Extended replicative lifespan of primary resting T cells by CRISPR/dCas9-based epigenetic modifiers and transcriptional activators.

Extension of the replicative lifespan of primary cells can be achieved by activating human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths. In this work, we utilize CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T cells in the peripheral blood mononuclear cells (PBMCs) by rewiring the epigenetic marks of the TERT promoter. Importantly, we have successfully expanded resting T cells and delayed their cellular senescence for at least three months through TERT reactivation, without affecting the expression of a T-cell marker (CD3) or inducing an accelerated cell division rate. We have also demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. TERT reactivation and replicative senescence delay were achieved without inducing malignancy transformation, as shown in various cellular senescence assays, cell cycle state, proliferation rate, cell viability, and karyotype analyses. Our chromatin immunoprecipitation (ChIP)-qPCR data together with TERT mRNA and protein expression analyses confirmed the specificity of CRISPR-based transcription activators in modulating epigenetic marks of the TERT promoter, and induced telomerase expression. Therefore, the strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.

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.

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.

Biophysical Phenotyping and Modulation of ALDH+ Inflammatory Breast Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) have been shown to initiate tumorigenesis and cancer metastasis in many cancer types. Although identification of CSCs through specific marker expression helps define the CSC compartment, it does not directly provide information on how or why this cancer cell subpopulation is more metastatic or tumorigenic. In this study, the functional and biophysical characteristics of aggressive and lethal inflammatory breast cancer (IBC) CSCs at the single-cell level are comprehensively profiled using multiple microengineered tools. Distinct functional (cell migration, growth, adhesion, invasion and self-renewal) and biophysical (cell deformability, adhesion strength and contractility) properties of ALDH+ SUM149 IBC CSCs are found as compared to their ALDH- non-CSC counterpart, providing biophysical insights into why CSCs has an enhanced propensity to metastasize. It is further shown that the cellular biophysical phenotype can predict and determine IBC cells' tumorigenic ability. SUM149 and SUM159 IBC cells selected and modulated through biophysical attributes-adhesion and stiffness-show characteristics of CSCs in vitro and enhance tumorigenicity in in vivo murine models of primary tumor growth. Overall, the multiparametric cellular biophysical phenotyping and modulation of IBC CSCs yields a new understanding of IBC's metastatic properties and how they might develop and be targeted for therapeutic interventions.

Protein-Substrate Adhesion in Microcontact Printing Regulates Cell Behavior.

Microcontact printing (µCP) is widely used to create patterns of biomolecules essential for studies of cell mechanics, migration, and tissue engineering. However, different types of µCPs may create micropatterns with varied protein-substrate adhesion, which may change cell behaviors and pose uncertainty in result interpretation. Here, we characterize two µCP methods for coating extracellular matrix (ECM) proteins (stamp-off and covalent bond) and demonstrate for the first time the important role of protein-substrate adhesion in determining cell behavior. We found that, as compared to cells with weaker traction force (e.g., endothelial cells), cells with strong traction force (e.g., vascular smooth muscle cells) may delaminate the ECM patterns, which reduced cell viability as a result. Importantly, such ECM delamination was observed on patterns by stamp-off but not on the patterns by covalent bonds. Further comparisons of the displacement of the ECM patterns between the normal VSMCs and the force-reduced VSMCs suggested that the cell traction force plays an essential role in this ECM delamination. Together, our results indicated that µCPs with insufficient adhesion may lead to ECM delamination and cause cell death, providing new insight for micropatterning in cell-biomaterial interaction on biointerfaces.

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.

Microengineered Conductive Elastomeric Electrodes for Long-Term Electrophysiological Measurements with Consistent Impedance under Stretch.

In this research, we develop a micro-engineered conductive elastomeric electrode for measurements of human bio-potentials with the absence of conductive pastes. Mixing the biocompatible polydimethylsiloxane (PDMS) silicone with other biocompatible conductive nano-particles further provides the material with an electrical conductivity. We apply micro-replica mold casting for the micro-structures, which are arrays of micro-pillars embedded between two bulk conductive-PDMS layers. These micro-structures can reduce the micro-structural deformations along the direction of signal transmission; therefore the corresponding electrical impedance under the physical stretch by the movement of the human body can be maintained. Additionally, we conduct experiments to compare the electrical properties between the bulk conductive-PDMS material and the microengineered electrodes under stretch. We also demonstrate the working performance of these micro-engineered electrodes in the acquisition of the 12-lead electrocardiographs (ECG) of a healthy subject. Together, the presented gel-less microengineered electrodes can provide a more convenient and stable bio-potential measurement platform, making tele-medical care more achievable with reduced technical barriers for instrument installation performed by patients/users themselves.

Automated long-term monitoring of parallel microfluidic operations applying a machine vision-assisted positioning method.

As microfluidics has been applied extensively in many cell and biochemical applications, monitoring the related processes is an important requirement. In this work, we design and fabricate a high-throughput microfluidic device which contains 32 microchambers to perform automated parallel microfluidic operations and monitoring on an automated stage of a microscope. Images are captured at multiple spots on the device during the operations for monitoring samples in microchambers in parallel; yet the device positions may vary at different time points throughout operations as the device moves back and forth on a motorized microscopic stage. Here, we report an image-based positioning strategy to realign the chamber position before every recording of microscopic image. We fabricate alignment marks at defined locations next to the chambers in the microfluidic device as reference positions. We also develop image processing algorithms to recognize the chamber positions in real-time, followed by realigning the chambers to their preset positions in the captured images. We perform experiments to validate and characterize the device functionality and the automated realignment operation. Together, this microfluidic realignment strategy can be a platform technology to achieve precise positioning of multiple chambers for general microfluidic applications requiring long-term parallel monitoring of cell and biochemical activities.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.