A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response

External forces are increasingly recognized as major regulators of cellular structure and function, yet the underlying mechanism by which cells sense forces and transduce them into intracellular biochemical signals and behavioral responses ('mechanotransduction') is largely undetermined. To aid in the mechanistic study of mechanotransduction, herein we devised a cell stretching device that allowed for quantitative control and real-time measurement of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached to the microposts. Using the mPAM, we studied the live-cell subcellular dynamic responses of contractile forces in vascular smooth muscle cells (VSMCs) to a sustained static equibiaxial cell stretch. Our data showed that in response to a sustained cell stretch, VSMCs regulated their cytoskeletal (CSK) contractility in a biphasic manner: they first acutely enhanced their contraction to resist rapid cell deformation ('stiffening') before they allowed slow adaptive inelastic CSK reorganization to release their contractility ('softening'). The contractile response across entire single VSMCs was spatially inhomogeneous and force-dependent. Our mPAM device and live-cell subcellular contractile measurements will help elucidate the mechanotransductive system in VSMCs and thus contribute to our understanding of pressure-induced vascular disease processes.     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

Engineering of a microfluidic cell culture platform embedded with nanoscale features

Cells residing in a microenvironment interact with the extracellular matrix (ECM) and neighboring cells. The ECM built from biomacromolecules often includes nanotopography. Through the ECM, interstitial flows facilitate transport of nutrients and play an important role in tissue maintenance and pathobiology. To create a microenvironment that can incorporate both nanotopography and flow for studies of cell-matrix interactions, we fabricated microfluidic channels endowed with nanopatterns suitable for dynamic culture. Using polymer thin film technology, we developed a versatile stitching technique to generate a large area of nanopatterned surface and a simple microtransfer assembly technique to assemble polydimethylsiloxane-based microfluidics. The cellular study showed that both nanotopography and fluid shear stress played a significant role in adhesion, spreading, and migration of human mesenchymal stem cells. The orientation and deformation of cytoskeleton and nuclei were regulated through the interplay of these two cues. The nanostructured microfluidic platform provides a useful tool to promote the fundamental understanding of cell-matrix interactions and may be used to regulate the fate of stem cells.     

A microfluidic flow-stretch chip for investigating blood vessel biomechanics

This microfluidic flow-stretch chip integrates fluid shear stress (FSS) and cyclic stretch (CS), two major mechanical stimulations in cardiovascular systems, for cultured cells. The model chip can deliver FSS and CS simultaneously or independently to vascular cells to mimic the haemodynamic microenvironment of blood vessels in vivo. By imposing FSS-only, CS-only, and FSS+CS stimulation on rat mesenchymal stem cells and human umbilical vein endothelial cells, we found the alignment of the cellular stress fibers varied with cell type and the type of stimulation. The flow-stretch chip is a reliable tool for simulating the haemodynamic microenvironment.     

Traction force microscopy on-chip: shear deformation of fibroblast cells

We develop here a microfabrication compatible force measurement technique termed as ultrasoft polydimethylsiloxane-based traction force microscopy (UPTFM). This technique is devised for mapping the cellular traction forces imparted on the adhering substrate, so as to depict the physiological state of the cells surviving in the micro-confinement. We subsequently integrate the technique with a microfluidic platform for evaluating different states of stress in adherent mouse skin fibroblast L929 cells. Utilizing this technique, we monitor the spatio-temporal evolution of cellular traction forces for static incubation periods with no media replenishment as well as for dynamic flow conditions that inherently induce cell deformation and detachment. While the studies conducted on a quiescent fluid medium enable us to obtain an optimal static cell incubation period, those executed under dynamic flow conditions provide us with the minuscule details of the cellular response, deformation and detachment processes. We elucidate the correlation between shear activated cytosolic calcium ion release profile and the local traction forces as an attempt to apply UPTFM in the domain of functional biological purposes. Pertinently, we map the centroidal displacement and the maximum traction stress in characterizing the critical shear rate conditions for the onset of the cell peeling-off process, and demonstrate their contrasting features in comparison to the vesicle lift off processes in a shear flow. Theoretically, these deviations can only be explained by taking physiologically relevant cell adhesion models into consideration, which, while retaining the intrinsic simplicity, are able to reproduce the key experimental outcomes at least with qualitative agreement. We execute further theoretical investigations with variable magnitudes of membrane stiffness, viscosity and adhesion strength, so as to come up with interesting biophysical confluences.     

Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.     

A passive pump-assisted microfluidic assay for quantifying endothelial wound healing in response to fluid shear stress

There as an urgent need to quantify the endothelial wound-healing process in response to fluid shear stress to improve the biological and clinical understanding of healing mechanisms, which is of great importance for preventing healing impairment, chronic wounds, and postoperative in-stent restenosis. However, current experimental platforms not only require expensive, cumbersome, and powered pumping devices (to, e.g., generate cell scratches and load shear stress stimulation) but also lack quantitative controls for quantitative analysis. In this paper, a passive pump-assisted microfluidic assay is developed to quantify endothelial wound healing in response to fluid shear stress. Our assay consists of passive constant-flow pumps based on the siphon principle and a three-inlet microfluidic chip for cell wound-healing experiments. We also propose a method for quantitatively adjusting cell scratch size by controlling trypsin flow. Both numerical simulations and fluorescein experiments validate the effectiveness of this method. Moreover, we use the designed microfluidic assay to successfully generate cell scratches, load a 12-h shear stress of 5 dyn/cm² to the cells, and observe wound healing. The results indicate that the healing of a cell scratch is significantly accelerated under the stimulation of shear stress. In conclusion, our passive pump-assisted microfluidic assay shows versatility, applicability, and the potential for quantifying endothelial wound healing in response to fluid shear stress.     

Optical and electrochemical detection techniques for cell-based microfluidic systems

The ability to fabricate microfluidic systems with complex structures and with compatible dimensions between the microfluidics and biological cells have attracted significant attention in the development of microchips for analyzing the biophysical and biochemical functions of cells. Just as cell-based microfluidics have become a versatile tool for biosensing, diagnostics, drug screening and biological research, detector modules for cell-based microfluidics have also undergone major development over the past decade. This review focuses on detection methods commonly used in cell-based microfluidic systems, and provides a general survey and an in-depth look at recent developments in optical and electrochemical detection methods for microfluidic applications for biological systems, particularly cell analysis. Selected examples are used to illustrate applications of these detection systems and their advantages and weaknesses.     

UV-modulated substrate rigidity for multiscale study of mechanoresponsive cellular behaviors

Mechanical properties of the extracellular matrix (ECM) have profound effects on cellular functions. Here, we applied novel photosensitive polydimethylsiloxane (photoPDMS) chemistry to create photosensitive, biocompatible photoPDMS as a rigidity-tunable material for study of mechanoresponsive cellular behaviors. By modulating the PDMS cross-linker to monomer ratio, UV light exposure time, and postexposure baking time, we achieved a broad range of bulk Young's modulus for photoPDMS from 0.027 to 2.48 MPa. Biocompatibility of photoPDMS was assayed, and no significant cytotoxic effect was detected as compared to conventional PDMS. We demonstrated that the bulk Young's modulus of photoPDMS could impact cell morphology, adhesion formation, cytoskeletal structure, and cell proliferation. We further fabricated photoPDMS micropost arrays for multiscale study of mechanoresponsive cellular behaviors. Our results suggested that adherent cells could sense and respond to changes of substrate rigidity at a subfocal adhesion resolution. Together, we demonstrated the potential of photoPDMS as a photosensitive and rigidity-tunable material for mechanobiology studies.     

Rapid spatial and temporal controlled signal delivery over large cell culture areas

Controlled chemical delivery in microfluidic cell culture devices often relies on slowly evolving diffusive gradients, as the spatial and temporal control provided by fluid flow results in significant cell-perturbation. In this paper we introduce a microfluidic device architecture that allows for rapid spatial and temporal soluble signal delivery over large cell culture areas without fluid flow over the cells. In these devices the cell culture well is divided from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. This configuration requires chemical signals in the microchannel to only diffuse through the thin membrane into large cell culture area, rather than diffuse in from the sides. The spatial chemical pattern within the microfluidic channel was rapidly transferred to the cell culture area with good fidelity through diffusion. The cellular temporal response to a step-function signal showed that dye reached the cell culture surface within 45 s, and achieved a static concentration in under 6 min. Chemical pulses of less than one minute were possible by temporally alternating the signal within the microfluidic channel, enabling rapid flow-free chemical microenvironment control for large cell culture areas.     

Microfluidic platforms for lab-on-a-chip applications

We review microfluidic platforms that enable the miniaturization, integration and automation of biochemical assays. Nowadays nearly an unmanageable variety of alternative approaches exists that can do this in principle. Here we focus on those kinds of platforms only that allow performance of a set of microfluidic functions--defined as microfluidic unit operations-which can be easily combined within a well defined and consistent fabrication technology to implement application specific biochemical assays in an easy, flexible and ideally monolithically way. The microfluidic platforms discussed in the following are capillary test strips, also known as lateral flow assays, the "microfluidic large scale integration" approach, centrifugal microfluidics, the electrokinetic platform, pressure driven droplet based microfluidics, electrowetting based microfluidics, SAW driven microfluidics and, last but not least, "free scalable non-contact dispensing". The microfluidic unit operations discussed within those platforms are fluid transport, metering, mixing, switching, incubation, separation, droplet formation, droplet splitting, nL and pL dispensing, and detection.     

微流控芯片流体剪切力和肿瘤坏死因子-α共同作用对大鼠软骨细胞表型的影响

流体剪切力是生物体内普遍存在的一种生物力学形式,是细胞微环境的重要组成部分,对细胞多种生物学行为有重要调节作用。该研究以微流控芯片技术为基础,建立了一种基于流阻原理能同时产生4个不同大小流体剪切力的微流控芯片平台,用以研究低流速的流体剪切力对大鼠原代软骨细胞表型维持的影响。结果表明,流体剪切力可促进软骨细胞的表型维持。还加入了肿瘤坏死因子-α(TNF-α),考察流体剪切力和TNF-α共同作用对软骨细胞表型的影响。结果表明,在剪切力和TNF-α共同作用下,软骨细胞的Ⅱ型胶原和蛋白多糖表达明显下调。该研究为软骨组织工程和骨性关节炎的疾病研究提供有力的研究平台,为骨关节疾病治疗和防治提供了理论依据。     

A practical guide to microfluidic perfusion culture of adherent mammalian cells

Culturing cells at microscales allows control over microenvironmental cues, such as cell-cell and cell-matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.     

Microfluidic applications on circulating tumor cell isolation and biomimicking of cancer metastasis

The prognosis of malignant tumors is challenged by insufficient means to effectively detect tumors at early stage. Liquid biopsy using circulating tumor cells (CTCs) as biomarkers demonstrates a promising solution to tackle the challenge, because CTCs play a critical role in cancer metastatic process via intravasation, circulation, extravasation, and formation of secondary tumor. However, the effectiveness of the solution is compromised by rarity, heterogeneity, and vulnerability associated with CTCs. Among a plethora of novel approaches for CTC isolation and enrichment, microfluidics leads to isolation and detection of CTCs in a cost-effective and operation-friendly way. Development of microfluidics also makes it feasible to model the cancer metastasis in vitro using a microfluidic system to mimick the in vivo microenvironment, thereby enabling analysis and monitor of tumor metastasis. This paper aims to review the latest advances for exploring the dual-roles microfluidics has played in early cancer diagnosis via CTC isolation and investigating the role of CTCs in cancer metastasis; the merits and drawbacks for dominating microfluidics-based CTC isolation methods are discussed; biomimicking cancer metastasis using microfluidics are presented with example applications on modelling of tumor microenvironment, tumor cell dissemination, tumor migration, and tumor angiogenesis. The future perspectives and challenges are discussed.     

Patterning chemical stimulation of reconstructed neuronal networks

A spatially resolved delivery of substances integrated with cell culture substrates shows promise for application in pharmacological assays, bioanalytical studies on cell signaling pathways and cell-based biosensors, where control over the extracellular biochemical environment with a cellular resolution is desirable. In this work, we studied a biohybrid system where rat embryonic cortical neuronal networks are reconstructed on microstructured silicon chips and interfaced to microfluidics. The design of cell-cell and cell-medium interactions in confined geometries is presented. We developed an aligned microcontact printing technique (AμCP) for poly(lysine)-extracellular matrix proteins on microstructured chips, which allows a high degree of geometrical control over the network architecture and alignment of the neuronal network with the microfluidic features of a substrate. Spatially resolved on-chip delivery of compounds with a cellular resolution is demonstrated by chemical stimulation of patterned rat cortical neurons within a network with a number of solutions of excitatory neurotransmitter glutamate delivered via microfluidics. The combination of the system described with a patch-clamp technique allowed both modulation of the biochemical environment on a cellular level and the monitoring of electrophysiological properties in the reconstructed rat embryonic cortical networks changed by this microenvironment.     

Acoustofluidics 1: Governing equations in microfluidics

In Part 1 of the thematic tutorial series "Acoustofluidics--exploiting ultrasonic standing waves forces and acoustic streaming in microfluidic systems for cell and particle manipulation", we establish the governing equations in microfluidics. Examples of basic flow solutions are presented, and equivalent circuit modeling for determining flow rates in microfluidic networks is introduced.     

Mechanics of cell spreading within 3D-micropatterned environments

Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.     

Single-cell metabolite analysis on a microfluidic chip

Metabolites can directly reflect and modulate cell responses and phenotypical changes by influencing energy balances, intercellular signals, and many other cellular functions throughout the lifespan of cells.Taking into account the heterogeneity of cells, single-cell metabolite analysis offers an insight into the functional process within one cell. Microfluidics as a powerful tool has attracted significant interest in the single-cell metabolite analysis field. The microfluidic platform is possib...     

Immunoassays in microfluidic systems

Immunoassays have greatly benefited from miniaturization in microfluidic systems. This review, which summarizes developments in microfluidics-based immunoassays since 2000, includes four sections, focusing on the configurations of immunoassays that have been implemented in microfluidics, the main fluid handling modalities that have been used for microfluidic immunoassays, multiplexed immunoassays in microfluidic platforms, and the emergence of label-free detection techniques. The field of microfluidic immunoassays is continuously improving and has great promise for the future.     

Acoustofluidic platforms for particle manipulation

There is an increasing interest in acoustics for microfluidic applications. This field, commonly known as acoustofluidics involves the interaction of ultrasonic standing waves with fluids and dispersed microparticles. The combination of microfluidics and the so-called acoustic standing waves (ASWs) led to the development of integrated systems for contact-less on-chip cell and particle manipulation where it is possible to move and spatially localize these particles based on the different acoustophysical properties. While it was initially suggested that the acoustic forces could be harmful to the cells and could impact cell viability, proliferation, or function via phenotypic or even genotypic changes, further studies disproved such claims. This review is summarizing some interesting applications of acoustofluidics in the manipulations of biomaterials, such as cells or subcellular vesicles, in works published mainly within the last 5 years.