantify the interfacial bonding properties between epoxy/CNT and silicon and unveil the underlying reinforcement mechanisms. The results provide insights to develop polymer/nanoparticle composites with reinforced interfacial bonding integrity for more sustainable and reliable applications including microelectronics, surface coatings, and adhesive materials.

High-throughput deterministic pairing and coculturing of single cells in a microwell array using combined hydrodynamic and recirculation flow captures.

Single-cell level coculture facilitates the study of cellular interactions for uncovering unknown physiological mechanisms, which are crucial for the development of new therapies for diseases. However, efficient approaches for high-throughput deterministic pairing of single cells and traceable coculture remain lacking. In this study, we report a new microfluidic device, which combines hydrodynamic and recirculation flow captures, to achieve high-throughput and deterministic pairing of single cells in a microwell array for traceable coculture. Compared with the existing techniques, the developed device exhibits advantages with regard to pairing efficiency, throughput, determinacy, and traceability. Through repeating a two-step method, which sequentially captures single cells in a meandering channel and a microwell array, cell number and type can be easily controlled. Double and triple single-cell pairings have been demonstrated with an efficiency of 72.2% and 38.0%, respectively. Cellular engulfment using two breast cell lines is investigated on a developed microfluidic chip as a biological case study, in which the morphological characteristics and the incidence rate are analyzed. This research provides an efficient and reliable alternative for the coculture of single cells on the microfluidic platform for various biomedical applications, such as studying cellular engulfment and tumor sphere formation under single-cell pairing condition.

An in silico glioblastoma microenvironment model dissects the immunological mechanisms of resistance to PD-1 checkpoint blockade immunotherapy.

The PD-1 immune checkpoint-based therapy has emerged as a promising therapy strategy for treating the malignant brain tumor glioblastoma (GBM). However, patient response varies in clinical trials due in large to the tumor heterogeneity and immunological resistance in the tumor microenvironment. To further understand how mechanistically the niche interplay and competition drive anti-PD-1 resistance, we established an in-silico model to quantitatively describe the biological rationale of critical GBM-immune interactions, such as tumor growth and apoptosis, T cell activation and cytotoxicity, and tumor-associated macrophage (TAM) mediated immunosuppression. Such an in-silico experimentation and predictive model, based on the in vitro microfluidic chip-measured end-point data and patient-specific immunological characteristics, allowed for a comprehensive and dynamic analysis of multiple TAM-associated immunosuppression mechanisms against the anti-PD-1 immunotherapy. Our computational model demonstrated that the TAM-associated immunosuppression varied in severity across different GBM subtypes, which resulted in distinct tumor responses. Our prediction results indicated that a combination therapy co-targeting of PD-1 checkpoint and TAM-associated CSF-1R signaling could enhance the immune responses of GBM patients, especially those patients with mesenchymal GBM who are irresponsive to the single anti-PD-1 therapy. The development of a patient-specific in silico-in vitro GBM model would help navigate and personalize immunotherapies for GBM patients.

Label-free biosensor of phagocytosis for diagnosing bacterial infections.

Phagocytic cells recognize and phagocytose invading microbes for destruction. However, bacterial pathogens can remain hidden at low levels from conventional detection or replicate intracellularly after being phagocytosed by immune cells. Current phagocytosis-detection approaches involve flow cytometry or microscopic search for rare bacteria-internalized phagocytes among large populations of uninfected cells, which poses significant challenges in research and clinical settings. Hence it is imperative to develop a rapid, non-disruptive, and label-free phagocytosis detection approach. Using deformability assays and microscopic imaging, we have demonstrated for the first time that the presence of intracellular bacteria in phagocytic blood cells led to aberrant physical properties. Specifically, human monocytes with internalized bacteria of various species were stiffer and larger compared with uninfected monocytes. Taking advantage of these physical differences, a novel microfluidics-based biosensor platform was developed to passively sort, concentrate and quantify rare monocytes with internalized pathogens (MIP) from uninfected monocyte populations for phagocytosis detection. The clinical utility of the MIP platform was demonstrated by enriching and detecting bacteria-internalized monocytes from spiked human blood samples within 1.5 h. Patient-derived clinical isolates were used to validate the utility of the MIP platform further. This proof-of-concept presents a phagocytosis detection platform that could be used to rapidly diagnose microbial infections, especially in bloodstream infections (BSIs), thereby improving the clinical outcomes for point-of-care management.

Low-cost laser-cut patterned chips for acoustic concentration of micro- to nanoparticles and cells by operating over a wide frequency range.

Acoustofluidic platforms for cell manipulation benefit from being contactless and label-free at potentially low cost. Particle concentration in a droplet relies on augmenting spatial asymmetry in the acoustic field, which is difficult to reproduce reliably. Etching periodic patterns into a chip to create acoustic band gaps is an attractive approach to spatially modify the acoustic field. However, the sensitivity of acoustic band structures to geometrical tolerances requires the use of costly microfabrication processes. In this work, we demonstrate particle concentration across a range of periodic structure patterns fabricated with a laser-cutting tool, suitable for low-cost and low-volume rapid prototyping. The relaxation on precision is underscored by experimental results of equally efficient particle concentration outside band gaps and even in their absence, allowing operation over a range of frequencies independent of acoustic band gaps. These results are significant by indicating the potential of extending the proposed method from the microscale (e.g. tumor cells) to the nanoscale (e.g. bacteria) by scaling up the frequency without being limited by fabrication capabilities. We demonstrate the device's high degree of biocompatibility to illustrate the method's applicability in the biomedical field for applications such as basic biochemical analysis and in vitro diagnosis.

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.

Nondestructive quantification of single-cell nuclear and cytoplasmic mechanical properties based on large whole-cell deformation.

The mechanical properties of cell nuclei have been recognized to reflect and modulate important cell behaviors such as migration and cancer cell malignant tendency. However, these nuclear properties are difficult to characterize accurately using conventional measurement methods, which are often based on probing or deforming local sites over a nuclear region. The corresponding results are sensitive to the measurement position, and they are not decoupled from the cytoplasmic properties. Microfluidics is widely recognized as a promising technique for bioassay and phenotyping. In this report, we develop a simple and nondestructive approach for the single-cell quantification of nuclear elasticity based on microfluidics by considering different deformation levels of a live cell captured along a confining microchannel. We apply two inlet pressure levels to drive the flow of human nasopharyngeal epithelial cells (NP460) and human nasopharyngeal cancerous cells (NPC43) into the microchannels. A model considering the essential intracellular components (cytoplasm and nucleus) for describing the mechanics of a cell deforming along the confining microchannel is used to back-calculate the cytoplasmic and nuclear properties. On the other hand, we also apply a widely used chemical nucleus extraction technique to examine its possible effects (e.g., reduced nuclear modulus and reduced lamin A/C expression). To determine if the decoupled nuclear properties are representative of cancer-related attributes, we classify the NP460 and NPC43 cells using the decoupled physical properties as classification factors, resulting in an accuracy of 79.1% and a cell-type specificity exceeding 74%. It should be mentioned that the cells can be recollected at the device outlet after the nondestructive measurement. Hence, the reported cell elasticity measurement can be combined with downstream genetic and biochemical assays for general cell research and cancer diagnostic applications.

Microfluidic Viscometer Using a Suspending Micromembrane for Measurement of Biosamples.

The viscosity of biofluids such as blood and saliva can reflect an individual's health conditions, and viscosity measurements are therefore considered in health monitoring and disease diagnosis. However, conventional viscometers can only handle a larger liquid volume beyond the quantity that can be extracted from a person. Though very effective, micro-sensors based on electrokinetic, ultrasonic, or other principles often have strict requirements for the supporting equipment and complicated procedures and signal processing. Sample contamination is always an important issue. In this paper, we report a microfluidic viscometer requiring a small volume of biosamples (<50 µL) and straightforward operation procedures. It is fabricated with low-cost and biocompatible polymeric materials as one-time-use devices, such that contamination is no longer the concern. It contains a suspending micromembrane located along a microchannel. Under a steady driving pressure, the membrane displacement is a function of viscosity of the liquid sample being tested. We derived a simple analytical relation and perform a simulation for converting the membrane displacement to the sample viscosity. We conducted experiments with liquids (water and mineral oil) with defined properties to verify such a relation. We further applied the micro-viscometer to measure bovine blood samples with different hematocrit levels. It can be concluded that the microfluidic viscometer has a high compatibility with a broad range of biomedical applications.

A two-chip acoustofluidic particle manipulation platform with a detachable and reusable surface acoustic wave device.

This work describes a two-chip acoustofluidic platform for two-dimensional (2D) manipulation of microparticles in a closed microchamber on a reusable surface acoustic wave (SAW) device. This platform comprises two microfabricated chips: (1) a detachable silicon superstrate enclosed by a PDMS microfluidic chamber and (2) a reusable SAW device for generating standing SAW (SSAW), which is typically an expensive component. Critical to such a two-chip acoustofluidic platform is the selection of a suitable coupling agent at the interface of the SAW device and superstrate. To this end, we applied a polymer thin film as a coupling agent that balances between acoustic coupling efficiency, stability over time, and reusability. Recycling of the SAW device lowers the cost-barrier for acoustofluidic particle manipulation. The SSAW is transmitted into the silicon superstrate via the coupling agent to form a standing Lamb wave (SLW) to trap and move microparticles. The reported two-chip strategy enables the single-use microfluidic superstrates to avoid chemical and biological contaminations, while maintaining the merits of acoustofluidic manipulation of being noncontact and label-free and applicable to a wide range of microparticles with different shapes, density, polarity, and electrical properties.

Early Committed Clockwise Cell Chirality Upregulates Adipogenic Differentiation of Mesenchymal Stem Cells.

Cell chirality is observed with diverse forms and coordinates various left-right (LR) asymmetry in tissue morphogenesis. To give rise to such diversity, cell chirality may be coupled with cell differentiation. Here, using micropatterned human mesenchymal stem cells (hMSCs), an early committed clockwise (CW) cell chirality that can itself upregulate the adipogenic differentiation is reported. hMSC chirality enables a positively tilted chiral orientation on micropatterned stripes. When cultured as single cells on circular micropatterns, an anticlockwise (ACW)-biased nucleus rotation and swirling pattern of actin filament are observed. Interestingly, with adipogenic induction for 3-6 days, such chirality is reversed to negative chiral orientation and CW-biased rotation, which is earlier than the maturation of other differentiation markers, and consistently expressed in terminally differentiated adipocytes. Using latrunculin A (LatA), cytochalasin D (CD), and nocodazole (Noco) that forces a CW-biased actin filament and nucleus rotation resembling the early differentiated chirality upon adipogenic induction, an upregulation of adipogenic differentiation is found. The result demonstrates that the early differentiated chirality may serve as a mechanical precursor to engage the lineage commitment, suggesting a feedback mechanism of chiral actin in regulating cell differentiation and LR morphogenesis.