A microfluidic platform for 3-dimensional cell culture and cell-based assays

This paper reports a novel microfluidic platform introducing peptide hydrogel to make biocompatible microenvironment as well as realizing in situ cell-based assays. Collagen composite, OPLA and Puramatrix scaffolds are compared to select good environment for human hepatocellular carcinoma cells (HepG2) by albumin measurement. The selected biocompatible self-assembling peptide hydrogel, Puramatrix, is hydrodynamically focused in the middle of main channel of a microfluidic device, and at the same time the cells are 3-dimensionally immobilized and encapsulated without any additional surface treatment. HepG2 cells have been 3-dimensionally cultured in a poly(dimethylsiloxane) (PDMS) microfluidic device for 4 days. The cells cultured in micro peptide scaffold are compared with those cultured by conventional petri dish in morphology and the rate of albumin secretion. By injection of different reagents into either side of the peptide scaffold, the microfluidic device also forms a linear concentration gradient profile across the peptide scaffold due to molecular diffusion. Based on this characteristic, toxicity tests are performed by Triton X-100. As the higher toxicant concentration gradient forms, the wider dead zone of cells in the peptide scaffold represents. This microfluidic platform facilitates in vivo-like 3-dimensional microenvironment, and have a potential for the applications of reliable cell-based screening and assays including cytotoxicity test, real-time cell viability monitoring, and continuous dose-response assay.     

Automatic identification of subcellular phenotypes on human cell arrays

Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells.     

On-chip testing device for electrochemotherapeutic effects on human breast cells

A microfabricated cell-based testing device for electrochemotherapy (ECT) has been developed by miniaturizing the widely used clinical electroporator with a two-needle array into two-dimensional planar electrodes while keeping the similarity of the electric field strength distribution. In this device, all the biological processes from cell culture to electroporation and final cell-based assays were carried out on a chip using a conventional 2D cell culture method, and the multiple electrochemotherapeutic assays could be realized by exploiting the six electroporation sites in a single device. With the proposed platform, the electroporation rate was evaluated with propidium iodide and cell proliferation after 48 h of electrochemotherapy with bleomycin was determined with T47D human breast ductal carcinoma cell line in various electric field strengths and drug concentrations. This microsystem has several advantages over conventional cuvette type electroporation assay, such as multiple assays on a chip, on-chip based operation from cell culture to final assay, and having similar electric field distribution as that of the clinical electroporator. As the clinical trials of electrochemotherapy are being carried out, this new platform is expected to have valuable applications in basic in vitro ECT studies, drug discovery, and development of clinical ECT equipment.     

Automated live cell screening system based on a 24-well-microplate with integrated micro fluidics

In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.     

基于图像处理的菌落自动计数系统

目的针对现有菌落计数仪结构复杂、便携性差等缺点,本文设计了一种基于图像处理技术和Android平台来完成菌落自动计数的系统。方法该系统由硬件拍照设备采集菌落图像,以智能手机为主要操作载体,对图像进行光谱阈值分割、中值滤波、洪水填充、开值运算、八邻域边界跟踪等多种算法处理后实现自动计数。结果与菌落计数仪相比,该系统使用移动端手机APP实时获取菌落图像并得到计数结果,可直接在手机上对结果进行手动校正,具有成本低、操作简单、便携性好等优点。结论基于图像处理的菌落自动计数系统与人工计数结果的相对误差不超过5%,满足菌落计数的要求。     

A microfluidic cell culture platform for real-time cellular imaging

This study reports a new microfluidic cell culture platform for real-time, in vitro microscopic observation and evaluation of cellular functions. Microheaters, a micro temperature sensor, and micropumps are integrated into the system to achieve a self-contained, perfusion-based, cell culture microenvironment. The key feature of the platform includes a unique, ultra-thin, culture chamber with a depth of 180 μm, allowing for real-time, high-resolution cellular imaging by combining bright field and fluorescent optics to visualize nanoparticle-cell/organelle interactions. The cell plating, culturing, harvesting and replenishing processes are performed automatically. The developed platform also enables drug screening and real-time, in situ investigation of the cellular and sub-cellular delivery process of nano vectors. The mitotic activity and the interaction between cells and the nano drug carriers (conjugated quantum dots-epirubicin) are successfully monitored in this device. This developed system could be a promising platform for a wide variety of applications such as high-throughput, cell-based studies and as a diagnostic cellular imaging system.     

Sequential array cytometry: multi-parameter imaging with a single fluorescent channel

Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases.     

A 3-D microfluidic combinatorial cell array

We present the development of a three-dimensional (3-D) combinatorial cell culture array device featured with integrated three-input, eight-output combinatorial mixer and cell culture chambers. The device is designed for cell-based screening of multiple compounds simultaneously on a microfluidic platform. The final assembled device is composed of a porous membrane integrated in between a Parylene 3-D microfluidic chip and a PDMS microfluidic chip. The membrane turned the cell culture chambers into two-level configuration to facilitate cell loading and to maintain cells in a diffusion dominated space during device operation. Experimentally, we first characterized the combined compound concentration profile at each chamber using a fluorescence method. We then successfully demonstrated the functionality of the quantitative cell-based assay by culturing B35 rat neuronal cells on this device and screening the ability of three compounds (1,5-dihydroxyisoquinoline, deferoxamine, and 3-aminobenzoic acid) to attenuate cell death caused by cytotoxic hydrogen peroxide. In another experiment, we assayed for the combinatorial effects of three chemotherapeutic compound exposures (vinorelbine, paclitaxel, and γ-linolenic acid) on human breast cancer cells, MDA-MB-231. The same technology will enable the construction of inexpensive lab-on-a-chip devices with high-input combinatorial mixer for performing high-throughput cell-based assay and highly parallel and combinatorial chemical or biochemical reactions.     

Cell proliferation measurement in cecum and colon of rats using scanned images and fully automated image analysis: Validation of method

The purpose of this study was to establish and validate fully automatic measurement of cell proliferation on scanned images of rat cecum and colon. Tissue slides were taken from a 4-week mechanistic study and processed for BrdU immunohistochemistry. Four sections of the cecum and colon per slide were scanned with the Zeiss MIRAX SCAN and transferred to the Definiens eCognition Analyst LS5.0 system for evaluation. Two rule sets for automatic counting of BrdU-positive and negative nuclei from mucosal cells on the image tiles were created by Definiens, one for cecum, one for colon. For validation, manual counting of 16 randomly selected tiles from five different slides of colon and cecum was performed. Negative and positive cell nuclei were counted in each image tile by four different people. Comparison of results from manual counting with the automatic counting showed that the sum as well as single tile data and labeling index (LI) from automatic counting were within the range of manual counting results ±10%. Automatic counting included only cell nuclei within the mucosa whereas muscularis and lymphoid tissue as well as wrinkles from tissue preparation were excluded. In addition, two data sets from automatic counting of the same image tile were compared: (1) data where image tiles with incorrect detection of mucosa were excluded from further calculation of LI and area, and (2) data where no visual check was performed and all measurements were included. Results were very similar for both data sets. The necessity of the manual correction may therefore be doubted.     

High-throughput imaging of bacterial colonies grown on filter plates with application to serum bactericidal assays

The ability to accurately enumerate viable bacteria has applications in antibiotic screening assays, toxicology testing, and serological assays for functional antibodies. An impediment to high-throughput bacterial assays is the requirement to grow bacteria as individual colonies on semisolid media containing agar. We have now developed a method for growth, staining, and counting of bacterial colonies in 96-well filter plates. A unique feature of the method is that colony size is inversely proportional to the number of colonies in each well, presumably due to nutrient depletion. As a result, as many as 300 colony-forming units (cfu) can be detected as discrete colonies within a single assay well. The resulting colonies can be counted automatically using an imaging system originally developed for ELISPOT assays. The method has been applied to the measurement of serum bactericidal activity (SBA) in human sera.     

Micropatterns of Matrigel for three-dimensional epithelial cultures

Three-dimensional (3D) epithelial culture models are widely used to promote a physiologically relevant microenvironment for the study of normal and aberrant epithelial organization. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hindered by the lack of existing platforms for large-scale 3D cellular studies. Current 3D standard culture does not allow for single spheroid or 'acinus' analysis required for high-throughput systems. Here, we present general strategies for creating bulk micropatterns of Matrigel that can be used as a platform for 3D epithelial culture and cell-based assays at the single acinus level. Both buried and free-standing micropatterns of Matrigel were created using modified soft lithography techniques such as microtransfer molding (microTM) and dry lift-off technique. Surface modification of poly(dimethylsiloxane) (PDMS) with oxygen plasma followed by treatment with poly(2-hydroxy-ethylmethacrylate) (poly-HEMA) was sufficient to promote deformation-free release of Matrigel patterns. In addition, a novel dual-layer dry lift-off technique was developed to simultaneously generate patterns of Matrigel and poly-HEMA on a single substrate. We also demonstrate that the micropatterned Matrigel can support 3D culture originating from a single normal human mammary epithelial (MCF-10A) cell or a human breast cancer cell (MDA-MB-231) with comparable phenotypes to standard 3D culture techniques. Culture of normal MCF-10A cells on micropatterned Matrigel resulted in formation of structures with the characteristic apoptosis of centrally located cells and formation of hollow lumens. Moreover, the carcinoma cell line showed their characteristic formation of disorganized invasive cellular clusters, lacking the normal epithelial architecture on micropatterned Matrigel. Hence, micropatterned Matrigel can be used as a 3D epithelial cell-based platform for a wide variety of applications in epithelial and cancer biology, tissue engineering, as well as gene/drug screening technology.     

Using living radical polymerization to enable facile incorporation of materials in microfluidic cell culture devices

High throughput screening tools are expediting cell culture studies with applications in drug discovery and tissue engineering. This contribution demonstrates a method to incorporate 3D cell culture sites into microfluidic devices and enables the fabrication of high throughput screening tools with uniquely addressable culture environments. Contact lithographic photopolymerization (CLiPP) was used to fabricate microfluidic devices with two types of 3D culture sites: macroporous rigid polymer cell scaffolds and poly(ethylene glycol) (PEG) encapsulated cell matrices. Cells were cultured on-device with both types of culture sites, demonstrating material cytocompatibility. Multilayer microfluidic devices were fabricated with channels passing the top and bottom sides of a series of rigid porous polymer scaffolds. Cells were seeded and cultured on device, demonstrating the ability to deliver cells and culture cells on multiple scaffolds along the length of a single channel. Flow control through these rigid porous polymer scaffolds was demonstrated. Finally, devices were modified by grafting of PEG methacrylate from surfaces to prevent non-specific protein adsorption and ultimately cell adhesion to channel surfaces. The living radical component of this CLiPP device fabrication platform enables facile incorporation of 3D culture sites into microfluidic cell culture devices, which can be utilized for high throughput screening of cell-material interactions.     

Optical-electronic device for rapid counting and volume determination of suspended particles of 15–1000 μm in diameter

An optical-electronic counting device for the rapid counting of suspended particles from 15 to 1000 μm in diameter is described. The device was used to count and size cell colonies, grown in agar, in concentrations ranging from 5 to 3000 per millilitre. The volume distribution of these particles was studied with a pulse-height analyser. At its lower range of sensitivity, the device is able to count large single cells. The counting device is composed of the following: (i) a hydraulic system, constraining the flow of particles to the central core of a region of cylindrical water flow, (ii) an optical system which allows colonies to be counted by interrupting a light beam, (iii) an electronic counting device quantitating the electronic pulses generated by the light output. Besides applications in the biological laboratory, the device is also potentially useful in areas such as the study of water quality in the environment, and the monitoring of particulate densities in industrial processes.     

A microfabricated platform probing cytoskeleton dynamics using multidirectional topographical cues

Cell migration, which involves complicated coordination of cytoskeleton elements and regulatory molecules, plays a central role in a large variety of biological processes from development, immune response to tissue regeneration. However, conventional methods to study in vitro cell migration are often limited to stimulating a cell along a single direction or at a single location. This restriction prevents a deeper understanding of the fundamental mechanisms that control the spatio-temporal dynamics of cytoskeleton. Here we report a novel microfabricated platform that enables a multi-directional stimulation to a cell using topographical cues. In this device, cells were seeded on a grid-patterned topographically structured surface composed of 2 μm wide and 2 μm high straight ridges. Because the size of a unit grid was smaller than a single cell, each cell was simultaneously experiencing contact guidance leading to different directions. The device showed that healthy cells preferred to align and migrate in the direction of the longer side of the grid. But cells with impaired intracelluar tension force generation exhibited multiple uncoordinated cell protrusions along guiding ridges in all directions. Our results demonstrate the importance of actomyosin network in long-range communication and regulation of local actin polymerization activities. This platform will find wide applications in investigations of signal transduction and regulation process in cell migration.     

Microfluidics for Mammalian Cell Chemotaxis

The emerging field of micro-technology has opened up new possibilities for exploring cellular chemotaxis in real space and time, and at single cell resolution. Chemotactic cells sense and move in response to chemical gradients and play important roles in a number of physiological and pathological processes, including development, immune responses, and tumor cell invasions. Due to the size proximity of the microfluidic device to cells, microfluidic chemotaxis devices advance the traditional macro-scale chemotaxis assays in two major directions: one is to build well defined and stable chemical gradients at cellular length scales, and the other is to provide a platform for quantifying cellular responses at both cellular and molecular levels using advanced optical imaging systems. Here, we present a critical review on the designing principles, recent development, and potential capabilities of the microfluidic chemotaxis assay for solving problems that are of importance in the biomedical engineering field.     

A uniaxial bioMEMS device for quantitative force-displacement measurements

There is a need for experimental techniques that allow the simultaneous imaging of cellular cystoskeletal components with quantitative force measurements on single cells. A bioMEMS device has been developed for the application of strain to a single cell while simultaneously quantifying its force response. The prototype device presented here allows the mechanical study of a single, adherent cell in vitro. The device works in a fashion similar to a displacement-controlled uniaxial tensile machine. The device is calibrated using an AFM cantilever and shows excellent agreement with the calculated spring constant. The device is demonstrated on a single fibroblast. The force response of the cell is seen to be linear until the onset of de-adhesion with the de-adhesion from the cell platform occurring at a force of approximately 1500 nN.     

Design of a microfluidic strategy for trapping and screening single cells

Traditionally, in vitro investigations on biology and physiology of cells rely on averaging the responses eliciting from heterogeneous cell populations, thus being unsuitable for assessing individual cell behaviors in response to external stimulations. In the last years, great interest has thus been focused on single cell analysis and screening, which represents a promising tool aiming at pursuing the direct and deterministic control over cause-effect relationships guiding cell behavior. In this regard, a high-throughput microfluidic platform for trapping and culturing adherent single cells was presented. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. A round-shaped culture chamber (Φ  =  250 µm, h  =  25 µm) was conceived presenting two connections with a main fluidic path: (i) an upper wide opening, and (ii) a bottom trapping junction which modulates the hydraulic resistance. Starting from eight different layouts, the chamber geometry was computationally optimized for maximizing the single cell trapping efficacy and then integrated in a polydimethylsiloxane (PDMS) microfluidic device. The final platform consists in (i) 288 chambers for trapping single cells organized in six culture units, independently addressable through the lines of (ii) a chaotic-mixer based serial dilution generator (SDG), designed for creating spatio-temporally controlled patterns of both soluble factors and non-diffusive particles. The device was experimentally validated by trapping polystyrene microspheres, featuring diameters comparable to cell size (Φ  =  10 µm).     

A microchip approach for practical label-free CD4+ T-cell counting of HIV-infected subjects in resource-poor settings

Simple affordable CD4 cell counting is urgently needed to stage and monitor HIV-infected patients in resource-limited settings. To address the limitations of current approaches, we designed a simple, label-free, and cost-effective CD4 cell counting device using microfluidic technology. We previously described the fabrication of a microfluidic system for high-efficiency isolation of pure populations of CD4+ T cells based on cell affinity chromatography operated under controlled flow. Here, we compare the performance of a microfluidic CD4 cell counting device against standard flow cytometry in 49 HIV-positive subjects over a wide range of absolute CD4 cell counts. We observed a close correlation between CD4 cell counts from the microchip device and measurements by flow cytometry, using unprocessed whole blood from HIV-positive adult subjects. Sensitivities for distinguishing clinically relevant thresholds of 200, 350, and 500 cells/microL are 0.86, 0.90, and 0.97, respectively. Specificity is 0.94 or higher at all thresholds. This device can serve as a functional cartridge for fast, accurate, affordable, and simple CD4 cell counting in resource-limited settings.     

Fast and efficient screening system for new biomaterials in tissue engineering: a model for peripheral nerve regeneration

The use of three-dimensional biodegradable matrices is one major issue in tissue engineering. Numerous materials, fabrication techniques, and modifications have been used and tested in different areas of tissue engineering recently. But nevertheless, technology is far from being optimized and optimal constructs with bioidentical and mechanical properties have not been described in the literature so far. Hence, there is great demand of new suitable biomaterials for tissue engineering applications. In this study, a fast and efficient screening system for initial testing of biomaterials for cell culture application was developed. The set up for the screening system and the decision criteria applied for the determination of suitability of new materials are presented. Hep-G2 and PC-12 cells were seeded onto different matrices and cultured over a period of 2 weeks. The viability of the cells was monitored via the MTT assay. Cell spreading was investigated by DAPI-staining of cell nuclei. Furthermore, the adhesion of the cells on the different matrices was examined by counting the number of attached cells. With these general assays a classification of materials is possible with regard to their suitability. Optimal cell models must be chosen for the defined applications and at least two cell lines are necessary for a differentiating interpretation.