Negative enrichment of target cells by microfluidic affinity chromatography

A three-dimensional microfluidic channel was developed for high-purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of nontarget cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multistep processes in microfluidic systems. In previous work (Li, P.; Tian, Y.; Pappas, D. Anal. Chem.2011, 83, 774-781), we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel system showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity and ease of fabrication and use is suitable for cell separations when subsequent analysis of target cells is required.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

A Microfluidic Strategy for Controllable Generation of Water‐in‐Water Droplets as Biocompatible Microcarriers

Droplet microfluidics has been widely applied in functional microparticles fabricating, tissue engineering, and drug screening due to its high throughput and great controllability. However, most of the current droplet microfluidics are dependent on water‐in‐oil (W/O) systems, which involve organic reagents, thus limiting their broader biological applications. In this work, a new microfluidic strategy is described for controllable and high‐throughput generation of monodispersed water‐in‐water (W/W) droplets. Solutions of polyethylene glycol and dextran are used as continuous and dispersed phases, respectively, without any organic reagents or surfactants. The size of W/W droplets can be precisely adjusted by changing the flow rate of dispersed and continuous phases and the valve switch cycle. In addition, uniform cell‐laden microgels are fabricated by introducing the alginate component and rat pancreatic islet (β‐TC6) cell suspension to the dispersed phase. The encapsulated islet cells retain high viability and the function of insulin secretion after cultivation for 7 days. The high‐throughput droplet microfluidic system with high biocompatibility is stable, controllable, and flexible, which can boost various chemical and biological applications, such as bio‐oriented microparticles synthesizing, microcarriers fabricating, tissue engineering, etc.     

Microfluidic Templated Multicompartment Microgels for 3D Encapsulation and Pairing of Single Cells

Controlled encapsulation and pairing of single cells within a confined 3D matrix can enable the replication of the highly ordered cellular structure of human tissues. Microgels with independently controlled compartments that can encapsulate cells within separately confined hydrogel matrices would provide precise control over the route of pairing single cells. Here, a one‐step microfluidic method is presented to generate monodisperse multicompartment microgels that can be used as a 3D matrix to pair single cells in a highly biocompatible manner. A method is presented to induce microgels formation on chip, followed by direct extraction of the microgels from oil phase, thereby avoiding prolonged exposure of the microgels to the oil. It is further demonstrated that by entrapping stem cells with niche cells within separate but adjacent compartments of the microgels, it can create complex stem cell niche microenvironments in a controlled manner, which can serve as a useful tool for the study of cell–cell interactions. This microfluidic technique represents a significant step toward high‐throughput single cells encapsulation and pairing for the study of intercellular communications at single cell level, which is of significant importance for cell biology, stem cell therapy, and tissue engineering.     

Screening Therapeutic Agents Specific to Breast Cancer Stem Cells Using a Microfluidic Single‐Cell Clone‐Forming Inhibition Assay

Screens of cancer stem cells (CSCs)‐specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single‐cell assay for screening breast cancer stem cell–specific agents. This assay takes advantage of the single‐cell clone‐forming capability of CSCs, which can be specifically inhibited by CSC‐targeting agents. The single‐cell assay is performed on a microfluidic chip with an array of 3840 cell‐capturing units; the single‐cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell‐capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long‐term suspension culture, only a minority of cells survive and form tumorspheres. The clone‐formation rate of MCF‐7, MDA‐MB‐231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone‐forming inhibition assay is conducted by exposing the single‐cell arrays to a set of anticancer agents. The CSC‐targeting agents show complete inhibition of single‐cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single‐cell assay with the potential to screen CSC‐specific agents with high efficiency provides new tools for individualized tumor therapy.     

Rapid Production of Cell‐Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform

This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)‐diacrylate, poly(ethylene glycol)‐fibrinogen, and gelatin methacrylate. Cell‐laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale‐up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time‐consuming and costly. Using a new molding technique, the microfluidic device employs a modified T‐junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10–60 million cells mL^?1) and a wide range of diameters (300–1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long‐term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor‐based cell expansion and differentiation, and high throughput tissue sphere‐based drug testing assays.     

Cover Picture: Multipotent Polymer Coatings Based on Chemical Vapor Deposition Copolymerization (Adv. Mater. 12/2006)

The cover shows that chemical vapor deposition can be used to prepare copolymer thin films, on varying substrates, that can bind two different ligands with high selectivity. In work reported by Lahann and co‐workers on p. 1521, the actual ligand ratios on the surface can be controlled by varying the copolymer composition. This technology may find applications in biomedical devices, high‐throughput screening platforms, microfluidic analysis devices, and diagnostic platforms.     

Optimization of microfluidic fuel cells using transport principles

Microfluidic fuel cells exploit the lack of convective mixing at low Reynolds number to eliminate the need for a physical membrane to separate the fuel from the oxidant. Slow transport of reactants in combination with high catalytic surface-to-volume ratios often inhibit the efficiency of a microfluidic fuel cell. The performance of microfluidic devices that rely on surface electrochemical reactions is controlled by the interplay between reaction kinetics and the rate of mass transfer to the reactive surfaces. This paper presents theoretical and experimental work to describe the role of flow rate, microchannel geometry, and location of electrodes within a microfluidic fuel cell on its performance. A transport model, based on the convective-diffusive flux of reactants, is developed that describes the optimal conditions for maximizing both the average current density and the percentage of fuel utilized. The results show that the performance can be improved when the design of the device includes electrodes smaller than a critical length. The results of this study advance current approaches to the design of microfluidic fuel cells and other electrochemically-coupled microfluidic devices.     

A Size‐Selective Intracellular Delivery Platform

Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 μm wide constriction is effective for delivery of cargo to primary human T‐cells that have an average diameter of 6.7 μm. In contrast, a 6 or 7 μm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 μm) and PANC‐1 (12.3 μm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T‐cells. The application of this approach is demonstrated by selectively delivering dextran‐conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.     

High Throughput‐Per‐Footprint Inertial Focusing

Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re‐scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput‐per‐footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect‐ratio rectangular microfluidic channels that are readily fabricated with a post‐CMOS integratable process, suggesting at least a 100‐fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost‐effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.     

Microfluidics for Biosynthesizing: from Droplets and Vesicles to Artificial Cells

Fabrication of artificial biomimetic materials has attracted abundant attention. As one of the subcategories of biomimetic materials, artificial cells are highly significant for multiple disciplines and their synthesis has been intensively pursued. In order to manufacture robust “alive” artificial cells with high throughput, easy operation, and precise control, flexible microfluidic techniques are widely utilized. Herein, recent advances in microfluidic‐based methods for the synthesis of droplets, vesicles, and artificial cells are summarized. First, the advances of droplet fabrication and manipulation on the T‐junction, flow‐focusing, and coflowing microfluidic devices are discussed. Then, the formation of unicompartmental and multicompartmental vesicles based on microfluidics are summarized. Furthermore, the engineering of droplet‐based and vesicle‐based artificial cells by microfluidics is also reviewed. Moreover, the artificial cells applied for imitating cell behavior and acting as bioreactors for synthetic biology are highlighted. Finally, the current challenges and future trends in microfluidic‐based artificial cells are discussed. This review should be helpful for researchers in the fields of microfluidics, biomaterial fabrication, and synthetic biology.     

A titanium dioxide nanorod array as a high-affinity nano-bio interface of a microfluidic device for efficient capture of circulating tumor cells

Nanomaterials show promising opportunities to address clinical problems (such as insufficient capture of circulating tumor cells;CTCs) via the high surface area-to-volume ratio and high affinity for biological cells.However,how to apply these nanomaterials as a nano-bio interface in a microfluidic device for efficient CTC capture with high specificity remains a challenge.In the present work,we first found that a titanium dioxide (TiO2) nanorod array that can be conveniently prepared on multiple kinds of substrates has high affinity for tumor cells.Then,the TiO2 nanorod array was vertically grown on the surface of a microchannel with hexagonally patterned Si micropillars via a hydrothermal reaction,forming a new kind of a micro-nano 3D hierarchically structured microfluidic device.The vertically grown TiO2 nanorod array was used as a sensitive nano-bio interface of this 3D hierarchically structured microfluidic device,which showed high efficiency of CTC capture (76.7％ ± 7.1％) in an artificial whole-blood sample.     

DNA‐SMART: Biopatterned Polymer Film Microchannels for Selective Immobilization of Proteins and Cells

A novel SMART module, dubbed “DNA‐SMART” (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single‐stranded DNA capture strands, microthermoformed into 3D structures, and postmodified with complementary DNA‐protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.     

Bioinspired Patterning with Extreme Wettability Contrast on TiO2 Nanotube Array Surface: A Versatile Platform for Biomedical Applications

Binary wettability patterned surfaces with extremely high wetting contrasts can be found in nature on living creatures. They offer a versatile platform for microfluidic management. In this work, a facile approach to fabricating erasable and rewritable surface patterns with extreme wettability contrasts (superhydrophilic/superhydrophobic) on a TiO₂ nanotube array (TNA) surface through self‐assembly and photocatalytic lithography is reported. The multifunctional micropatterned superhydrophobic TNA surface can act as a 2D scaffold for site‐selective cell immobilization and reversible protein absorption. Most importantly, such a high‐contrast wettability template can be used to construct various well‐defined 3D functional patterns, such as calcium phosphate, silver nanoparticles, drugs, and biomolecules in a highly selective manner. The 3D functional patterns would be a versatile platform in a wide range of applications, especial for biomedical devices (e.g., high‐throughput molecular sensing, targeted antibacterials, and drug delivery). In a proof‐of‐concept study, the surface‐enhanced Raman scattering and antibacterial performance of the fabricated 3D AgNP@TNA pattern, and the targeted drug delivery for site‐specific and high‐sensitivity cancer cell assays was investigated.     

Microfluidic‐Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high‐efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label‐free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor‐intensive steps of labeling molecular signatures of cells. In general, microfluidic‐based cell sorting approaches can separate cells using “intrinsic” (e.g., fluid dynamic forces) versus “extrinsic” external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label‐free microfluidic‐based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic‐based cell separation methods are discussed.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

Capture and Culturing of Living Cells on Microstructured DNA Substrates

A modular system for the DNA‐directed immobilization of antibodies was applied to capture living cells on microstructured DNA surfaces. It is demonstrated in two different set‐ups, static incubation and hydrodynamic flow, that this approach is well suited for specific capture and selection of cells from culture medium. The adhered cells show intact morphology and they can be cultivated to grow to dense monolayers, restricted to the lateral dimensions of DNA spots on the surface. Owing to the modularity of surface biofunctionalization, the system can readily be configured to serve as a matrix for adhesion and growth of different cells, as demonstrated by specific binding of human embryonic kidney cells (HEK293) and Hodgkin lymphoma L540cy cells onto patches bearing appropriate recognition moieties inside a microfluidic channel. We therefore anticipate that the systems described here should be useful for fundamental research in cell biology or applications in biomedical diagnostics, drug screening, and nanobiotechnology.     

Optimal conditions for protein array deposition using continuous flow

Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.     

Microfluidic Single‐Cell Omics Analysis

The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.     

Combining Multiplex SERS Nanovectors and Multivariate Analysis for In Situ Profiling of Circulating Tumor Cell Phenotype Using a Microfluidic Chip

Isolating and in situ profiling the heterogeneous molecular phenotype of circulating tumor cells are of great significance for clinical cancer diagnosis and personalized therapy. Herein, an on‐chip strategy is proposed that combines size‐based microfluidic cell isolation with multiple spectrally orthogonal surface‐enhanced Raman spectroscopy (SERS) analysis for in situ profiling of cell membrane proteins and identification of cancer subpopulations. With the developed microfluidic chip, tumor cells are sieved from blood on the basis of size discrepancy. To enable multiplex phenotypic analysis, three kinds of spectrally orthogonal SERS aptamer nanovectors are designed, providing individual cells with composite spectral signatures in accordance with surface protein expression. Next, to statistically demultiplex the complex SERS signature and profile the cellular proteomic phenotype, a revised classic least square algorithm is employed to obtain the 3D phenotypic information at single‐cell resolution. Combined with categorization algorithm partial least square discriminate analysis, cells from different human breast cancer subtypes can be reliably classified with high sensitivity and selectivity. The results demonstrate that this platform can identify cancer subtypes with the spectral information correlated to the clinically relevant surface receptors, which holds great potential for clinical cancer diagnosis and precision medicine.