Microfluidic generation of transient cell volume exchange for convectively driven intracellular delivery of large macromolecules

Efficient intracellular delivery of target macromolecules remains a major obstacle in cell engineering and other biomedical applications. We discovered a unique cell biophysical phenomenon of transient cell volume exchange using microfluidics to rapidly and repeatedly compress cells. This behavior consists of brief, mechanically induced cell volume loss followed by rapid volume recovery. We harness this behavior for high-throughput, convective intracellular delivery of large polysaccharides (2000?kDa), particles (100?nm), and plasmids while maintaining high cell viability. Successful proof of concept experiments in transfection and intracellular labeling demonstrated potential to overcome the most prohibitive challenges in intracellular delivery for cell engineering.     

Mechanical consequences of cellular force generation

The discovery that adherent tissue cells actively sustain internal tension and exert mechanical forces on their surroundings has opened new vistas in the field of cell and tissue mechanics. Cellular forces, generated by acto-myosin contractility, play a central role in numerous aspects of cell behavior and function. Apart from the various specific functions that cells perform by applying forces (e.g., wound healing, remodeling of the extracellular matrix and muscle contraction), cells also apply stresses as a generic means for sensing and responding to the mechanical nature of their environment. In addition, the internal tension plays a role in actively controlling the elastic moduli and shape stability of the cell. In this review, we survey recent theoretical and experimental studies of the physical consequences of cell mechanical activity including its role in cell morphology, adhesion strength, stress-fiber polarization, and the elastic properties of cells. We also discuss the role of cell mechanics in orienting cellular assemblies and in the response of cells to external loads.     

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.     

Cell and molecular mechanics of biological materials

Living cells can sense mechanical forces and convert them into biological responses. Similarly, biological and biochemical signals are known to influence the abilities of cells to sense, generate and bear mechanical forces. Studies into the mechanics of single cells, subcellular components and biological molecules have rapidly evolved during the past decade with significant implications for biotechnology and human health. This progress has been facilitated by new capabilities for measuring forces and displacements with piconewton and nanometre resolutions, respectively, and by improvements in bio-imaging. Details of mechanical, chemical and biological interactions in cells remain elusive. However, the mechanical deformation of proteins and nucleic acids may provide key insights for understanding the changes in cellular structure, response and function under force, and offer new opportunities for the diagnosis and treatment of disease. This review discusses some basic features of the deformation of single cells and biomolecules, and examines opportunities for further research.     

High Throughput Screening of Cell Mechanical Response Using a Stretchable 3D Cellular Microarray Platform

Cells in vivo are constantly subjected to multiple microenvironmental mechanical stimuli that regulate cell function. Although 2D cell responses to the mechanical stimulation have been established, these methods lack relevance as physiological cell microenvironments are in 3D. Moreover, the existing platforms developed for studying the cell responses to mechanical cues in 3D either offer low‐throughput, involve complex fabrication, or do not allow combinatorial analysis of multiple cues. Considering this, a stretchable high‐throughput (HT) 3D cell microarray platform is presented that can apply dynamic mechanical strain to cells encapsulated in arrayed 3D microgels. The platform uses inkjet‐bioprinting technique for printing cell‐laden gelatin methacrylate (GelMA) microgel array on an elastic composite substrate that is periodically stretched. The developed platform is highly biocompatible and transfers the applied strain from the stretched substrate to the cells. The HT analysis is conducted to analyze cell mechano‐responses throughout the printed microgel array. Also, the combinatorial analysis of distinct cell behaviors is conducted for different GelMA microenvironmental stiffnesses in addition to the dynamic stretch. Considering its throughput and flexibility, the developed platform can readily be scaled up to introduce a wide range of microenvironmental cues and to screen the cell responses in a HT way.     

Microbuckling of fibrin provides a mechanism for cell mechanosensing

Biological cells sense and respond to mechanical forces, but how such a mechanosensing process takes place in a nonlinear inhomogeneous fibrous matrix remains unknown. We show that cells in a fibrous matrix induce deformation fields that propagate over a longer range than predicted by linear elasticity. Synthetic, linear elastic hydrogels used in many mechanotransduction studies fail to capture this effect. We develop a nonlinear microstructural finite-element model for a fibre network to simulate localized deformations induced by cells. The model captures measured cell-induced matrix displacements from experiments and identifies an important mechanism for long-range cell mechanosensing: loss of compression stiffness owing to microbuckling of individual fibres. We show evidence that cells sense each other through the formation of localized intercellular bands of tensile deformations caused by this mechanism.     

Probing Cell Deformability via Acoustically Actuated Bubbles

An acoustically actuated, bubble‐based technique is developed to investigate the deformability of cells suspended in microfluidic devices. A microsized bubble is generated by an optothermal effect near the targeted cells, which are suspended in a microfluidic chamber. Subsequently, acoustic actuation is employed to create localized acoustic streaming. In turn, the streaming flow results in hydrodynamic forces that deform the cells in situ. The deformability of the cells is indicative of their mechanical properties. The method in this study measures mechanical biomarkers from multiple cells in a single experiment, and it can be conveniently integrated with other bioanalysis and drug‐screening platforms. Using this technique, the mean deformability of tens of HeLa, HEK, and HUVEC cells is measured to distinguish their mechanical properties. HeLa cells are deformed upon treatment with Cytochalasin. The technique also reveals the deformability of each subpopulation in a mixed, heterogeneous cell sample by the use of both fluorescent markers and mechanical biomarkers. The technique in this study, apart from being relevant to cell biology, will also enable biophysical cellular diagnosis.     

Cell Encapsulation in Soft,Anisometric Poly(ethylene) Glycol Microgels Using a Novel Radical‐Free Microfluidic System

Complex 3D artificial tissue constructs are extensively investigated for tissue regeneration. Frequently, materials and cells are delivered separately without benefitting from the synergistic effect of combined administration. Cell delivery inside a material construct provides the cells with a supportive environment by presenting biochemical, mechanical, and structural signals to direct cell behavior. Conversely, the cell/material interaction is poorly understood at the micron scale and new systems are required to investigate the effect of micron‐scale features on cell functionality. Consequently, cells are encapsulated in microgels to avoid diffusion limitations of nutrients and waste and facilitate analysis techniques of single or collective cells. However, up to now, the production of soft cell‐loaded microgels by microfluidics is limited to spherical microgels. Here, a novel method is presented to produce monodisperse, anisometric poly(ethylene) glycol microgels to study cells inside an anisometric architecture. These microgels can potentially direct cell growth and can be injected as rod‐shaped mini‐tissues that further assemble into organized macroscopic and macroporous structures post‐injection. Their aspect ratios are adjusted with flow parameters, while mechanical and biochemical properties are altered by modifying the precursors. Encapsulated primary fibroblasts are viable and spread and migrate across the 3D microgel structure.     

Material forces for inelastic models at large strains: application to fracture mechanics

Elastomeric materials show a wide range of different elastic and inelastic properties. Additionally, this class of materials is subjected to large deformations. Considering all these effects, fracture mechanical investigations are very challenging tasks and cannot be performed with standard approaches. Effects of inhomogeneities and discontinuities such as cracks can be investigated with the so-called material force approach in an efficient and elegant way. For comprehensive investigations of inelastic materials, the complete balance of the material motion problem has to be formulated. In this case, the material volume forces depend on the internal history variables which are required for the inelastic constitutive model. This paper derives a general formulation for rate-dependent and rate-independent inelastic materials based on a multiplicative split of the deformation gradient to cover viscoelastic and elastoplastic materials at finite deformations.     

Hypersonic Poration: A New Versatile Cell Poration Method to Enhance Cellular Uptake Using a Piezoelectric Nano‐Electromechanical Device

Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for modern biotechnology. Herein, a new type of chemical‐free cell poration method—hypersonic poration—is developed to improve the cellular uptake, especially the nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric nano‐electromechanical resonator, which directly induces normal/shear stress and “molecular bombardment” effects on the bilayer membranes, and creates reversible temporal nanopores improving the membrane permeability. Both theory analysis and cellular uptake experiments of exogenous compounds prove the high delivery efficiency of hypersonic poration. Since target molecules in cells are accumulated with the treatment, the delivered amount can be controlled by tuning the treatment time. Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery at a confined spatial location and tunable arrays of the resonators that are compatible with multiwell plate can be achieved. The hypersonic poration method shows great delivery efficacy combined with advantage of scalability, tunable throughput, and simplification in operation and provides a potentially powerful strategy in the field of molecule delivery, cell transfection, and gene therapy.     

硫化橡胶动态力学性能的分数阶微分Kelvin模型

研究炭黑填充硫化橡胶的动态粘弹性,采用Gabo Eplexor 500N对材料进行不同频率时的温度扫描测试,得到材料玻璃化转变温度Tg随频率的变化规律。在Tg~Tg+50℃范围内进行不同温度的频率扫描测试,得到材料存储模量、损耗模量和损耗因子。采用分数阶微分Kelvin模型对动态粘弹特性进行分析,确定了模型参数。结果表明,分数阶微分Kelvin模型可以较好地描述材料在不同温度和较宽频率范围内的动态粘弹性力学行为。当温度高于Tg时,随着温度的升高,材料从Tg附近的粘弹态向高温时的橡胶态转变,模型中的微分阶数相应地逐渐减小。     

基材属性对铝蜂窝共面力学性能的影响

目的研究不同属性的基体材料对铝蜂窝共面压缩力学性能的影响。方法在保持正六边形蜂窝结构参数不变的情况下,改变基材属性,基体材料模型分别选择不同应变强化参数的双线性各向同性强化模型和理想弹塑性模型,建立相关可靠的有限元模型并进行大量的模拟计算。获得相应的变形模式和应力-应变曲线,对曲线进一步处理得到蜂窝共面静动态峰应力,并将结果以图表形式展示并分析。结果随着冲击速度的增加,样品依次出现了"X","V","一"字型3种变形模式,基体材料的应变强化效应使变形趋于均匀化;基体材料的应变强化效应显著增加了蜂窝的静态峰应力,对动态峰应力增量的影响可以忽略,对计算数据处理后得到了应变强化参数与动态峰应力的计算公式。结论基材具有强化特性的蜂窝,其共面静态力学性能优于基材为弹性理想塑性材料模型的蜂窝;在利用数值模拟的方法来研究蜂窝结构共面静态力学行为时,需要考虑基体材料的强化效应。     

Loss of Vimentin Enhances Cell Motility through Small Confining Spaces

The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F‐actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin‐driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild‐type and vimentin‐null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto‐myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.     

Viscoelastic and thermal behavior of woven hemp fiber reinforced poly(lactic acid) composites

This study examined the physical behavior of hemp/poly(lactic acid) (PLA) composites, particularly the thermal properties and viscoelastic behavior. Twill and plain woven hemp fabrics were used as reinforcements and hemp fabrics-reinforced PLA composites were produced using a film stacking method. The coefficient of thermal expansion of the composites decreased sharply with increasing the volume fraction of fiber. The twill structure was found to be suitable for reinforcing a PLA resin with higher impact strength and better mechanical properties than the plain woven. The viscoelastic properties of the composites including the storage modulus, loss modulus and loss tangent were also examined by dynamic mechanical analysis. In addition, morphological analysis was performed using scanning electron microscopy.     

混合非线性黏弹性阻尼器非线性特征与力学模型研究

随着黏弹性材料的不断发展,出现了具有更大耗能能力和变形能力的新型黏弹性阻尼器,但同时不可避免的丧失了其线性特征。该文基于一种混合非线性黏弹性阻尼器,揭示了其多种非线性特征的来源及规律,并基于此提出和验证了能够全面考虑其非线性特征的力学模型。结果表明,该种混合非线性黏弹性阻尼器的非线性来源主要包括五方面:相位差非线性引起滞回曲线形状的改变、初次加载大应变速率引起的初始刚度、升温效应和疲劳性能引起的软化、马林斯效应导致的大应变幅值下的软化和捏拢效应导致的大应变幅值下的硬化。提出的力学模型数学表达简洁,能够呈现其多种非线性因素和规律,无需在不同工况下分别进行参数识别,与试验结果吻合良好,能够精确模拟其滞回行为。     

An XFEM‐based numerical strategy to model mechanical interactions between biological cells and a deformable substrate

Contractile cells are known to constantly probe and respond to their mechanical environment through mechanosensing. Although the very mechanisms responsible for this behavior are still obscure, it is now clear that cells make full use of cross‐talks between mechanics, chemistry, and transport to organize their structure and generate forces. To investigate these processes, it is important to derive mathematical and numerical models that can accurately capture the interactions between cells and an underlying deformable substrate. The present paper therefore introduces a computational framework, based on the extended FEM (XFEM) and the level set method, to model the evolution of two‐dimensional (plane stress) cells lying on an elastic substrate whose properties can be varied. Cells are modeled with a continuum mixture approach previously developed by the authors to describe key phenomena of cell sensing, such as stress fiber formation, mechanosensitive contraction, and molecular transport whereas cell–substrate adhesion is formulated with a linear elastic cohesive model. From a numerical viewpoint, cell and substrate are discretized on a single, regular finite element mesh, whereas the potentially complex cell geometry is defined in terms of a level set function that is independent of discretization. Field discontinuities across the cell membrane are then naturally enforced using enriched shape functions traditionally used in the XFEM formulation. The resulting method provides a flexible platform that can handle complex cell geometries, can avoid expensive meshing techniques, and can potentially be extended to study cell growth and migration on an elastic substrate. In addition, the XFEM formalism facilitates the consideration of the cell's cortical elasticity, a feature that is known to be important during cell deformation. The proposed method is illustrated with a few biologically relevant examples of cell–substrate interactions. Generally, the method is able to capture some key phenomena observed in biological systems and displays numerical versatility and accuracy at a moderate computational cost.Copyright © 2012 John Wiley & Sons, Ltd.     

Mechanical and electronic-structure properties of compressed CdSe tetrapod nanocrystals

The coupling of mechanical and optical properties in semiconductor nanostructures can potentially lead to new types of devices. This work describes our theoretical examination of the mechanical properties of CdSe tetrapods under directional forces, such as may be induced by AFM tips. In addition to studying the general behavior of the mechanical properties under modifications of geometry, nanocrystal-substrate interaction, and dimensional scaling, our calculations indicate that mechanical deformations do not lead to large changes in the band-edge state eigenenergies, and have only a weak effect on the oscillator strengths of the lowest energy transitions.