Three-dimensional microfiber devices that mimic physiological environments to probe cell mechanics and signaling

Many physiological systems are regulated by cells that alter their behavior in response to changes in their biochemical and mechanical environment. These cells experience this dynamic environment through an endogenous biomaterial matrix that transmits mechanical force and permits chemical exchange with the surrounding tissue. As a result, in vitro systems that mimic three-dimensional, in vivo cellular environments can enable experiments that reveal the nuanced interplay between biomechanics and physiology. Here we report the development of a minimal-profile, three-dimensional (MP3D) experimental microdevice that confines cells to a single focal plane, while allowing the precise application of mechanical displacement to cells and concomitant access to the cell membrane for perfusion with biochemical agonists. The MP3D device--an ordered microfiber scaffold erected on glass--provides a cellular environment that induces physiological cell morphologies. Small manipulations of the scaffold's microfibers allow attached cells to be mechanically probed. Due to the scaffold's minimal height profile, MP3D devices confine cells to a single focal plane, facilitating observation with conventional epifluorescent microscopy. When examining fibroblasts within MP3D devices, we observed robust cellular calcium responses to both a chemical stimulus as well as mechanical displacement of the cell membrane. The observed response differed significantly from previously reported, mechanically-induced calcium responses in the same cell type. Our findings demonstrate a key link between environment, cell morphology, mechanics, and intracellular signal transduction. We anticipate that this device will broadly impact research in fields including biomaterials, tissue engineering, and biophysics.     

Chemical Physics in Living Cells-using Light to Visualize and Control Intracellular Signal Transduction?

Cells are crowded microenvironments filled with macromolecules undergoing constant physical and chemical interactions. The physicochemical makeup of the cells affects various cellular responses, determines cell-cell interactions and influences cell decisions. Chemical and physical properties differ between cells and within cells. Moreover, these properties are subject to dynamic changes in response to environmental signals, which often demand adjustments in the chemical or physical states of intracellular molecules. Indeed, cellular responses such as gene expression rely on the faithful relay of information from the outside to the inside of the cell, a process termed signal transduction. The signal often traverses a complex path across subcellular spaces with variable physical chemistry, sometimes even influencing it. Understanding the molecular states of such signaling molecules and their intracellular environments is vital to our understanding of the cell. Exploring such intricate spaces is possible today largely because of experimental and theoretical tools. Here, we focus on one tool that is commonly used in chemical physics studies-light. We summarize recent work which uses light to both visualize the cellular environment and also control intracellular processes along the axis of signal transduction. We highlight recent accomplishments in optical microscopy and optogenetics, an emerging experimental strategy which utilizes light to control the molecular processes in live cells. We believe that optogenetics lends unprecedented spatiotemporal precision to the manipulation of physicochemical properties in biological contexts. We hope to use this work to demonstrate new opportunities for chemical physicists who are interested in pursuing biological and biomedical questions.     

Mechanical stimulation of epithelial cells using polypyrrole microactuators

The importance of mechanotransduction for physiological systems is becoming increasingly recognized. The effect of mechanical stimulation is well studied in organs and tissues, for instance by using flexible tissue culture substrates that can be stretched by external means. However, on the cellular and subcellular level, dedicated technology to apply appropriate mechanical stimuli is limited. Here we report an organic electronic microactuator chip for mechanical stimulation of single cells. These chips are manufactured on silicon wafers using traditional microfabrication and photolithography techniques. The active unit of the chip consists of the electroactive polymer polypyrrole that expands upon the application of a low potential. The fact that polypyrrole can be activated in physiological electrolytes makes it well suited as the active material in a microactuator chip for biomedical applications. Renal epithelial cells, which are responsive to mechanical stimuli and relevant from a physiological perspective, are cultured on top of the microactuator chip. The cells exhibit good adhesion and spread along the surface of the chip. After culturing, individual cells are mechanically stimulated by electrical addressing of the microactuator chip and the response to this stimulation is monitored as an increase in intracellular Ca(2+). This Ca(2+) response is caused by an autocrine ATP signalling pathway associated with mechanical stimulation of the cells. In conclusion, the present work demonstrates a microactuator chip based on an organic conjugated polymer, for mechanical stimulation of biological systems at the cellular and sub-cellular level.     

Molecular Encryption and Reconfiguration for Remodeling of Dynamic Hydrogels

Dynamic materials have been widely studied for regulation of cell adhesion that is important to a variety of biological and biomedical applications. These materials can undergo changes mainly through one of the two mechanisms: ligand release in response to chemical, physical, or biological stimuli, and ligand burial in response to mechanical stretching or the change of electrical potential. This study demonstrates an encrypted ligand and a new hydrogel that are capable of inducing and inhibiting cell adhesion, which is controlled by molecular reconfiguration. The ligand initially exhibits an inert state; it can be reconfigured into active and inert states by using unblocking and recovering molecules in physiological conditions. Since molecular reconfiguration does not require the release of the ligand from the hydrogels, inhibiting and inducing cell adhesion on the hydrogels can be repeated for multiple cycles.     

Polyelectrolyte multilayers with a tunable Young's modulus: influence of film stiffness on cell adhesion

Mechanical properties of model and natural gels have recently been demonstrated to play an important role in various cellular processes such as adhesion, proliferation, and differentiation, besides events triggered by chemical ligands. Understanding the biomaterial/cell interface is particularly important in many tissue engineering applications and in implant surgery. One of the final goals would be to control cellular processes precisely at the biomaterial surface and to guide tissue regeneration. In this work, we investigate the substrate mechanical effect on cell adhesion for thin polyelectrolyte multilayer (PEM) films, which can be easily deposited on any type of material. The films were cross linked by means of a water-soluble carbodiimide (EDC), and the film elastic modulus was determined using the AFM nanoindentation technique with a colloidal probe. The Young's modulus could be varied over 2 orders of magnitude (from 3 to 400 kPa) for wet poly(L-lysine)/hyaluronan (PLL/HA) films by changing the EDC concentration. The chemical changes upon cross linking were characterized by means of Fourier transform infrared spectroscopy (FTIR). We demonstrated that the adhesion and spreading of human chondrosarcoma cells directly depend on the Young's modulus. These data indicate that, besides the chemical properties of the polyelectrolytes, the substrate mechanics of PEM films is an important parameter influencing cell adhesion and that PEM offer a new way to prepare thin films of tunable mechanical properties with large potential biomedical applications including drug release.     

High‐Frequency Mechanostimulation of Cell Adhesion

Cell adhesion is regulated by molecularly defined protein interactions and by mechanical forces, which can activate a dynamic restructuring of adhesion sites. Previous attempts to explore the response of cell adhesion to forces have been limited to applying mechanical stimuli that involve the cytoskeleton. In contrast, we here apply a new, oscillatory type of stimulus through push–pull azobenzenes. Push–pull azobenzenes perform a high‐frequency, molecular oscillation upon irradiation with visible light that has frequently been applied in polymer surface relief grating. We here use these oscillations to address single adhesion receptors. The effect of molecular oscillatory forces on cell adhesion has been analyzed using single‐cell force spectroscopy and gene expression studies. Our experiments demonstrate a reinforcement of cell adhesion as well as upregulated expression levels of adhesion‐associated genes as a result of the nanoscale “tickling” of integrins. This novel type of mechanical stimulus provides a previously unprecedented molecular control of cellular mechanosensing.     

A biological breadboard platform for cell adhesion and detachment studies

The dynamic nature of cell adhesion and detachment, which plays a critical role in a variety of physiological and pathological phenomena, still remains unclear. This motivates the pursuit of controllable manipulation of cell adhesion and detachment for a better understanding of cellular dynamics. Here we present an addressable, multifunctional, and reusable platform, termed the biological breadboard (BBB), for spatiotemporal manipulation of cell adhesion and detachment at cellular and subcellular levels. The BBB, composed of multiple gold electrodes patterned on a Pyrex substrate, is surface-modified with arginine-glycine-aspartic acid terminated thiol (RTT) and polyethylene glycol (PEG) to achieve a cell-adhesive surface on the gold electrodes and a cell-resistive surface on the Pyrex substrate, respectively. Cell adhesion is regulated by the steric repulsion of PEG chains, while cell detachment is controlled by the reductive desorption of a gold-thiol self-assembled monolayer (SAM) at an activation potential of -0.90 to -1.65 V. Experimental characterizations using NIH 3T3 fibroblasts are presented to demonstrate the utility of our device.     

Cellular immobilization within microfluidic microenvironments: dielectrophoresis with polyelectrolyte multilayers

The development of biomimetic microenvironments will improve cell culture techniques by enabling in vitro cell cultures that mimic in vivo behavior; however, experimental control over attachment, cellular position, or intercellular distances within such microenvironments remains challenging. We report here the rapid and controllable immobilization of suspended mammalian cells within microfabricated environments using a combination of electronic (dielectrophoresis, DEP) and chemical (polyelectrolyte multilayers, PEMS) forces. While cellular position within the microsystem is rapidly patterned via intermittent DEP trapping, persistent adhesion after removal of electronic forces is enabled by surface treatment with PEMS that are amenable to cellular attachment. In contrast to DEP trapping alone, persistent adhesion enables the soluble microenvironment to be systematically varied, facilitating the use of soluble probes of cell state and enabling cellular characterization in response to various soluble stimuli.     

High-throughput tracking of single yeast cells in a microfluidic imaging matrix

Time-lapse live cell imaging is a powerful tool for studying signaling network dynamics and complexity and is uniquely suited to single cell studies of response dynamics, noise, and heritable differences. Although conventional imaging formats have the temporal and spatial resolution needed for such studies, they do not provide the simultaneous advantages of cell tracking, experimental throughput, and precise chemical control. This is particularly problematic for system-level studies using non-adherent model organisms such as yeast, where the motion of cells complicates tracking and where large-scale analysis under a variety of genetic and chemical perturbations is desired. We present here a high-throughput microfluidic imaging system capable of tracking single cells over multiple generations in 128 simultaneous experiments with programmable and precise chemical control. High-resolution imaging and robust cell tracking are achieved through immobilization of yeast cells using a combination of mechanical clamping and polymerization in an agarose gel. The channel and valve architecture of our device allows for the formation of a matrix of 128 integrated agarose gel pads, each allowing for an independent imaging experiment with fully programmable medium exchange via diffusion. We demonstrate our system in the combinatorial and quantitative analysis of the yeast pheromone signaling response across 8 genotypes and 16 conditions, and show that lineage-dependent effects contribute to observed variability at stimulation conditions near the critical threshold for cellular decision making.     

Stress induced plant resistance and enzyme activity varying in cucumber

When pathogens penetrate plant cells, some chemical secretions are elicited, and the mechanical signals in plant cell may be induced by the simultaneous physical pressure to change. Based on the previous cognitions, we investigated the plant resistance and the variation of anti-disease enzyme activity in cucumber leaves after mechanical stress loading. Results showed that the appropriate mechanical stimulation could significantly improve plant resistance and alter the activity of phenylalanine ammonial lyases (PAL) and POD, leading to synthesis of lignin. However, we found that the effects of the stress on these cellular fundamental events were eliminated when the adhesion between plasma membrane and cell wall was disrupted. We speculated that mechanical signal transduction in plants depend on the adhesion of plasma membrane–cell wall.     

Nanoscale topography reduces fibroblast growth, focal adhesion size and migration-related gene expression on platinum surfaces

Controlling cellular responses on biomaterial surfaces is crucial in biomedical applications such as tissue engineering and implantable prosthetics. Since cells encounter various nanoscale topographic features in their natural environment, it has been postulated that surface nanotopography may be an alternative route to fabricate biomaterials with a desirable cellular response. In this framework, we investigated the responses of primary human fibroblasts to platinum substrates with different levels of surface roughness at the nanoscale. The nanorough surfaces were fabricated by using the glancing angle deposition technique (GLAD). We found that levels of cellular responses depended on the surface roughness and the size of the nanoscale features. We showed that in response to nanotopography cells spread less and have an elongated morphology, displaying signs of actin cytoskeleton impairment and reduced formation of focal adhesion complexes. Although cell growth and adhesion were impaired on the nanorough substrates, cell viability was not affected by topography. To a minor extent our results also indicate that cell migration might be reduced on the nanorough surfaces, since a significantly lower gene expression of migration related genes were found on the roughest surfaces as compared to the flat reference. The results presented here demonstrate that surface nanotopography influences fibroblasts responses on platinum, which may be used to reduce cellular adhesion on platinum implant surfaces such as implantable neural electrodes.     

Monitoring the status of T-cell activation in a microfluidic system

Leukocyte adhesion to the endothelium through surface molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1) is a critical cellular event reflecting the physiological status of both cell types. Here we present a microfluidic system that can not only easily monitor the interaction between leukocytes and endothelial cells under physiological conditions, but also screen drug candidates for potential modulation of this interaction. Shear stress, which is an important factor for the binding of activated T cells to tumor necrosis factor-alpha (TNF-α)-treated human umbilical vein endothelial cells (HUVECs), was easily controlled by adjusting the flow rate in the microfluidic system. Whole blood of patients with systemic lupus erythematosus (SLE) who have auto-reactive T cells were infused into the activated HUVECs which subsequently showed a higher level of binding compared to a control blood sample from a person without SLE. When these autoreactive T cells were treated with immunosuppressors tacrolimus and cyclosporin A, the binding of the T cells to HUVECs was dramatically decreased. Therefore, this microfluidic system is capable of differentiating the physiological status of T cells or endothelial cells representing different disease conditions, as well as being useful for the identification of novel reagents that modulate the functions of leukocytes or endothelial cells.     

Chemically programmed cell adhesion with membrane-anchored oligonucleotides

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane-anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, and tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells that are chemically immobilized on a passivated glass surface.     

Organs on microfluidic chips: A mini review

Microfluidic technology provides opportunities to create in vitro models with physiological microenvironment for cell study. Introducing the identified key aspects, including tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical forces, of living organs into the microfluidic system, “organs-on-chips” display an unprecedented application potential in a lot of biological fields such as fundamental physiological and pathophysiological research, drug efficacy and toxicity testing, and clinical diagnosis. Here, we review the recent development of organs-on-chips and briefly discuss their future challenges.     

Evaluation of substrata effect on cell adhesion properties using freestanding poly(L-lactic acid) nanosheets

Investigation of the interactions between cells and material surfaces is important not only for the understanding of cell biology but also for the development of smart biomaterials. In this study, we investigated the substrate-related effects on the interaction between cell and polymeric ultrathin film (nanosheet) by modulating the mechanical properties of the nanosheet with a metal substrate or mesh. A freestanding polymeric nanosheet with tens-of-nanometers thickness composed of poly(L-lactic acid) (PLLA nanosheet) was fabricated by combination of a spin-coating technique and a water-soluble sacrificial layer. The freestanding PLLA nanosheet was collected on a stainless steel mesh (PLLA-mesh) and subsequently used for cell adhesion studies, comparing the results to the ones on a control SiO(2) substrate coated with an ultrathin layer of PLLA (PLLA-substrate). The adhesion of rat cardiomyocytes (H9c2) was evaluated on both samples after 24 h of culture. The PLLA-mesh with the tens-of-nanometers thick nanosheets induced an anisotropic adhesion of H9c2, while H9c2 on the PLLA-substrate showed an isotropic adhesion independent from the nanosheet thickness. Interestingly, an increment in the nanosheet thickness in the PLLA-mesh samples reduced the cellular anisotropy and led to a similar morphology to the PLLA-substrate. Considering the huge discrepancy of Young's modulus between PLLA nanosheet (3.5-4.2 GPa) and metal substrate (hundreds of GPa), cell adhesion was mechanically regulated by the Young's modulus of the underlying substrate when the thickness of the PLLA nanosheet was tens of nanometers. Modulation of the stiffness of the polymeric nanosheet by utilizing a rigid underlying material will allow the constitution of a unique cell culture environment.     

Optical and Electrochemical Probes for Monitoring Cytochrome c in Subcellular Compartments During Apoptosis

Cytochrome c (Cyt. c) is a key initiator of the caspases that activate cell apoptosis. The spatiotemporal evaluation of the contents of Cyt. c in cellular compartments and the detection of Cyt. c delivery between cellular compartments upon apoptosis is important for probing cell viabilities. We introduce an optical probe and an electrochemical probe for the quantitative assessment of Cyt. c in cellular compartments at the single cell level. The optical or electrochemical probes are functionalized with photoresponsive o-nitrobenzylphosphate ester-caged Cyt. c aptamer constituents. These are uncaged by light stimuli at single cell compartments, allowing the spatiotemporal detection of Cyt. c through the formation of Cyt. c/aptamer complexes at non-apoptotic or apoptotic conditions. The probes are applied to distinguish the contents of Cyt. c in cellular compartments of epithelial MCF-10A breast cells and malignant MCF-7 and MDA-MB-231 breast cells under apoptotic/non-apoptotic conditions.     

Real-time characterization of cytotoxicity using single-cell impedance monitoring

Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.     

纳米粒子与细胞相互作用的力学-化学偶联研究进展

纳米粒子在生物医学和大气环境领域的广泛研究使得其生物安全性越来越受到重视。目前已经有许多研究关注纳米粒子与细胞的相互作用及细胞毒性问题。本综述从细胞力学-化学偶联的角度总结了近五年来有关纳米粒子与细胞相互作用的研究进展。首先介绍了与细胞力学-化学偶联性质相关的分子基础以及目前检测细胞机械性质的纳米技术,然后重点讨论了纳米粒子对细胞粘附、骨架、刚度和迁移性质的影响。在此基础上,进一步指出了纳米生物力学-化学偶联的挑战与展望。     

Single cell 3-D platform to study ligand mobility in cell-cell contact

Lateral mobility and dimensionality have both been shown to influence cellular behavior, but have yet to be combined and applied in a single in vitro platform to address, e.g., cell adhesion in a setting mimicking the three-dimensional environment of neighboring cells in a reductionist way. To study the effect of the lateral mobility of cell adhesive ligands in three dimensions we present and characterize a platform, which enables patterning of single cells into microwells presenting a cell membrane mimetic interface pre-patterned to its walls. Soluble E-cadherin extracellular domains coupled through an optimized streptavidin-antibody linkage to lipids in a supported lipid bilayer (SPB) were presented on the microwell walls as either laterally mobile or immobile ligands. The fluidity was controlled through a small change in temperature by choosing phospholipids for the SPB with a lipid phase transition temperature around 30 °C. The platform thus enabled the investigation of cell adhesion to either laterally immobile or mobile E-cadherin ligands presented on the same cell membrane mimetic surface. Chinese hamster ovary (CHO) cells engineered to express E-cadherin that were cultured on the platform demonstrated that enhanced cadherin lateral mobility significantly decreased the formation of actin bundles and resulted in more diffuse actin organization, while constraining the cell shape to that of the microwell. This example highlights the potential to use in vitro cell culture platforms to mimic direct cell-cell interaction in a controlled environment that nevertheless captures the dynamic nature of the native cell environment.     

Ion implantation into collagen-coated surfaces for the development of small diameter artificial grafts

Ion implantation into collagen (Type I) coated inner surfaces of test tubes with a length of 50 mm and an inner diameter of 2 and 3 mm were performed to develop hybrid type small diameter artificial vascular grafts. To obtain information about the cellular response and chemical and physical structure of those collagen surfaces, several experiments such as platelets adhesion test, endothelial cell culture, analysis of amino acids and animal study were performed. He(+) ion implanted collagen coated specimen exhibited cell attachment and inhibit platelet adhesion. From these results, it was assumed that He(+) ions broke the ligands that correspond to platelet, and the ligands that correspond to endothelial cell adhesion still existed after ion implantation. It was suggested that platelets and cell attachment could be control individually by ion implantation into collagen.