Abstract
Laboratory Automation and High-Throughput Chemistry
Circulating Tumor Cells (CTCs): Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell
Contemporary cancer diagnostics are becoming increasing reliant on sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare CTCs require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability for use in a clinical setting, or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.
Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy. This platform uses high-throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore, the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (coefficient of variation, <25%) down to at least 9 CTC/mL. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Nonspecific recovery of background cells is independent of target cell density and averages 55 cells/mL. Ten percent purity can be achieved with as low as 6 CTCs/mL, and better than 1% purity can be achieved with 1 CTC/mL.
The significance of this study is that the LiquidBiopsy platform is an automated validated platform that provides high-throughput molecular access to the CTC population. It can be validated and integrated into the lab flow, enabling CTC enumeration as well as recovery of consistently high-purity samples for molecular analysis such as quantitative PCR and next-generation sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs (Winer-Jones, J. P., et al., PloS One,
A Novel Platform for Automated High-Throughput Fluxome Profiling of Metabolic Variants
Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, Heux et al. developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for (13)C-labeling experiments and sampling of labeled material with nuclear magnetic resonance–based isotopic fingerprinting and automated data interpretation. As a proof of concept, this workflow is applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants are clearly discriminated, whereas pathways that support metabolic flexibility toward modulation of a single enzyme are elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources, and screening capacity are achieved. This will undoubtedly facilitate the development of efficient cell factories (Heux, S., et al., Metab. Eng.,
A Quantitative Liposome Microarray to Systematically Characterize Protein-Lipid Interactions
Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds, and signaling molecules, but they are still largely underrepresented in known biological networks. Saliba et al. describe a liposome microarray–based assay, a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed, and high-throughput manner (Saliba, A. E., et al., Nat. Methods,
Large-Scale De Novo DNA Synthesis: Technologies and Applications
For more than 60 y, the synthetic production of new DNA sequences has helped researchers understand and engineer biology. Kosuri and Church summarize methods and caveats for the de novo synthesis of DNA, with particular emphasis on recent technologies that allow for large-scale and low-cost production. In addition, they discuss emerging applications enabled by large-scale de novo DNA constructs, as well as the challenges and opportunities that lie ahead (Kosuri, S., and Church, G. M., Nat. Methods,
Large-Scale Cell Production of Stem Cells for Clinical Application Using the Automated Cell-Processing Machine
Cell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, Kami et al. focus on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported to date. The system AutoCulture used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. Kami et al. examine the feasibility of this system in a clinical setting.
The authors observe similar characteristics by both culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture is a feasible method for the cultivation of human cells for regenerative medicine. An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products (Kami, D., et al., BMC Biotechnol.,
New High-Throughput Screening Method for Drug Release Measurements
In the field of drug delivery systems, microparticles made of polymeric matrix appear as an attractive approach. The in vitro release kinetic profile is crucial information when developing new particulate formulations. These data are essential for batch-to-batch comparison, quality control, and anticipation of in vivo behavior to select the best formulation to go further in preclinical investigations. The methods available present common drawbacks such as the time and compound consumption that do not fit with formulation screening requirements in the early development stages. In this study, a new microscale high-throughput screening (HTS) method investigates drug release kinetics from piroxicam-loaded polylactic acid and polylactic-co-glycolic acid microparticles. The method is sample and separation based, in which separation is performed by filtration using 96-well microfilter plates. Ninety-six experiments can therefore be performed on one plate in one time in a fully automated way and with a very low sample and particle consumption. The influence of different parameters controlling release profiles is also investigated using this technique. The HTS method gives the same release profile as the standard dialysis method. Shaking, particle concentration, and the nature of the release medium are found to be of influence. The HTS method appears to be a reliable method for evaluating drug release from particles, with a smaller standard deviation and less consumption of material (Pelczarska, A., et al., Eur. J. Pharm. Biopharm.,
Microfluidic Chip Technology and Microreactor Technology
Accessing New Chemical Entities through Microfluidic Systems
Flow systems have been successfully used for a wide variety of applications in chemical research and development, including the miniaturization of (bio)analytical methods and synthetic (bio)organic chemistry. Rodrigues et al. are witnessing the growing use of microfluidic technologies for the discovery of new chemical entities. As a consequence, chemical biology and molecular medicine research are being reshaped by this technique. In this mini review, the authors portray the state of the art, including the most recent advances in the application of microchip reactors as well as the micro- and mesoscale coil reactor–assisted synthesis of bioactive small molecules and forecast the potential future use of this promising technology (Rodrigues, T., et al., Angew Chem. Int. Ed. Engl.,
A Novel Tool for Dynamic Cell Adhesion Studies: The De-Adhesion Number Investigator (DANI)
For an optimal implementation of materials, such as medical implants in living environments, a thorough characterization of cell adhesion, kinetics, and strength is required, as well as a prerequisite (e.g., for bone integration). Hartmann et al. present a miniaturized (~100 µL) lab-on-a-chip implant hybrid system that allows quantification of cell adhesion under dynamic conditions, mimicking those of physiological relevance. Surface acoustic waves are excited and used on optical transparent chips to induce micro acoustic streaming and to create a microfluidic shear spectrum ranging from 0 to ~35 s(–1). The authors demonstrate its potential for a time-efficient, dynamic screening test of new implant materials using a model of an osseointegration with SAOS-2 cells. The upside-down orientation also allows use of the micro reactor on nontransparent materials such as titanium and diamond-like carbon (Hartmann, A., et al., Lab Chip,
Microfluidic Bead Suspension Hopper
Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. Price et al., from the Scripps Research Institute, developed a bead suspension hopper that exploits sedimentation to load beads into a microfluidic droplet generator.
A suspension hopper continuously delivers synthesis resin beads (17 µm diameter, 112,000 over 2.67 h) functionalized with a photolabile linker and pepstatin A into picoliter-scale droplets of an HIV-1 protease activity assay to model ultraminiaturized compound screening. Likewise, trypsinogen template DNA-coated magnetic beads (2.8 µm diameter, 176,000 over 5.5 h) are loaded into droplets of an in vitro transcription/translation system to model a protein evolution experiment. The suspension hopper should effectively remove any barriers to using suspensions as sample inputs, paving the way for microfluidic automation to replace robotic library distribution (Price, A. K., et al., Anal. Chem.,
Multiplexed Analysis of Protein-Ligand Interactions by Fluorescence Anisotropy in a Microfluidic Platform
Homogeneous assay platforms for measuring protein-ligand interactions are highly valued because of their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive because of the large amounts of reagents required and the need for automation. Cheow et al. implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. The authors demonstrate the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different posttranslationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays yet requires two orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification (Cheow et al., Anal. Chem.,
Single-Cell Microfluidics: Opportunity for Bioprocess Development
Cell-to-cell heterogeneity in microbial biotechnological processes caused by biological (intrinsic) and environmental (extrinsic) fluctuations can have a severe impact on productivity. However, little is known about the complex interplay between environmental reactor dynamics and cellular activity. A few years ago, innovative microfluidic systems were introduced facilitating the spatiotemporal analysis of single cells under well-defined environmental conditions, allowing so far unachievable insights into population heterogeneity and bioreactor inhomogeneity. Examples of microfabricated systems include microfluidic cavities harboring micropopulations of several thousand cells down to femtoliter-size structures entrapping individual bacteria. In well-defined perfusion experiments, central questions in biotechnology regarding, for example, growth, productivity, and heterogeneity on the single-cell level have been addressed for the first time. Microfluidics will take its place as a single-cell analytical technique in biotechnological process and strain characterization (Grunberger, A., et al., Curr. Opin. Biotechnol.,
Single-Use Disposable Technologies for Biopharmaceutical Manufacturing
The manufacture of protein biopharmaceuticals is conducted under current good manufacturing practice and involves multiple unit operations for upstream production and downstream purification. Until recently, production facilities relied on the use of relatively inflexible, hard-piped equipment including large stainless-steel bioreactors and tanks to hold product intermediates and buffers. However, there is an increasing trend toward the adoption of single-use technologies across the manufacturing process. Technical advances have now made an end-to-end single-use manufacturing facility possible, but several aspects of single-use technology require further improvement and are continually evolving. This article provides a perspective on the current state of the art in single-use technologies and highlights trends that will improve performance and increase the market penetration of disposable manufacturing in the future (Shukla, A. A., and Gottschalk, U., Trends Biotechnol.,
Automation Systems
A Smartphone-Controlled Handheld Microfluidic Liquid-Handling System
Microfluidics and lab-on-a-chip technologies have made it possible to manipulate small-volume liquids with unprecedented resolution, automation, and integration. However, most current microfluidic systems still rely on bulky off-chip infrastructures such as compressed pressure sources, syringe pumps, and computers to achieve complex liquid manipulation functions. Li et al. present a handheld automated microfluidic liquid-handling system controlled by a smartphone, which is enabled by combining elastomeric on-chip valves and a compact pneumatic system. As a demonstration, the authors show that the system can automatically perform all the liquid-handling steps of a bead-based HIV1 p24 sandwich immunoassay on a multilayer PDMS chip without any human intervention. The footprint of the system is 6 × 10.5 × 16.5 cm, and the total weight is 829 g, including battery. Powered by a 12.8 V 1500 mAh Li battery, the system consumes 2.2 W on average during the immunoassay and lasts for 8.7 h. This handheld microfluidic liquid-handling platform is generally applicable to many biochemical and cell-based assays requiring complex liquid manipulation and sample preparation steps such as fluorescence in situ hybridization, PCR, flow cytometry, and nucleic acid sequencing. In particular, the integration of this technology with read-out biosensors may help enable the realization of the long-sought Tricorder-like handheld in vitro diagnostic systems (Li, B., et al., Lab Chip, 2014).
New Trends in Robotics for Agriculture: Integration and Assessment of a Real Fleet of Robots
Computer-based sensors and actuators such as global positioning systems, machine vision, and laser-based sensors have progressively been incorporated into mobile robots with the aim of configuring autonomous systems capable of shifting operator activities in agricultural tasks. However, the incorporation of many electronic systems into a robot impairs its reliability and increases its cost. Hardware minimization, as well as software minimization and ease of integration, is essential to obtain feasible robotic systems. A step forward in the application of automatic equipment in agriculture is the use of fleets of robots, in which a number of specialized robots collaborate to accomplish one or several agricultural tasks. This article strives to develop a system architecture for both individual robots and robots working in fleets to improve reliability, decrease complexity and costs, and permit the integration of software from different developers. Several solutions are studied, from a fully distributed to a whole integrated architecture in which a central computer runs all processes. This work also studies diverse topologies for controlling fleets of robots and advances other prospective topologies. The architecture presented in this article is being successfully applied in the RHEA fleet, which comprises three ground mobile units based on a commercial tractor chassis (Emmi, L., et al., Sci. World J.,
Next-Generation Sequencing Is a Credible Strategy for Blood Group Genotyping
Although several medium-/high-throughput tools have been engineered for molecular analysis of blood group genes, they usually rely on the targeting of single nucleotide polymorphisms, while other variants remain unidentified. To circumvent this limitation a strategy for genotyping blood group genes by next-generation sequencing (NGS) was set up. Libraries consisting of exons, flanking introns, and untranslated regions of 18 genes involved in 15 blood systems are generated by the Ion AmpliSeq Library Kit 2.0 and by fragmenting polymerase chain reaction products, normalized by two different approaches, mixed and sequenced by the Ion Torrent Personal Genome Machine Sequencer. As per the laboratory conditions in this study, defined to limit both intra- and intersample variability, sequences from mixed libraries are read in a single run for a total coverage of 86.03% of the coding DNA sequences, including all loci defining the most clinically relevant antigens in all genes, except ABO. Importantly, the challenging attempt to generate gene-specific data for the homologous genes is successful. This work, which combines two complementary approaches to generate libraries, defines technical conditions for genotyping blood group genes, illustrates that NGS is suitable for such an application, and suggests that, after automation, this novel tool could be used for molecular typing at the laboratory level (Fichou, Y., et al., Br J Haematol.,
Advances in Miniaturized Instruments
Advances in Miniaturized Instruments for Genomics
In recent years, a lot of demonstrations of miniaturized instruments were reported for genomic applications. They provide the advantages of miniaturization, automation, sensitivity, and specificity for the development of point-of-care diagnostics. The aim of this article is to report on recent developments on miniaturized instruments for genomic applications. Based on the mature development of microfabrication, microfluidic systems have been demonstrated for various genomic detections. Because one of the objectives of miniaturized instruments is for the development of a point-of-care device, impedimetric detection is found to be a promising technique for this purpose. An in-depth discussion of the impedimetric circuits and systems is included to provide total consideration of the miniaturized instruments and their potential application toward real-time portable imaging in the “-omics” era. The current excellent demonstrations suggest a solid foundation for the development of practical and widespread point-of-care genomic diagnostic devices (Gong, C. S., and Lei, K. F., Biomed. Res. Int.,
Development of a Miniaturized Multi-Turn Time-of-Flight Mass Spectrometer with a Pulsed Fast Atom Bombardment Ion Source
A miniaturized multi-turn time-of-flight (TOF) mass spectrometer with a pulsed fast atom bombardment (FAB) ion source (FAB-MULTUM) has been designed and constructed to overcome the drawbacks associated with magnetic sector–type instruments using a FAB ion source such as size and weight. This instrument consists of a pulsed FAB ion source, a multiturn TOF mass spectrometer, a detector, vacuum system, and electronic circuits. The size and weight of the system are less than H520 mm ×L580 mm × W230 mm and 45 kg (including vacuum pumps and electronic circuits). The achieved resolving power and mass accuracy of this instrument are >25,000 and about 1 ppm, respectively, which are equivalent to those of magnetic sector–type instruments, although the size and weight are much smaller than those of magnetic sector–type instruments. The experimental results lead the authors to conclude that this instrument enables accurate mass measurements and is a powerful tool for the confirmation of synthesized compounds (Nagao, H., et al., Eur. J. Mass Spectrom. (Chichester, Eng).,