Targeted cell adhesion on selectively micropatterned polymer arrays on a poly(dimethylsiloxane) surface

Herein, we introduce the fabrication of polymer micropattern arrays on a chemically inert poly(dimethylsiloxane) (PDMS) surface and employ them for the selective adhesion of cells. To fabricate the micropattern arrays, a mercapto-ester—based photocurable adhesive was coated onto a mercaptosilane—coated PDMS surface and photopolymerized using a photomask to obtain patterned arrays at the microscale level. Robust polymer patterns, 380 μm in diameter, were successfully fabricated onto a PDMS surface, and cells were selectively targeted toward the patterned regions. Next, the performance of the cell adhesion was observed by anchoring cell adhesive linker, an RGD oligopeptide, on the surface of the mercapto-ester—based adhesive-cured layer. The successful anchoring of the RGD linker was confirmed through various surface characterizations such as water contact angle measurement, XPS analysis, FT-IR analysis, and AFM measurement. The micropatterning of a photocurable adhesive onto a PDMS surface can provide high structural rigidity, a highly–adhesive surface, and a physical pathway for selective cell adhesion, while the incorporated polymer micropattern arrays inside a PDMS microfluidic device can serve as a microfluidic platform for disease diagnoses and high-throughput drug screening.     

RNAi microarray analysis in cultured mammalian cells

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time- and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function.     

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.     

Phenotype MicroArrays for High-Throughput Phenotypic Testing and Assay of Gene Function

The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes.     

A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens

The generation of monoclonal antibodies (mAb) of desired specificity to cell surface antigens can serve as a valuable tool to study protein expression and function. However, traditional approaches to mAb generation usually involve large-scale protein purification and intensive screening, and may not result in mAb specificities to the native protein of interest. We describe a simple, inexpensive, high-throughput method for the generation and screening of hybridomas secreting mAb specific for cell surface receptors. Intact reporter cells expressing a CD3zeta-fusion receptor of the protein of interest are plated in 96-well arrays of captured, plate-bound hybridoma supernatants. A mAb to the protein of interest generates a signal leading to reporter-cell expression of beta-galactosidase, and enzyme activity can be screened in a single day using a non-radioactive substrate. Importantly, a single cell line can be used for immunization, screening, semi-quantitative affinity comparisons, and subsequent screening for physiological ligand expression, if the protein of interest is a receptor. We describe an application of this approach to generate mAb specific for a protein of previously unknown expression and undocumented function.     

A robust high-throughput sandwich cell-based drug screening platform

Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatocytes are readily transferred to 96-well plate for high-throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.     

RNA干扰在哺乳动物细胞高通量基因功能研究中的应用

RNA干扰(RNAi)技术是近年来迅速发展起来的一种高效、特异、易操作的基因阻断技术,它已被应用于线虫、果蝇等低等生物的大规模功能丧失表型遗传学筛选中,并成功鉴定了大量的新基因。近期,在利用现有siRNA文库进行的小规模哺乳动物细胞功能基因筛选研究中,RNAi同样显示了卓越的性能。随着高复杂度的siRNA文库的构建和筛选策略的完善,RNAi在哺乳动物细胞高通量基因功能研究中的应用已日见成熟。     

Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei

In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.     

ELECTRON MICROSCOPIC SUB-CLASSIFICATION OF SMALL CELL CARCINOMA OF THE LUNG

An electron microscopic study of 28 small cell carcinomas of the lung is presented. Cytoplasmic secretory granules, characteristic of endocrine cells of the human foetal lung were observed in a variable number of tumor cells. Two groups of tumors could be distinguished based on the morphology of the cytoplasmic secretory granules. Twenty-three tumors showed cells with granules resembling type 1 or P₁ cells of the human fetal lung, and 5 tumors with granules resembling type 3 cells of the human fetal lung. No relationship was found between the light microscopic WHO classification of small cell carcinoma of the lung and the results obtained by electron microscopy. Increased serum calcitonin as well as inappropriate ADH secretion may be correlated with one of the two types of small cell carcinoma, but further investigations are needed.     

Electron microscopic sub-classification of small cell carcinoma of the lung

An electron microscopic study of 28 small cell carcinomas of the lung is presented. Cytoplasmic secretory granules, characteristic of endocrine cells of the human foetal lung were observed in a variable number of tumor cells. Two groups of tumors could be distinguished based on the morphology of the cytoplasmic secretory granules. Twenty-three tumors showed cells with granules resembling type 1 or P1 cells of the human fetal lung, and 5 tumors with granules resembling type 3 cells of the human fetal lung. No relationship was found between the light microscopic WHO classification of small cell carcinoma of the lung and the results obtained by electron microscopy. Increased serum calcitonin as well as inappropriate ADH secretion may be correlated with one of the two types of small cell carcinoma, but further investigations are needed.     

<Emphasis Type="Italic">In vitro</Emphasis> assays for determining the metastatic potential of melanoma cell lines with characterized <Emphasis Type="Italic">in vivo</Emphasis> invasiveness

The metastatic potential of cancer cells is an elusive property that is indicative of the later stages of cancer progression. The ability to distinguish between poorly and highly metastatic cells is invaluable for understanding the basic biology of cancer and to develop more treatments. In this paper, we exploit a A375 melanoma cell line series (A375P, A375MA1, A375MA2) that vary in metastatic potential, to demonstrate an in vitro screening assay using polydimethylsiloxane (PDMS) microbubble well arrays that can distinguish these cell lines by their growth characteristics in including morphology, migratory potential, and clonogenic potential. These cell lines cannot be distinguished by their growth characteristics when cultured on standard tissue culture plastic or planar PDMS. Results show that the more metastatic cell lines (A375MA1, A375MA2) have a higher proliferative potential and a distinctive radial spreading growth pattern out of the microbubble well. The A375MA2 cell line also has a higher tendency to form multicellular spheroids. The ability to successfully correlate the metastatic potential of cancer cells with their growth characteristics is essential first step toward developing a high-throughput screening assay to identify aggressive tumor cells in primary samples. The capability to culture and recover aggressive cells from microbubble wells will enable identification of candidate metastatic biomarkers which has immense clinical significance.     

线虫基因组功能的高通量研究

线虫是研究细胞生物学的一个理想模型,RNA i现象也是最早在线虫中发现的。由于RNA i技术不仅具有高度特异性靶定基因的特点,而且简便、易于操作,从而成为研究基因组功能的高通量方法。很多研究小组已经使用大规模RNA i系统地研究线虫各个基因的功能缺失表型。线虫独特的RNA i方法和以此为基础的基因组大规模分析有利于对线虫的各种生物学过程产生新的认识。     

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.     

分化抑制蛋白1在小鼠树突状细胞肉瘤细胞分化中的作用

目的 研究降低分化抑制蛋白1(Id.1)的表达在树突状细胞肉瘤(DCS)细胞分化中的作用.方法 通过RNA干扰技术来降低DCS细胞株中Id-l蛋白的表达,Western blot和real.timePCR分别检测该蛋白在蛋白水平和mRNA水平L的变化;倒置显微镜下观察降低Id.1蛋白表达后DCS细胞形态的变化;Casy细胞计数分析仪检测细胞大小的分布情况;流式细胞仪检测细胞周期的变化.通过Western blot和免疫组织化学检测树突状细胞的分化标志如Id-2、CD86、CDllc、MHC II的变化.生长曲线及四甲基偶氮唑盐(MTY)法检测DCS细胞的增殖情况的变化;通过TransweH和克隆形成率测定分别观察降低Id-1蛋白前后细胞侵袭、成瘤能力的变化.以不加siRNA的空白组和无关 对照siRNA作阴性对照,以诱导分化剂丁酸钠作为阳性对照.结果 降低Id-1蛋白表达后DCS细胞分枝增多,细胞体积明显增大,核质比下降,G0/G1期细胞增多,S期细胞减少(P<0.01),树突状细胞分化标志Id-2、CD86等表达上调,细胞增殖受到抑制,侵袭、成瘤能力减弱(P<0.01).结论 通过RNA干扰技术降低Id-1基因的表达,可以促进恶性实体肿瘤细胞的分化,降低恶性程度.     

Protein microarrays for the detection of biomarkers in head and neck squamous cell carcinomas

Protein microarrays are of increasing importance for high-throughput screening of fresh tissues. In our study, protein microarrays were generated by printing antibodies onto membranes to characterize protein profiles expressed by head and neck squamous cell carcinomas (HNSCCs). Cellular proteomes of 30 matched normal squamous epithelial cells and carcinoma specimens were analyzed after tissue microdissection using microarrays composed of 83 different antibodies. As controls, Western blot analysis and tissue microarrays (TMAs) containing 98 HNSCC specimens were used. Of the 83 proteins examined, 14 showed differential expression between HNSCCs and normal epithelium. The protein microarray approach revealed an upregulation of 8 proteins and a downregulation of 6 proteins. Bag-1, Cox-2, Hsp-70, Stat3, pescadillo, MMP-7 (matrilysin), IGF-2, and cyclin D1 were identified to be significantly upregulated, whereas suppressor of cytokine signaling 1, thrombospondin, TGF-beta1, Jun, Fos, and Fra-2 were downregulated. The differential expression of these proteins was confirmed using Western blot and TMA. Upon correlation of differentially regulated proteins with the clinicopathologic data of our patients, MMP-7 (matrilysin) was found to be associated with survival in univariate, but not multivariate, analysis. These data indicate that our protein arrays provide protein information in a systematic, reproducible, and also high-throughput fashion.     

裸鼠人早幼粒白血病突变株细胞(HL-60-AR)异种移植模型的建立

将HGPRT~-的人早幼粒白血病突变株细胞(HL-60-AR)接种于裸鼠皮下,能形成实体性白血病肉瘤。肿瘤组织经光镜形态学、细胞超微结构、细胞化学、LDH同工酶谱、染色体分析、细胞遗传标记、以及分化性状的检测等多项指标鉴定,均类似于原来体外长期培养的细胞株。移植瘤细胞能在裸鼠体内连续传代,迄今已传至第12代。这个裸鼠人白血病细胞异种移植模型的建立,将为研究人类白血病细胞在体内的增殖、分化以及对抗白血病药物治疗的反应,提供一个有价值的实验系统。     

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.