Abstract
Microfluidic Chip Technology
Microfluidic Processor Allows Rapid HER2 Immunohistochemistry of Breast Carcinomas and Significantly Reduces Ambiguous (2+) Readouts
Biomarkers analysis has become an established method for improving cancer detection and treatment. Depending on the presence or absence of specific biomarkers, certain predictions can be made regarding the severity of cancer as well as the success of different therapeutic regimens. One of the best examples of the success of biomarker analysis in cancer diagnosis and therapy is the identification of HER2+ breast cancers. HER2 amplification in breast cancer represents a significant subtype of breast cancer that can be treated with HER2-specific therapeutic antibodies. Conventional biomarker analysis by immunohistochemistry is time- and labor-consuming. Automation and miniaturization of this process significantly reduces the cost related to adding biomarker analysis to any cancer management plan. In this report, a glass/silicone microfluidic chamber for processing breast cancer tissue is capable of reducing tissue-staining time to only 2 min. Particularly interesting is the comparison of this microfluidic processor to conventional macroscopic methods analyzing a set of 76 clinical samples. Microfluidic-based methods do not result in any false negatives or false positives, and the number of ambiguous cases is reduced by 90%. As such, this report demonstrates a clear clinical advantage of microfluidic chip technology in improving the efficiency as well as accuracy of biomarker analysis in cancer diagnosis. (Cliftlik, A. T., et al., Proc Natl. Acad. Sci. U. S. A.
Mutation Scanning Using MUT-MAP, a High-Throughput, Microfluidic Chip–Based, Multianalyte Panel
Identification of mutated genes can serve as a prognostic biomarker method for diagnosing cancer as well as informing the best therapeutic options. Furthermore, identification of mutated genes can serve as a useful tool for identifying novel drug targets. Mutated genes are often identified by quantitative allele-specific polymerase chain reaction (AS-PCR). Complicating this analysis is that DNA from fixed tissue is often degraded or cross-linked by the fixation process that serves to preserve cancer tissue for multiple diagnostic studies. Traditional methods of detecting mutations also require significant amounts of DNA that may not necessarily be easily accessible by clinicians. Using microfluidic chip technology, these authors develop a high-throughput method for detecting multiple mutations in a single sample. Choosing six genes that are commonly mutated across a wide variety of cancers, they are able to analyze 71 different cancer-relevant mutations. This is accomplished with as little as 2 ng of genomic DNA compared with 0.5 to 1 µg of DNA by traditional methods. Analyzing both cancer cell lines and clinical samples, they are able to demonstrate the benefits of microfluidic chip technology in a clinical setting as it relates to multiple mutation analysis of cancer tissue. (Patel, R., et al., PLoS One
Inertial Focusing for Tumor Antigen-Dependent and Antigen-Independent Sorting of Rare Circulating Tumor Cells
Isolation of circulating tumor cells (CTCs) has gained intense interest recently as these rare populations of cells have been linked to metastasis and tumor-initiating cancer stem cells. Isolation of CTCs has the potential to allow clinicians to gain greater insight into the properties and identity of these tumor-initiating cancer stem cells that would inform the diagnosis and treatment of cancer. Because CTCs are so rare in blood, it is difficult to isolate CTCs in useable amounts by conventional methods.
Previously, the Toner group demonstrated a microfluidic approach to isolating CTCs that used microposts to isolate EpCAM-positive CTCs from whole blood. One drawback of this method was that the isolated CTCs were immobilized to the microchip and could not be readily dislodged and used for further analysis by standard macroscopic means.
To address this drawback, the authors incorporate a magnetic cell-sorting function into their microfluidic chip that allows for the isolation of CTCs in suspension in an antigen-dependent and antigen-independent manner. In tests with cancer cell lines that express high levels of EpCAM, this method allows for approximately 90% or greater recovery of cancer cells from whole-blood solutions. When applied to prostate cancer patient samples, this approach proves capable of isolating CTCs with a much greater sensitivity than current Food and Drug Administration (FDA)–approved methods. In addition to demonstrating the superior clinical ability of this technology, these authors use their chips to study the biological properties of these CTCs, identifying novel heterogeneity among CTCs within a single patient sample that have implications in identifying drug-resistant and tumor-initiating CTCs. (Ozkumur, E., et al., Sci. Transl. Med.
An Automated High-Throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood
Most current approaches to isolating CTCs rely on the expression of EpCAM on the surface of these cells. The FDA-approved method, CellSearch, requires the expression of this protein for detection of cancer cells. For some cancers, a single biomarker is insufficient for the detection of the entire population of CTCs. By first labeling whole blood with multiple antibodies, these authors are capable of detecting CTCs through expression of EpCAM in combination with other important cancer markers in a flow-through microfluidic chip. Using breast cancer patient samples, they are capable of identifying and counting EpCAM+/Her2+ as well as EpCAM+/CD44+ CTCs. Her2+ breast cancer cells are responsive to Her2-specific therapy, while CD44+ breast cancer cells have been identified as tumor-initiating cancer stem cells. To determine the clinical relevance of their technology, the authors have conducted a 2-year study analyzing 90 blood samples from 24 patients with stage IV metastatic breast cancer, comparing their approach with the FDA-approved CellSearch system. Using the standard method, only 22% of samples have CTCs based on EpCAM expression, while the authors’ approach results in the identification of CTCs in 60% of samples with additional identification of CD44+ CTCs in 30 samples. (Zhao, M., et al., Anal. Chem.
A Programmable Microenvironment for Cellular Studies via Microfluidics-Generated Double Emulsions
Miniaturization of reaction volumes of cellular studies is an important technological advance toward improving the cost and efficiency of high-throughput drug discovery and screening. Furthermore, microscale bioreactors can facilitate cellular studies under more controlled conditions than those available in macroscale studies. Many initial approaches to compartmentalized microenvironments for cellular studies involve the isolation of cells in arrays of microwells. A drawback of this approach is that it often requires continuous flow-through of chemicals and biological agents that are required to maintain the appropriate microenvironments. This flow could result in contamination among wells. One method for overcoming this limitation is compartmentalization in discrete aqueous droplets dispersed in oil. For certain cellular studies, however, oil emulsions are not desirable due to limitations in nutrient supplementation as well as the incompatibility of oil with certain analytical techniques such as flow cytometry.
The authors of this report use a microfluidic approach to add another aqueous layer to form stable and monodispersed water-in-oil-in-water emulsions. Furthermore, they are capable of encapsulating reporter gene–expressing bacteria in these double emulsions. Following entrapment, they are able to control the delivery of chemical nutrients to regulate reporter gene expression as well as study oscillations of bacterial density. This work demonstrates the ability of their microfluidic chips in fabricating reliable double emulsion microenvironments that should be effective in studying cellular functions in a high-throughput, automated manner. (Zhang, Y., et al., Biomaterials
Dissecting Genealogy and Cell Cycle as Sources of Cell-to-Cell Variability in MAPK Signaling Using High-Throughput Lineage Tracking
When conducting cellular studies, it is clear that cells with identical genetics will often respond differently to the same external stimuli. Furthermore, this heterogeneous response is inheritable in a nongenetic manner. The variability in responses has been attributed to a variety of factors, but the quantitative study of these factors such as cell cycle and activation of specific pathways is difficult to study in a large population of cells by normal cell culture.
The authors of this study develop a high-throughput and fully automated microfluidic live imaging system in which to concurrently quantify multiple cellular properties of multiple yeast strains under dynamically changing culture conditions. Using this system, the authors investigate how yeast response to pheromones is affected by cell cycle and nongenetic heritability. Specific lineages of yeast that respond uniquely to stimuli and that would normally be masked by the bulk mass could be identified and followed. In addition, quantitative analysis of cell cycle is able to be tracked and linked to heterogeneous populations that would have otherwise been hidden. (Ricicova, M., et al., Proc. Natl. Acad. Sci. U. S. A.
Suspended Microfluidics
Microfluidics has proven useful in increasing the efficiency and lowering the costs related to a number of biological and medical applications. There are, however, significant barriers to entry in terms of ease of design while maintaining high levels of functionality. Open microfluidic systems with their open air-water interface address this issue with the ability to provide complex functionality in an accessible system with simpler fabrication requirements compared with older microfluidic platforms. While promising, some areas of improvement prevent full acceptance of open microfluidic systems. Specifically, more robust design rules that relate to improving control of fluid flow are needed.
These authors describe the application of a microscale capillary flow system, termed suspended microfluidics, in which a wide range of fluids is able to flow through open microchannels. To achieve optimal microscale capillary flow through an open environment, the authors identify a number of design requirements, including maximum width of channel, critical channel depth, contact angle, and overall shape. Using two series of channels connected to an aperture, an array of nanoliter hydrogel deposits is created that can be fed with combinations of liquids for multiplexed high-throughput cellular studies. This array is then validated in the study of cell invasion in a unique membrane-free cell invasion assay. Furthermore, the ability to mix immiscible organic solvents from a top channel with aqueous cell culture media present in a lower chamber is applied to the study of metabolic response, further demonstrating the wide range of biological studies that can be done in a high-throughput manner with this system. (Casavant, B. P., et al., Proc. Natl. Acad. Sci. U. S. A.
Single-Cell Antimicrobial Susceptibility Testing by Confined Microchannels and Electrokinetic Loading
With the widespread use of antibiotics for both the prevention and treatment of bacterial infections of all types, a serious medical problem is emerging in the form of multidrug-resistant bacterial strains. The current approach to determining effective treatment against infection involves antimicrobial susceptibility testing (AST) of bacteria found in patient samples. This procedure is cumbersome as the samples are often processed offsite at a clinical microbiology laboratory with results requiring a wait time of 48 to 72 h.
Microfluidic chip technology has the potential to bring this analysis back to the clinic and potentially into the field as well, providing more efficient diagnosis and treatment against drug-resistant bacterial infections. In this report, the authors describe the fabrication of a single-cell AST system that uses confined microchannels and electrokinetic loading to isolate single uropathogenic Escherichia coli cells into specific locations on the chip for study. To achieve this, the authors conduct a number of experiments to determine the optimal applied voltage needed to maximize loading while not impairing bacterial function. Analyzing bacterial strains in the context of three antibiotics, the authors demonstrate an efficient AST of single bacterial cells within 1 h. As such, this work demonstrates the potential of the platform to bring fast and efficient AST to the clinic and beyond. (Lu, Y., et al., Anal. Chem.
Microfluidic Heart on a Chip for Higher Throughput Pharmacological Studies
A major hurdle in drug development is the potential for toxic effects on normal organs. In particular, cardiac toxicity remains a common hurdle for both drugs in clinical trials and existing drugs. Technology that improves our ability to screen for such toxic effects in vitro will streamline the development of drugs as well as make it more cost-effective by eliminating drug candidates at an earlier stage that would likely fail at later, more expensive clinical trial stages due to cardiac toxicity. The use of cardiac myocytes in standard tissue culture to determine toxicity is not truly biologically representative and is ineffective at predicting all toxic effects. A better method is to grow cardiac cells on an elastomeric substrate, known as the muscular thin-film (MTF) assay.
This assay allows for the measurement of contractibility and tissue structure when treated with candidate drugs. In this report, the authors develop an improved MTF assay through laser-based fabricated thin PDMS cantilevers. Furthermore, they design a fully enclosed microdevice for the chip, which allows for precise control over drug flow in and drug flow out. Other design considerations that aid in the development of a high-throughput cardiac function assay device include a heated metallic base as well as a transparent top for analysis. The authors are able to use their device to interrogate the effects of tissue structure on cardiac function as well as perform drug dose-response studies, thus demonstrating the potential of organ-on-a-chip application in drug development as well as biological research. (Agarwal, A., et al., Lab. Chip
Automated High-Throughput Screening
Automated Disposable Small-Scale Reactor for High-Throughput Bioprocess Development: A Proof-of-Concept Study
Biologics, including antibodies and therapeutic cytokines, continue to gain drug market share as an important option in the treatment of a wide range of diseases. Because of this, there is an ever-increasing interest in the development of novel biologics. One drawback to the development of biologics is the inordinate amount of work needed to develop the bioprocesses by which biologics are grown and purified. The development of automated high-throughput methods for identifying the optimal clones and growth conditions for making biologics is important to lowering the cost and time it takes to bring biologics to the market.
To achieve this, the authors describe the development of a prototype small-scale bioreactor that combines single-use disposable technology with high-throughput automation to lower the cost and time needed to evaluate bioprocesses that can be easily scaled up to large-scale bioreactors. Using a 250-mL small-scale disposable reactor, the authors are able to achieve equivalent performance with respect to antibody titer production when compared with 30-L stainless steel and 3-L glass reactors. Similar equivalent performance also is seen when the small-scale reactor is applied to recombinant protein production as well. (Bareither, R., et al., Biotechnol. Bioeng.
Clinical Performance of an Automated Stool DNA Assay for Detection of Colorectal Neoplasia
Early, noninvasive detection of cancer continues to be an important area of development in the fight against cancer. Stool DNA analysis has proven to be an effective method for detection of colon cancer, including early detection of colon cancer while at the curable stage. For stool DNA analysis to be effective from a public health perspective, the procedure must be widely accessible, cost-effective, and efficient.
The authors of this study describe the development of an automated system for stool DNA assays to detect colon cancer. In this system, the process of DNA isolation from stool as well as setting up the quantitative PCR plates is fully automated, with the only manual step requiring sealing and transfer of plates to the thermal cycler. The assay is able to detect gene mutations as well as aberrantly methylated genes that have been linked to colon cancer. Using this automated platform in the clinical setting, the authors can identify colon cancer–bearing individuals with 98% sensitivity. For some patients with stage II or stage IV colon cancer, the sensitivity is 100%. Thus, this study demonstrates the fidelity of an automated system for detection of colon cancer with wider implications in other areas of disease detection for stool and other easily accessible biological samples. (Lidgard, G. P., et al., Clin. Gastroenterol. Hepatol.
A High-Throughput Assay for the Detection of Tyr-Phosphorylated Proteins in Urine of Patients with Bladder Cancer
Aberrant tyrosine (Tyr) phosphorylation of proteins is a common contributor to cancer initiation and progression. As such, detection of aberrant Tyr phosphorylation as well as the development of inhibitors remains attractive areas of cancer diagnostic and therapeutic development.
Previously, these authors demonstrated that Tyr-phosphorylated proteins could be detected in the urine of patients with bladder cancer at a 5-fold greater level than seen in urine from normal healthy patients. The method by which this was done, however, was time- and labor-consuming as it was done by traditional benchtop molecular biological methods. To make this method of bladder cancer detection more viable, an automated high-throughput method that can reliably detect changes in phosphorylation of proteins at a sufficient level of sensitivity is required. Here, the authors describe the use of an immobilized metal affinity chromatography (IMAC) system that is miniaturized to a 96-well format and integrated with an automated, highly sensitive detection platform for a more high-throughput screening method. Using this system, the authors are able to achieve higher sensitivity with the miniaturized IMAC system compared with standard IMAC procedures. When applied to clinical samples, the system is able to achieve 87% sensitivity and 95% specificity, thus confirming the validity of Tyr-phosphorylated proteins as bladder cancer markers and the viability of an automated high-throughput platform in performing the test in a reliable and sensitive manner. (Khadjavi, A., et al., Biochim. Biophys. Acta
A Fully Unsupervised Compartment-on-Demand Platform for Precise Nanoliter Assays of Time-Dependent Steady-State Enzyme Kinetics and Inhibition
Large-scale combinatorial assays are often required when evaluating and understanding potential chemical and biologic drug candidates. These assays often provide the initial screening step of drug screening as it relates to enzymatic activity or other protein function. Previously, only companies with large-scale capital expenditures were capable of doing these studies in an effective and timely manner that involved a large amount of labor and equipment. Microfluidic technology has allowed for the miniaturization of these processes and has made these screening assays more accessible to those with labor and capital constraints. Specifically, droplet-based microfluidics has allowed for even further reductions in reaction volume.
In this report, the authors describe the development of compartment-on-demand platforms for the generation of water-in-oil emulsion droplets for the fabrication of precise nanoliter reaction volumes. Furthermore, this system is fully automated and capable of rapidly screening chemical and biological libraries in a high-throughput manner. Enzymatic kinetic parameters can be determined within 20 min in a fully automated manner. When compared with conventional methods, the automated droplet-based platform performs just as well despite a >104-fold reduction in reaction volume. (Gielen, F., et al., Anal. Chem.
An Automated System for High-Throughput Single-Cell–Based Breeding
Cell-based therapeutics is becoming increasingly important in drug development. Already, biologics are among the top-selling drugs and will continue to be for the foreseeable near future. In addition to producing biologics, cell-based therapeutics also includes the use of isolated cells themselves for therapeutics, including bone marrow transplants, stem cell–based therapy, and other applications. Colony cell-based screening has proven to be flawed, as there is now definitive evidence of heterogeneity within these colonies that requires single-cell screening and expansion for optimal development of cell-based therapeutics. Currently, the cost to develop and implement single-cell–based therapeutics is too high as the screening and expansion of therapeutically competent cells is inefficient and time- and labor-consuming. To identify single cells within a colony, flow cytometry is often used, which requires a skilled technician to handle a large number of cells while the procedure itself may damage rare valuable cells through mechanical stress.
The authors of this study report the use of an automated system for high-throughput single-cell breeding. Consisting of a microchamber array chip paired to an automated micromanipulator and a fluorescent microscope, this robot is capable of identifying a single positive embryonic stem (ES) cell from 5.0 × 104 cells. Daughter cells from this single ES cell demonstrate greater markers of pluripotency potential than those from the original unsorted parental population. Further demonstrating the potential of this system, the authors screen hybdridomas in this platform and are able to identify single-cell hybdridomas with the highest antibody production capability within 1 day. Thus, it is clear that automated high-throughput approaches are capable of improving single-cell screening for the purposes of cell-based therapeutics and identification of optimal biologics-producing clones. (Yoshimoto, N., et al., Sci. Rep.
Efficient Drug Screening and Gene Correction for Treating Liver Disease Using Patient-Specific Stem Cells
A goal of stem cell–based therapy is the use of patient-specific stem cells to screen and identify novel therapeutics that will be more effective for that specific patient and those like them. Induced pluripotent stem cells (iPSCs) have further extended that promise with the ability to provide a source of stem cells that would otherwise be rare and difficult to collect. The contribution of iPSCs to medicine, especially in the field of drug screening, remains elusive because of the inefficiencies in the conventional methods of cell growth, screening, and validation.
In this report, the authors apply high-throughput screening technology to iPSCs derived from a patient with a mutation that is commonly associated with liver disorders. To accomplish this, the authors differentiate iPSCs into hepatocytes prior to plating into a 96-well format. Following differentiation and growth of iPSC-derived hepatocytes, a high-throughput immunofluorescence reader analyzes the 96-well plates. When applied to a drug screen of clinically ready drugs, 43 promising candidates are identified, allowing for more efficient identification of the most promising candidate by confirmatory assays. In addition to drug screening, high-throughput immunofluorescent screening is applied to gene correction experiments of iPSCs to identify iPSCs that have undergone proper gene correction. As such, these researchers demonstrate the validity of high-throughput screening of iPSC-derived cells for both drug development and gene corrections. Integration of this approach in early stage drug development and stem cell–based therapeutics should help iPSCs achieve the clinical potential that was so evident when they were first identified. (Choi, S. M., et al., Hepatology