Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).     

Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons

Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.     

Multiphase electropatterning of cells and biomaterials

Tissues formed by cells encapsulated in hydrogels have uses in biotechnology, cell-based assays, and tissue engineering. We have previously presented a 3D micropatterning technique that rapidly localizes live cells within hydrogels using dielectrophoretic (DEP) forces, and have demonstrated the ability to modulate tissue function through the control of microscale cell architecture. A limitation of this method is the requirement that a single biomaterial must simultaneously harbor biological properties that support cell survival and function and material properties that permit efficient dielectrophoretic patterning. Here, we resolve this issue by forming multiphase tissues consisting of microscale tissue sub-units in a 'local phase' biomaterial, which, in turn, are organized by DEP forces in a separate, mechanically supportive 'bulk phase' material. We first define the effects of medium conductivity on the speed and quality of DEP cell patterning. As a case study, we then produce multiphase tissues with microscale architecture that combine high local hydrogel conductivity for enhanced survival of sensitive liver progenitor cells with low bulk conductivity required for efficient DEP micropatterning. This approach enables an expanded range of studies examining the influence of 3D cellular architecture on diverse cell types, and in the future may improve the biological function of inhomogeneous tissues assembled from a variety of modular tissue sub-units.     

二维平面细胞微图案化技术及其生物学应用

可以控制细胞粘附形状、大小的方法统称为细胞图案化技术.这些方法结合微纳米制备、表面化学、电化学和光化学等手段可以动态控制细胞的粘附、迁移、分化及其相互作用,为细胞生物学研究提供了一个新平台.本文介绍了二维平面细胞图案化的各种方法,并对其优缺点进行了总结,评述了细胞图案化技术在细胞生物学基础研究、组织工程以及基于细胞的生物传感器领域的应用.     

A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels

In tissues, cell microenvironment geometry and mechanics strongly impact on cell physiology. Surface micropatterning allows the control of geometry while deformable substrates of tunable stiffness are well suited for the control of the mechanics. We developed a new method to micropattern extracellular matrix proteins on poly-acrylamide gels in order to simultaneously control cell geometry and mechanics. Microenvironment geometry and mechanics impinge on cell functions by regulating the development of intra-cellular forces. We measured these forces in micropatterned cells. Micropattern geometry was streamlined to orient forces and place cells in comparable conditions. Thereby force measurement method could be simplified and applied to large-scale experiment on chip. We applied this method to mammary epithelial cells with traction force measurements in various conditions to mimic tumoral transformation. We found that, contrary to the current view, all transformation phenotypes were not always associated to an increased level of cell contractility.     

Loss of epithelial polarity is accompanied by differential association of proteins with intracellular membranes

Cellular membranes play an important role in the formation and maintenance of epithelial polarity, which is lost early during carcinogenesis. We set out to identify membrane proteins which are altered during loss of cell polarity in mammary epithelium. As a model system we used murine mammary epithelial cells expressing the conditional oncoprotein c-JunER, which induces a reversible loss of polarity upon beta-estradiol-driven activation [1]. When grown either in the absence or presence of hormone, these cells exhibit a polarized or unpolarized phenotype, respectively. Different membrane fractions of polarized or unpolarized cells were analyzed by two-dimensional electrophoresis (2-DE) and differentially expressed membrane proteins were identified. To distinguish between transmembrane orientation and peripheral attachment of these proteins, were performed extractions with carbonate at high pH or with Triton X-114. In addition, cytosolic proteins of both states were analyzed to investigate their differential association with distinct membrane fractions. We found ten protein spots preferentially or exclusively in polarized cells and 17 other proteins as being upregulated during loss of polarity. Some of the peripheral membrane proteins were identified by microsequencing. The resident Golgi protein nucleobindin and fructose-bisphosphate aldolase were preferentially associated with membranes of polarized cells, whereas alphaB crystallin was detected exclusively and in high amounts in unpolarized cells.     

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.     

Facile cell patterning on an albumin-coated surface

Fabrication of micropatterned surfaces to organize and control cell adhesion and proliferation is an indispensable technique for cell-based technologies. Although several successful strategies for creating cellular micropatterns on substrates have been demonstrated, a complex multistep process and requirements for special and expensive equipment or materials limit their prevalence as a general experimental tool. To circumvent these problems, we describe here a novel facile fabrication method for a micropatterned surface for cell patterning by utilizing the UV-induced conversion of the cell adhesive property of albumin, which is the most abundant protein in blood plasma. An albumin-coated surface was prepared by cross-linking albumin with ethylene glycol diglycidyl ether and subsequent casting of the cross-linked albumin solution on the cell culture dish. While cells did not attach to the albumin surface prepared in this way, UV exposure renders the surface cell-adhesive. Thus, surface micropatterning was achieved simply by exposing the albumin-coated surface to UV light through a mask with the desired pattern. Mouse fibroblast L929 cells were inoculated on the patterned albumin substrates, and cells attached and spread in a highly selective manner according to the UV-irradiated pattern. Although detailed investigation of the molecular-level mechanism concerning the change in cell adhesiveness of the albumin-coated surface is required, the present results would give a novel facile method for the fabrication of cell micropatterned surfaces.     

Detection of non‐small cell lung cancer cells based on microfluidic polarization microscopic image analysis

In early diagnosis of lung cancer, a polarization microscopy is a powerful tool to obtain the optical information of biological tissues. In this paper, a new microfluidic polarization imaging and analysis method was proposed for the detection and classification of cancer‐associated fibroblasts and the two kinds of non‐small cell lung cancer cells, A549 and H322. A polarizing microscopy system was constructed based on a commercial microscope to obtain 3*3 Mueller matrix of cells. Based on the Muller matrix decomposition algorithm and analysis in spatial domain and frequency domain, appropriate classification parameters were selected for the characterization of different polarization characteristics of cells. Finally, the logistic regression models based on machine learning were applied to determine optimal feature parameters and classify cells. This method integrated the morphological information of the cells, and the polarization characteristics of the cells in different polarization states. It is for the first time that the polarization microscopic image analysis method has been applied to the detection and classification of non‐small cell lung cancer cells. The results show that the presented microfluidic polarization microscopic image analysis method could classify cells effectively. Compared with the Muller matrix measurement and calculation methods, the method proposed in this paper was greatly simplified in both the acquisition of polarized images and the analysis and processing of polarized images.     

Induced hydrophobic recovery of oxygen plasma-treated surfaces

Plasma treatment is a widely used method in microfabrication laboratories and the plasticware industry to functionalize surfaces for device bonding and preparation for mammalian cell culture. However, spatial control of plasma treatment is challenging because it typically requires a tedious masking step that is prone to alignment errors. Currently, there are no available methods to actively revert a surface from a treated hydrophilic state to its original hydrophobic state. Here, we describe a method that relies on physical contact treatment (PCT) to actively induce hydrophobic recovery of plasma-treated surfaces. PCT involves applying brushing and peeling processes with common wipers and tapes to reverse the wettability of hydrophilized surfaces while simultaneously preserving hydrophilicity of non-contacted surfaces. We demonstrate that PCT is a user-friendly method that allows 2D and 3D surface patterning of hydrophobic regions, and the protection of hydrophilic surfaces from unwanted PCT-induced recovery. This method will be useful in academic and industrial settings where plasma treatment is frequently used.     

Plasma stencilling methods for cell patterning

In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability.

Tissue-Specific stem cell differentiation in an in vitro airway model

The respiratory tract is the primary site of exposure to airborne compounds, with the bronchial epithelium providing one of the first lines of defence. A growing need exists for an accurate in vitro model of the bronchial epithelium. Here, normal human bronchial epithelial (NHBE) cells cultured at an air/liquid interface create a fully differentiated, in-vivo-like model of the human bronchial epithelium. Developmental characterisation includes (i) trans-epithelial electrical resistance, (ii) morphology and (iii) bronchial cell specific stains/markers. It is concluded that the basal/progenitor cells create a pseudo-stratified, mucociliary NHBE model containing basal, serous, Clara, goblet and ciliated cells, reflective of the normal human bronchial epithelium (days 24-33 ALI culture).     

Micropatterning of polystyrene nanoparticles and its bioapplications

Micropatterning of biomolecules forms the basis of cell culture, biosensor and microarray technology. Currently, the most widely used techniques are photoresist lithography, soft lithography or using robots which all involve multi-step surface modification directly on a planar substrate. Here we report a method to pattern biomolecules through self-assembling polystyrene nanoparticles in arrayed microwells on a solid surface to form well-ordered patterning, followed by attaching biomolecules to the assembled nanoparticles. The formation of colloidal patterns depends on capillary force, surface wettability and physical confinement. This method can be used for micropatterning a variety of biomolecules such as protein and antibody.     

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Introduction Thehumancornea,animportantopticalcompo nentthatfocusesthelightontheiris,isatransparent avasculartissuethatalsoplaysaprotectiveroleforthe eye.Therefore,anyinjury,diseaseorcelldamagethat canleadtoopacificationofthecornea,canimpairthe vision,and…     

Biocompatible micropatterning of two different cell types

The spatial arrangement of individual cell types can now be routinely controlled using soft-lithography-based micropatterning of complementary cell-adhesive and cell-resistant patterns. However, the application of these tools in tissue engineering to recreate tissue complexity in vitro has been hampered by the challenge of finding noncytotoxic procedures for converting complementary cell-resistant regions that define the arrangement of the first cell type into cell-adhesive regions to allow for the attachment of other cell types. A polyelectrolyte assembly approach is presented here for the first time, which allows for this noncytotoxic conversion and, thus, micropatterning of two different cell types, for example, endothelial cells and fibroblasts, on biodegradable substrates. The flexibility of this approach is further demonstrated by inducing organized capillary formation by endothelial cells on micropatterned lines followed by subsequent assembly of fibroblasts.     

E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion

Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.     

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.     

Discrimination of cancerous and non-cancerous cell lines by headspace-analysis with PTR-MS

Proton transfer reaction mass spectrometry (PTR-MS) has been used to analyze the volatile organic compounds (VOCs) emitted by in-vitro cultured human cells. For this purpose, two pairs of cancerous and non-cancerous human cell lines were selected:1. lung epithelium cells A-549 and retinal pigment epithelium cells hTERT-RPE1, cultured in different growth media; and 2. squamous lung carcinoma cells EPLC and immortalized human bronchial epithelial cells BEAS2B, cultured in identical growth medium. The VOCs in the headspace of the cell cultures were sampled: 1. online by drawing off the gas directly from the culture flask; and 2. by accumulation of the VOCs in PTFE bags connected to the flask for at least 12 h. The pure media were analyzed in the same way as the corresponding cells in order to provide a reference. Direct comparison of headspace VOCs from flasks with cells plus medium and from flasks with pure medium enabled the characterization of cell-line-specific production or consumption of VOCs. Among all identified VOCs in this respect, the most outstanding compound was m/z = 45 (acetaldehyde) revealing significant consumption by the cancerous cell lines but not by the non-cancerous cells. By applying multivariate statistical analysis using 42 selected marker VOCs, it was possible to clearly separate the cancerous and non-cancerous cell lines from each other.     

Simple and biocompatible micropatterning of multiple cell types on a polymer substrate by using ion implantation

A noncytotoxic procedure for the spatial organization of multiple cell types remains as a major challenge in tissue engineering. In this study, a simple and biocompatible micropatterning method of multiple cell types on a polymer surface is developed by using ion implantation. The cell-resistant Pluronic surface can be converted into a cell-adhesive one by ion implantation. In addition, cells show different behaviors on the ion-implanted Pluronic surface. Thus this process enables the micropatterning of two different cell types on a polymer substrate. The micropatterns of the Pluronic were formed on a polystyrene surface. Primary cells adhered to the spaces of the bare polystyrene regions separated by the implanted Pluronic patterns. Secondary cells then adhered onto the implanted Pluronic patterns, resulting in micropatterns of two different cells on the polystyrene surface.     

Asymmetric elastic properties of Dictyostelium discoideum in relation to chemotaxis

In this study we used an AFM to investigate the cytoskeletal properties of live Dictyostelium discoideum cells by measuring the local stiffness across individual living cells. We have examined differences in elastic properties of polarized and unpolarized AX3 wild type and the mutant DAip1- cells, as well as the differences in the front and rear of the cells in relation to organization of the actin cytoskeleton. We found that the average Young's modulus increases upon polarization for the thin regions of the cell and that in polarized cells, the cell front was stiffer than the cell back. We also found that AX3 cells were stiffer than DAip1- cells. This finding suggests that actin polymerization is one of the major determinants of cell motility in Dictyostelium. In addition, a thin agarose film was studied as a model system to examine the influence of the substrate of thin materials probed with the AFM.