Pavak K. Shah

Micro total analysis systems: Fundamental advances and applications in the laboratory, clinic, and field

Applications of micro total analysis systems (μTAS) span basic-science research, clinical medicine, and field work. Assay devices designed for these applications offer improvements to existing methods or provide fundamentally new strategies. Both mature methods and novel techniques have benefited from the increased throughput, integration and miniaturization afforded by μTAS. Traditional assays such Western blots and binding assays are recapitulated in a μTAS format but with reduced reagent usage, decreased performance times and added capabilities. An increasingly vibrant area is the performance of drug screening and toxicology assays on-chip, enabling the efficient screening of very large numbers of molecules. Similarly, recent μTAS reactors demonstrate greater chemical synthetic yields and novel product synthesis compared to macro-systems, often as a result of accurate control over reaction conditions including precision reagent dispensing. These exciting systems are now enabling on-site production of short-lived radioactive compounds for medical applications. The greatest impact of μTAS may very well be the ability to perform massively parallel laboratory experiments, for example, the use millions of reaction vessels or the analysis of hundreds of thousands of single cells. Another strength of μTAS lies in the creation of multicellular communities, for example, the combination of many cell types into an interacting system to explore intercellular communication. Devices with multiple layers of co-cultured tissues benefit from precise placement of molecules, such as extracellular matrices or growth factors, in both space and time. Similarly, the complexity and variety of organ-on-chip and organism-on-chip technologies continues to escalate rapidly. Impressively, the types of organisms cultured on-chip now range from the simplest bacteria to complex animals such as fish. Automation, reliability, and integration must all increase as a device moves from the specialist environment of a lab to usage by non-expert personnel in the outside world, for example, at a clinical point-of-care or in environmental monitoring. Key innovations in recent months result in devices that operate with minimal external equipment, error-free operation, and unambiguous readouts, all critical for operation by untrained personnel. Lightweight, portable devices are increasingly used to identify chemical and biological toxins in water, air and soil with applications in public health, defense, and homeland security. Perhaps most exciting is the development of μTAS with sufficient robustness for operation in challenging environments, such as the ocean and outer space. A central component of these systems is the ability to withstand the unexpected. These systems push the boundaries of current integration principles and spur rapid growth of new design philosophies. This review focuses on advances in the area of μTAS or “lab-on-a-chip” systems over the time span of May 2011 through September 2012 with a focus on applications in basic research, clinical medicine and field usage. A range of journals with 2011 impact factors from 2.0 to 36.3 were screened to cover publications with highly specialized content as well as those directed at multidisciplinary audiences. These publications included discipline-specific journals such as Analytical Chemistry and Lab on a Chip as well as general scientific publications, e.g. Science and Nature. To identify material beyond the individually examined journals, extensive key word searches in databases such as PubMed, SciFinder, and Web of Science were performed. Recent reviews in the area of μTAS were also examined for appropriate references. Care was taken to identify impactful and exciting work from across the globe. Well over a thousand papers in the three target areas were identified and discussed. Due to space limitations, we were unable to include all papers but instead incorporated those most fitting into the review scheme and those reporting innovations in basic microdevice technology as well as in applications to biological, physical and engineering sciences. We apologize in advance for omitted papers and welcome feedback regarding any oversights on our part.

Trapping cells on a stretchable microwell array for single–cell analysis

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.

Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells.

Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). The analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but ongoing degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically relevant analytes and time-dependent processes in large numbers of single cells.

Scalable synthesis of a biocompatible, transparent and superparamagnetic photoresist for microdevice fabrication

The functionalization of photoresists with colloids has enabled the development of novel active and passive components for microfabricated devices. Incorporation of colloidal particles often results in undesirable reductions in photolithographic fidelity and device transparency. We present a novel photoresist composite incorporating poly(methyl methacrylate-co-methacrylic acid) (PMMA/MMA), the epoxy resin 1002F and colloidal maghemite nanoparticles to produce a stable, transparent and biocompatible photoresist. The composite photoresist was prepared in a scalable fashion in batches up to 1 kg with the particles remaining dispersed during room-temperature storage for at least 6 months. Following photolithography to form films, the nanoparticle size remained well below that of visible-light wavelengths as demonstrated by electron microscopy. Structures fabricated from the photoresist by conventional photolithography displayed aspect ratios greater than ten. When grown on the photoresist, the metabolic rate of HeLa cells was unchanged relative to cells grown on glass. Primary murine mesenchymal stem cells also displayed a normal morphology on the resist surface. The ability to manipulate microstructures formed from the composite was demonstrated by magnetically collecting clonal colonies of HeLa cells from a micropallet array. The transparency, biocompatibility, scalable synthesis and superparamagnetic properties of the novel composite address key limitations of existing magnetic composites.

Small sample sorting of primary adherent cells by automated micropallet imaging and release

Primary patient samples are the gold standard for molecular investigations of tumor biology yet are difficult to acquire, heterogeneous in nature and variable in size. Patient‐derived xenografts (PDXs) comprised of primary tumor tissue cultured in host organisms such as nude mice permit the propagation of human tumor samples in an in vivo environment and closely mimic the phenotype and gene expression profile of the primary tumor. Although PDX models reduce the cost and complexity of acquiring sample tissue and permit repeated sampling of the primary tumor, these samples are typically contaminated by immune, blood, and vascular tissues from the host organism while also being limited in size. For very small tissue samples (on the order of 103 cells) purification by fluorescence‐activated cell sorting (FACS) is not feasible while magnetic activated cell sorting (MACS) of small samples results in very low purity, low yield, and poor viability. We developed a platform for imaging cytometry integrated with micropallet array technology to perform automated cell sorting on very small samples obtained from PDX models of pancreatic and colorectal cancer using antibody staining of EpCAM (CD326) as a selection criteria. These data demonstrate the ability to automate and efficiently separate samples with very low number of cells.

Erratum to: Trapping cells on a stretchable microwell array for single-cell analysis

The authors wish to correct an error in the manuscript “Trapping cells on a stretchable microwell array for singlecell analysis” published in Analytical and Bioanalytical Chemistry (2012) 402:1065-1072. In the article it was stated that the LiveCell ArrayTM was a product of the NUNC Company and that it was no longer commercially available. We have since learned that the LiveCell ArrayTM is produced by Molecular Cytomics, Inc. and can now be purchased through their website and other distributors.

Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening

Wnt/β-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/β-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/β-catenin signaling were screened over short (<6) and long (>25) generational timescales. To characterize phenotypic divergence over these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1119 clonal colonies in parallel. This screen revealed phenotypic divergence after <6 generations at a similar scale to that observed in monoclonal cell lines cultured for >25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provides insight into the pathways mechanisms of positive feedback and self-inhibition.