Application of resequencing microarrays in microbial detection and characterization

Microarrays are powerful, highly parallel assays that are transforming microbiological diagnostics and research. The adaptation of microarray-based resequencing technology for microbial detection and characterization resulted in the development of a number assays that have unique advantages over other existing technologies. This technological platform seems to be especially useful for sensitive and high-resolution multiplexed diagnostics for clinical syndromes with similar symptoms, screening environmental samples for biothreat agents, as well as genotyping and whole-genome analysis of single pathogens.     

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Development and validation of an automated high-throughput system for zebrafish in vivo screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism.     

A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro

Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.     

Optical coding of mammalian cells using semiconductor quantum dots

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.     

Novel fluorescent technology platform for high throughput cytotoxicity and proliferation assays

We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays. This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations. Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death. The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs. When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems. Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria. In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times. To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines. In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC(50) values were found with the Oxygen BioSensor for five different toxins or drugs. This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays.     

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-μmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.     

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway^® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community.     

Unique morphology of HeLa cell attachment, spreading and detachment from microcarrier beads covalently coated with a specific and non-specific substratum

A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific.     

A comprehensive classification system for lipids

Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, "lipidomics," in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid. This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.     

Sensitive and accurate quantification of human leukocyte migration using high-content Discovery-1 imaging system and ATPlite assay

Migration is a fundamental aspect of leukocyte behavior and represents a significant therapeutic target clinically. However, most migration assays used in research are relatively low throughput and not easily compatible with rapid analysis or high-throughput screening (HTS) protocols required for drug screening assays. We therefore investigated the quantification of the migration of human leukocytes using the Molecular Devices high-content Discovery-1 platform or PerkinElmer ATPlite assay compared to manual counting. We have conducted extensive assay validation, investigating the detection limits, sensitivity, and precision of each method to count human leukocytes. Leukocyte migration assays were conducted using 96-well HTS-Transwell plates and the potent chemokine stromal cell-derived factor-1 (SDF-1). We reveal that the Discovery-1 and ATPlite methods developed here provide useful approaches to quantify leukocyte migration in an HTS manner with high levels of detection, sensitivity, and precision.     

微载体规模化培养细胞的研究

通过实验探索使用微载体进行动物细胞规模化培养,以期达到建立规模化生产病毒疫苗的目的。实验研究了Vero细胞的生长曲线,以及对细胞生长过程中影响细胞生长的葡萄糖、氨含量两个主要因素的变化规律以及微载体浓度与细胞密度的关系。通过实验发现微载体规模化培养细胞易于操作,比传统转瓶培养的细胞密度高,封闭式的培养方式不但减少了污染几率,而且可以充分保证疫苗的质量。最终找出适宜疫苗培养的微载体使用浓度为2.5g/L,适宜的细胞接种浓度为:1~5×105cell/m l。     

Engineering stem cell fate with biochemical and biomechanical properties of microcarriers

Microcarriers have been widely used for various biotechnology applications because of their high scale‐up potential, high reproducibility in regulating cellular behavior, and well‐documented compliance with current Good Manufacturing Practices (cGMP). Recently, microcarriers have been emerging as a novel approach for stem cell expansion and differentiation, enabling potential scale‐up of stem cell‐derived products in large bioreactors. This review summarizes recent advances of using microcarriers in mesenchymal stem cell (MSC) and pluripotent stem cell (PSC) cultures. From the reported data, efficient expansion and differentiation of stem cells on microcarriers rely on their ability to modulate cell shape (i.e. round or spreading) and cell organization (i.e. aggregate size). Nonetheless, current screening of microcarriers remains empirical, and accurate understanding of how stem cells interact with microcarriers still remains unknown. This review suggests that accurate characterization of biochemical and biomechanical properties of microcarriers is required to fully exploit their potential in regulating stem cell fate decision. Due to the variety of microcarriers, such detailed analyses should lead to the rational design of application‐specific microcarriers, enabling the exploitation of reproducible effects for large scale biomedical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1354–1366, 2013     

Sigpep: calculating unique peptide signature transition sets in a complete proteome background

We have created a new software platform called sigpep that analyzes transition redundancy in selected reaction monitoring assays. Building on this platform, we also created a web application to generate transition sets with unique signatures for targeted peptides. The platform has been made available under the permissive Apache 2.0 open-source license, and the web application can be accessed from http://iomics.ugent.be/sigpep.     

A cellular automaton model for microcarrier cultures

In order to achieve high cell densities anchoragedependent cells are commonly cultured on microcarriers, where spatial restrictions to cell growth complicates the determination of the growth kinetics. To design and operate large-scale bioreactors for microcarrier cultures, the effect of this spatial restriction to growth, referred to as contact inhibition, must be decoupled from the growth kinetics. In this article, a cellular automaton approach is recommended to model the growth of anchorage-dependent cells on microcarriers. The proposed model is simple to apply yet provides an accurate representation of contact-inhibited cell growth on microcarriers. The distribution of the number of neighboring cells per cell, microcarrier surface areas, and inoculation densities are taken into account with this model. When compared with experimental data for Vero and MRC-5 microcarrier cultures, the cellular automaton predictions were very good. Furthermore, the model can be used to generate contact-inhibition growth curves to decouple the effect of contact-inhibition from growth kinetics. With this information, the accurate determination of kinetic parameters, such as nutrient uptake rates, and the effects of other environmental factors, such as toxin levels, may be determined. (c) 1994 John Wiley & Sons, Inc.     

Serial propagation of mammalian cells on microcarriers

For the large-scale operation of microcarrier culture to be successful, a technically feasible method for sequential inoculation is essential. Using human foreskin fibroblasts, FS-4, we have achieved this by detaching cells viably from microcarriers employing a selection pH trypsinization technique. Cells thus detached are able to reattach to microcarriers and grow normally after subsequent reinoculation into new cultures. However, after reinoculation cells attach to new microcarriers at a higher rate than to used microcarriers on which cells have previously grown. The effect of this differential cell attachment was analyzed and overcome by employing a low inoculum concentration. FS-4 cells could thus be serially propagated on microcarriers and subsequently used for beta-interferon production. This technique has also been applied to the cultivation of a monkey kidney cell line, Vero. We have also shown that Vero cells directly inoculated from a seed microcarrier culture could be used for virus production.     

TipChip: a modular,MEMS‐based platform for experimentation and phenotyping of tip‐growing cells

Large‐scale phenotyping of tip‐growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high‐throughput execution of more sophisticated assays, an experimental platform, the TipChip, was developed based on microfluidic and microelectromechanical systems (MEMS) technology. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels equipped with microscopic experimental set‐ups. The tip‐growing cells (pollen tubes, filamentous yeast or fungal hyphae) may be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform, we were able to answer several outstanding questions on pollen tube growth. We established that, unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air, raising the question of how and where water is taken up by the cell. The platform opens new avenues for more efficient experimentation and large‐scale phenotyping of tip‐growing cells under precisely controlled, reproducible conditions.