Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.     

Transfected cell microarrays: an efficient tool for high-throughput functional analysis

Transfected cell microarrays are considered to be a breakthrough methodology for high-throughput and high-content functional genomics. Here, recent advances in the cell microarray field are reviewed, along with its potential to increase the speed of determining gene function. These advances, combined with an increasing number and diversity of gene perturbing systems, such as RNAi and ectopic gene expression, provide tools for expanding our understanding of biology at the systems level.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.     

Development of a novel,high-throughput screening tool for efficient perfusion-based cell culture process development

Perfusion technology has been successfully used for the commercial production of biotherapeutics, in particular unstable recombinant proteins, for more than a decade. However, there has been a general lack of high-throughput cell culture tools specifically for perfusion-based cell culture processes. Here, we have developed a high-throughput cell retention operation for use with the ambr® 15 bioreactor system. Experiments were run in both 24 and 48 reactor configurations for comparing perfusion mimic models, media development, and clone screening. Employing offline centrifugation for cell retention and a variable volume model developed with MATLAB computational software, the established screening model has demonstrated cell culture performance, productivity, and product quality were comparable to bench scale bioreactors. The automated, single use, high-throughput perfusion mimic is a powerful tool that enables us to have rapid and efficient process development of perfusion-based cell culture processes.     

DNA fingerprinting—a valuable new technique for the characterisation of cell lines

DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic “snap-shot” of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.     

A novel cell-based system for evaluating skeletal muscle cell hypertrophy-inducing agents

Skeletal muscle is a tissue that adapts to increased use by increasing contractile protein gene expression and ultimately skeletal muscle mass (hypertrophy). To identify hypertrophy-inducing agents that may be potentially useful in the treatment of age-related muscle loss (sarcopenia) and to better understand hypertrophy signal transduction pathways, we have created a skeletal muscle cell-based hypertrophy-responsive system. This system was created by permanently modifying the relatively undifferentiated C2C12 cell line so that it contains the beta-myosin heavy chain (beta-MHC) gene promoter and enhancer regions fused to a luciferase reporter gene. This cell line responds, by increasing luciferase expression, to a variety of skeletal muscle hypertrophy-inducing agents, including insulin, insulin-like growth factor I, testosterone, and the beta-adrenergic receptor agonist isoproterenol, in both the undifferentiated and differentiated states. This cell-based system should be useful for identifying novel hypertrophy-inducing agents as well as understanding hypertrophy signal transduction.     

A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity

Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 10⁶ variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.     

A high-throughput cell-based assay for inhibitors of ABCG2 activity

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 microM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z' 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (> or = 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 microM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.     

Quantitative, high-throughput cell-based assays for inhibitors of trkA receptor

Two quantitative, high-throughput cell-based assays for evaluating inhibitors of NGF-stimulated trkA phosphorylation in trkA-transfected NIH3T3 cells have been established. Both assays involve capture of the trkA receptor from cell lysates in microtiter plates coated with an anti-trk antibody. The amount of trkA phosphorylation is then measured using either an anti-phosphotyrosine antibody with a colorimetric readout or a lanthanide (europium)-labeled anti-phosphotyrosine antibody with a fluorometric detection. The two assay formats exhibited at least a fivefold increase in phosphorylated trkA signal in trkA-transfected cells compared to vector control. Inhibition plots generated for trkA kinase inhibitors using the two detection systems yielded comparable IC(50) values. Overall, the two assays represent a marked improvement over the standard gel-based/western blot method in terms of throughput, quantitation, and amenability to automation.     

A novel multiplex cell viability assay for high-throughput RNAi screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.     

A method for establishing cell lines fromDrosophila melanogaster embryos

Summary A simple method is presented for establishing continuous cell lines fromDrosophila melanogaster embryos. Subculturing is performed after the first 8 weeks and at 2-week intervals therafter. Initial plating densities of 5×10⁴ to 5×10⁵ cells per cm² are required for maintaining the subcultures. Cell lines were established from wild-type embryos, from embryos bearing chromosomal rearrangements and from embryos bearing recessive mutations. Permanent lines have doubling times of 24 to 48 hr and have been maintained for as long as 13 months and 25 subcultures. Supported in part by NSF grant BMS75-02138 and NIH grant NS09330 to. R. Seecof.     

细胞周期相关蛋白cyclin D1、p53和p21WAF1/Cip1在食管鳞癌中的表达改变及其病理学意义

食管鳞状细胞癌(Esophageal squamous cell carcinoma, ESCC)是我国常见的恶性肿瘤之一, 虽然临床诊治手段正逐步改进, 但中晚期患者5年生存率仍然很低。目前认为细胞周期调控异常与肿瘤发生发展关系密切, 然而相关周期调节蛋白在食管癌患者中的表达改变、临床意义及其应用价值还没有明确结论。文章应用组织微阵列联合免疫组织化学技术(TMA-IHC), 对148例食管鳞癌组织标本中细胞G₁/S期调控蛋白cyclin D1、p53和p21^WAF1/Cip1的表达进行检测, 分析其与临床病理参数之间的相关性。结果显示, cyclin D1与p53蛋白在食管癌细胞中表达升高, p53表达阳性率与区域淋巴结转移显著相关(P = 0.001)。p21^WAF1/Cip1蛋白在肿瘤组织中表达降低, 且p21WAF1/Cip1表达阴性患者的术后生存时间显著短于表达阳性的患者(P = 0.001)。多因素生存分析显示p21WAF1/Cip1是一个独立的预后因素(相对危险度为0.418, P<0.001)。微阵列比较基因组杂交(array-CGH)检测进一步表明45.4%的食管癌患者存在cyclin D1基因扩增。以上结果提示食管鳞癌中存在细胞周期G1/S期调控异常, p21^WAF1/Cip1蛋白可能是一个有应用价值的预后因子。     

Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.     

A packed Cytodex microbead array for three-dimensional cell-based biosensing

A packed Cytodex 3 microbead array was fabricated as a simple three-dimensional (3-D) cell-based biosensing format. Resting membrane potentials and voltage-gated calcium channel (VGCC) function of SH-SY5Y human neuroblastoma cells cultured on the microbead array versus collagen-coated flat (2-D) substrates were evaluated by confocal microscopy with a potentiometric dye, tetramethylrhodamine methyl ester, and a calcium fluorescent indicator, Calcium Green-1. SH-SY5Y cells, differentiated with 1mM dibutyryl cAMP and 2.5 microM 5-bromodeoxyuridine, showed significant resting membrane potential establishment on the topographical scaffolds in a period of 13 days into differentiation, in contrast to the previously reported insignificant resting membrane potential establishment of the same cells within collagen hydrogels. On days 2, 8 and 13 into differentiation, cells on collagen-coated flat substrates developed resting membrane potentials of -6.0+/-19.5 mV (n=198), -30.5+/-19.9 mV (n=191) and -21.7+/-18.9 mV (n=308), in contrast to values for cells on 3-D scaffolds of -25.8+/-14.7 mV (n=112), -37.6+/-13.1 mV (n=120) and -28.7+/-12.2 mV (n=158), respectively. The development of VGCC function, as measured by percentage of cells responsive to 50 mM high K(+) depolarization, was significantly slower for cells on 3-D scaffolds (20.0% on day 13 into differentiation) than for cells on 2-D substrates (30.7% on day 8 into differentiation). The exaggerated 2-D cell calcium dynamics, in comparison with those of 3-D cells, is consistent with previous 2-D/3-D comparative studies. This study established the rationale and feasibility of the microbead array format for 3-D cell-based biosensing.     

Stem cell-based cell therapy for neuroprotection in stroke: A review

Neurological disorders, such as stroke, are triggered by a loss of neurons and glial cells. Ischemic stroke remains a substantial problem for industrialized countries. Over the previous few decades our understanding about the pathophysiology of stroke has enhanced, nevertheless, more awareness is required to advance the field of stroke recovery. Existing therapies are incapable to adequately relief the disease outcome and are not appropriate to all patients. Meanwhile, the majority of patients continue to show neurological deficits even subsequent effective thrombolysis, recuperative therapies are immediately required that stimulate brain remodeling and repair once stroke damage has happened. Cell therapy is emergent as a hopeful new modality for increasing neurological recovery in ischemic stroke. Numerous types of stem cells from various sources have been identified and their possibility and efficiency for the treatment of stroke have been investigated. Stem cell therapy in patients with stroke using adult stem cells have been first practiced in clinical trials since 15 years ago. Even though stem cells have revealed a hopeful role in ischemic stroke in investigational studies besides early clinical pilot studies, cellular therapy in human is still at a primary stage. In this review, we summarize the types of stem cells, various delivery routes, and clinical application of stem cell-based therapy for stroke treatment.     

A novel technique for viable cell determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 micrograms/ml; acridine orange, 1-5.0 micrograms/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.