Microfluidic high-throughput single-cell mechanotyping:Devices and applications

The mechanical behavior of individual cells plays an important role in regulating various biological activities at the molecular and cellular levels.It can serve as a promising label-free marker of cells’physiological states.In the past two decades,several techniques have been developed for understanding correlations between cellular mechanical changes and human diseases.However,numerous technical challenges remain with regard to realizing high-throughput,robust,and easy-to-perform measurements of single-cell mechanical properties.In this paper,we review the emerging tools for single-cell mechanical characterization that are provided by microfluidic technology.Different techniques are benchmarked by considering their advantages and limitations.Finally,the potential applications of microfluidic techniques based on cellular mechanical properties are discussed.

Noise in nanopore sensors: Sources, models, reduction, and benchmarking

Label-free nanopore sensors have emerged as a new generation technology of DNA sequencing and have been widely used for single molecule analysis.Since the firstα-hemolysin biological nanopore,various types of nanopores made of different materials have been under extensive development.Noise represents a common challenge among all types of nanopore sensors.The nanopore noise can be decomposed into four components in the frequency domain(1/f noise,white noise,dielectric noise,and amplifier noise).In this work,we reviewed and summarized the physicalmodels,origins,and reduction methods for each of these noise components.For the first time,we quantitatively benchmarked the root mean square(RMS)noise levels for different types of nanopores,demonstrating a clear material-dependent RMS noise.We anticipate this review article will enhance the understanding of nanopore sensor noises and provide an informative tutorial for developing future nanopore sensors with a high signal-to-noise ratio.

A tip-coupled, two-cantilever, non-resonant microsystem for direct measurement of liquid viscosity

We report a non-resonant piezoelectric microelectromechanical cantilever system for the measurement of liquid viscosity.The system consists of two PiezoMEMS cantilevers in-line,with their free ends facing each other.The system is immersed in the fluid under test for viscosity measurement.One of the cantilevers is actuated using the embedded piezoelectric thin film to oscillate at a pre-selected non-resonant frequency.The second cantilever,the passive one,starts to oscillate due to the fluid-mediated energy transfer.The relative response of the passive cantilever is used as the metric for the fluid's kinematic viscosity.The fabricated cantilevers are tested as viscosity sensors by carrying out experiments in fluids with different viscosities.The viscometer can measure viscosity at a single frequency of choice,and hence some important considerations for frequency selection are discussed.A discussion on the energy coupling between the active and the passive cantilevers is presented.The novel PiezoMEMS viscometer architecture proposed in this work will overcome several challenges faced by state-of-the-art resonance MEMS viscometers,by enabling faster and direct measurement,straightforward calibration,and the possibility of shear rate-dependent viscosity measurement.

Microfluidic deformability-activated sorting of single particles

Mechanical properties have emerged as a significant label-free marker for characterizing deformable particles such as cells.Here,we demonstrated the first single-particle-resolved,cytometry-like deformability-activated sorting in the continuous flow on a microfluidic chip.Compared with existing deformability-based sorting techniques,the microfluidic device presented in this work measures the deformability and immediately sorts the particles one-by-one in real time.It integrates the transit-time-based deformability measurement and active hydrodynamic sorting onto a single chip.We identified the critical factors that affect the sorting dynamics by modeling and experimental approaches.We found that the device throughput is determined by the summation of the sensing,buffering,and sorting time.A total time of~100 ms is used for analyzing and sorting a single particle,leading to a throughput of 600 particles/min.We synthesized poly(ethylene glycol)diacrylate(PEGDA)hydrogel beads as the deformability model for device validation and performance evaluation.A deformability-activated sorting purity of 88%and an average efficiency of 73%were achieved.We anticipate that the ability to actively measure and sort individual particles one-by-one in a continuous flow would find applications in cell-mechanotyping studies such as correlational studies of the cell mechanical phenotype and molecular mechanism.

Active cell-matrix coupling regulates cellular force landscapes of cohesive epithelial monolayers

Epithelial cells can assemble into cohesive monolayers with rich morphologies on substrates due to competition between elastic,edge,and interfacial effects.Here we present a molecularly based thermodynamic model,integrating monolayer and substrate elasticity,and force-mediated focal adhesion formation,to elucidate the active biochemical regulation over the cellular force landscapes in cohesive epithelial monolayers,corroborated by microscopy and immunofluorescence studies.The predicted extracellular traction and intercellular tension are both monolayer size and substrate stiffness dependent,suggestive of cross-talks between intercellular and extracellular activities.Our model sets a firm ground toward a versatile computational framework to uncover the molecular origins of morphogenesis and disease in multicellular epithelia.

Anti-oxidant anti-inflammatory and antibacterial tannin-crosslinked citrate-based mussel-inspired bioadhesives facilitate scarless wound healing

The revolutionary role of tissue adhesives in wound closure,tissue sealing,and bleeding control necessitates the development of multifunctional materials capable of effective and scarless healing.In contrast to the use of traditionally utilized toxic oxidative crosslinking initiators(exemplified by sodium periodate and silver nitrate),herein,the natural polyphenolic compound tannic acid(TA)was used to achieve near instantaneous(<25s),hydrogen bond mediated gelation of citrate-based mussel-inspired bioadhesives combining anti-oxidant,anti-inflammatory,and antimicrobial activities(3A-TCMBAs).The resulting materials were self-healing and possessed low swelling ratios(<60%)as well as considerable mechanical strength(up to~1.0 MPa),elasticity(elongation~2700%),and adhesion(up to 40 kPa).The 3A-TCMBAs showed strong in vitro and in vivo anti-oxidant ability,favorable cytocompatibility and cell migration,as well as photothermal antimicrobial activity against both Staphylococcus aureus and Escherichia coli(>90%bacterial death upon near-infrared(NIR)irradiation).In vivo evaluation in both an infected full-thickness skin wound model and a rat skin incision model demonstrated that 3A-TCMBAs+NIR treatment could promote wound closure and collagen deposition and improve the collagen Ⅰ/Ⅲ ratio on wound sites while simultaneously inhibiting the expression of pro-inflammatory cytokines.Further,phased angiogenesis was observed via promotion in the early wound closure phases followed by inhibition and triggering of degradation&remodeling of the extracellular matrix(ECM)in the late stage(supported by phased CD31(platelet endothelial cell adhesion molecule-1)PDGF(platelet-derived growth factor)and VEGF(vascular endothelial growth factor)expression as well as elevated matrix metalloprotein-9(MMP-9)expression on day 21),resulting in scarless wound healing.The significant convergence of material and bioactive properties elucidated above warrant further exploration of 3A-TCMBAs as a significant,new class of bioadhesive.

Wearable bioelectronics fabricated in situ on skins

In recent years,wearable bioelectronics has rapidly expanded for diagnosing,monitoring,and treating various pathological conditions from the skin surface.Although the devices are typically prefabricated as soft patches for general usage,there is a growing need for devices that are customized in situ to provide accurate data and precise treatment.In this perspective,the stateof-the-art in situ fabricated wearable bioelectronics are summarized,focusing primarily on Drawn-on-Skin(DoS)bioelectronics and other in situ fabrication methods.The advantages and limitations of these technologies are evaluated and potential future directions are suggested for the widespread adoption of these technologies in everyday life.

Fabricating 3-dimensional human brown adipose microtissues for transplantation studies

Transplanting cell cultured brown adipocytes(BAs)represents a promising approach to prevent and treat obesity(OB)and its associated metabolic disorders,including type 2 diabetes mellitus(T2DM).However,transplanted BAs have a very low survival rate in vivo.The enzymatic dissociation during the harvest of fully differentiated BAs also loses significant cells.There is a critical need for novel methods that can avoid cell death during cell preparation,transplantation,and in vivo.Here,we reported that preparing BAs as injectable microtissues could overcome the problem.We found that 3D culture promoted BA differentiation and UCP-1 expression,and the optimal initial cell aggregate size was 100μm.The microtissues could be produced at large scales via 3D suspension assisted with a PEG hydrogel and could be cryopreserved.Fabricated microtissues could survive in vivo for long term.They alleviated body weight and fat gain and improved glucose tolerance and insulin sensitivity in high-fat diet(HFD)-induced OB and T2DM mice.Transplanted microtissues impacted multiple organs,secreted protein factors,and influenced the secretion of endogenous adipokines.To our best knowledge,this is the first report on fabricating human BA microtissues and showing their safety and efficacy in T2DM mice.The proposal of transplanting fabricated BA microtissues,the microtissue fabrication method,and the demonstration of efficacy in T2DM mice are all new.Our results show that engineered 3D human BA microtissues have considerable advantages in product scalability,storage,purity,safety,dosage,survival,and efficacy.

GABAergic Abnormalities Associated with Sensorimotor Corticostriatal Community Structural Deficits in ErbB4 Knockout Mice and First-Episode Treatment-Naive Patients with Schizophrenia

The current study was designed to explore how disruption of specific molecular circuits in the cerebral cortex may cause sensorimotor cortico-striatal community structure deficits in both a mouse model and patients with schizophrenia.We used prepulse inhibition(PPI)and brain structural and diffusion MRI scans in 23 mice with conditional ErbB4 knockout in parvalbumin interneurons and 27 matched controls.Quantitative real-time PCR was used to assess the differential levels of GABA-related transcripts in brain regions.Concurrently,we measured structural and diffusion MRI and the cumulative contribution of risk alleles in the GABA pathway genes in firstepisode treatment-naı¨ve schizophrenic patients(n=117)and in age-and sex-matched healthy controls(n=86).We present the first evidence of gray and white matter impairment of right sensorimotor cortico-striatal networks and reproduced the sensorimotor gating deficit in a mouse model of schizophrenia.Significant correlations between gray matter volumes(GMVs)in the somatosensory cortex and PPI as well as glutamate decarboxylase 1 mRNA expression were found in controls but not in knockout mice.Furthermore,these findings were confirmed in a human sample in which we found significantly decreased gray and white matter in sensorimotor cortico-striatal networks in schizophrenic patients.The psychiatric risk alleles of the GABA pathway also displayed a significant negative correlation with the GMVs of the somatosensory cortex in patients.Our study identified that ErbB4 ablation in parvalbumin interneurons induced GABAergic dysregulation,providing valuable mechanistic insights into the sensorimotor cortico-striatal community structure deficits associated with schizophrenia.

Heart regeneration with human pluripotent stem cells: Prospects and challenges

Cardiovascular disease,ranging from congenital heart disease to adult myocardial infarction,is the leading cause of death worldwide.In pursuit of reliable cardiovascular regenerative medicine,human pluripotent stem cells(hPSCs),including human embryonic stem cells(hESCs)and human induced pluripotent stem cells(hiPSCs),offer plenty of potential cell-based applications.HPSCs are capable of proliferating indefinitely in an undifferentiated state,and are also pluripotent,being able to differentiate into virtually any somatic cell types given specific stepwise cues,thus representing an unlimited source to generate functional cardiovascular cells for heart regeneration.Here we recapitulated current advances in developing efficient protocols to generate hPSCderived cardiovascular cell lineages,including cardiomyocytes,endothelial cells,and epicardial cells.We also discussed applications of hPSC-derived cells in combination with compatible bioactive materials,promising trials of cell transplantation in animal models of myocardial infarction,and potential hurdles to bring us closer to the ultimate goal of cell-based heart repair.

Robust genome and RNA editing via CRISPR nucleases in PiggyBac systems

CRISPR/Cas-mediated genome editing in human pluripotent stem cells(hPSCs)offers unprecedented opportunities for developing in vitro disease modeling,drug screening and cell-based therapies.To efficiently deliver the CRISPR components,here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively.These vectors utilized the PiggyBac transposon system,which allows stable expression of CRISPR components in hPSCs.The Cas9 vector PB-CRISPR exhibited high efficiency(up to 99%)of inducing gene knockout in both protein-coding genes and long non-coding RNAs.The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown(98%knockdown for CD90)with optimized gRNA designs.Taken together,our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.