Operation Of Microfluidic Devices Research Articles

Experimental Investigations on Geometry Modulated Solute Mixing in Viscoelastic Media

In this study, geometrically modified microchannels fabricated using a stereolithography technique are employed to analyze micromixing of polymeric solutions. Experimental and numerical analyses were conducted to evaluate the qualitative and quantitative validity of the Newtonian fluid flow inside the geometrically modified channels. An in-house image processing code was used to analyze the dye concentration distributions resulting from complex fluid transport and mixing within the modified channels. Our analysis illustrates the existence of inertio-elastic instability in the geometrically modified microchannels for the transport of viscoelastic fluids. In addition, the presence of inertio-elastic instability significantly enhances the mixing efficiency as a result of the interaction between viscoelasticity and modified channel geometry. This analysis provides important physical insights into the cost-effective design and operation of microfluidic devices that handle viscoelastic fluids, which could be useful in designing and analyzing a passive micromixer that transports bio/polymeric fluids.

3D printing-based microfluidic devices in fabric

Fabric-based microfluidics is a growing sub-field of porous materials-based microfluidics. 3D printing has been demonstrated as a useful fabrication method for open channel microfluidic devices, and also in the context of porous substates such as cellulose. In the current report, we describe a straightforward method for 3D printing fabric-based microfluidic devices. We demonstrate the ability to create both full and partial barriers in fabric, characterizing minimum channel and barrier widths, as well as reproducibility of the method using the metric of flow time repeatability through the channels. We discuss considerations specific to 3D printing in fabric including fabric anisotropy, stretching, and nonuniformity. Further, we highlight our fabrication method via the implementation of a colorimetric urea assay.

Controlling bead and cell mobility in a recirculating hanging-drop network.

Integrating flowing cells, such as immune cells or circulating tumour cells, within a microphysiological system is crucial for body-on-a-chip applications. However, ensuring unimpeded recirculation of cells is a significant challenge. Closed microfluidic devices have a no-slip boundary condition along channel walls and a defined chip geometry (laminar flow) that hinders the ability to freely control cell flow. Open microfluidic devices, where the bottom device boundary is an air-liquid interface (ALI), e.g., hanging drop networks (HDNs), offer the advantage of an easily-actuatable fluid-phase geometry, where cells can either flow or stagnate. In this paper, we optimized a hanging-drop-integrated pneumatic-pump system for closed-loop recirculation of particles (i.e., beads or cells). Experiments with both beads and cells in cell culture medium initially resulted in particle stagnation, which was suggestive of a pseudo-no-slip boundary condition at the ALI. Transmission electron microscopy and dynamic light scattering measurements of the ALI suggested that aggregation of submicron-scale cell-culture-medium components is the cause of the pseudo-no-slip boundary condition. We used the finite element method to study the forces on particles at the ALI and to optimize HDN design (drop aperture) and operation (drop height) parameters. Based on this analysis, we report a phase diagram delineating the conditions for free flow or stagnation of particles at the ALI of hanging drops. Using our experimental setup with 3.5 mm drop apertures, we conducted particle flow experiments while actuating drop heights. We confirmed the ability to control the flow or stagnation of particles by actuating the height of hanging drops: a drop height over 300 μm led to particle stagnation and a drop height under 300 μm allowed for particle flow. This particle-flow control, combined with the ease of integrating scaffold-free organ models (microtissues or organoids) in HDNs, constitutes the basis for an experimental setup enabling the control of the residence time of single cells around 3D organ models.

An open microfluidic coculture model of fibroblasts and eosinophils to investigate mechanisms of airway inflammation.

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling.

Electrotransfer of Immunoprobes through Thin-Layer Polyacrylamide Gels.

Hydrogels are important structural and operative components of microfluidic systems, finding diverse utility in biological sample preparation and interrogation. One inherent challenge for integrating hydrogels into microfluidic tools is thermodynamic molecular partitioning, which reduces the in-gel concentration of molecular solutes (e.g., biomolecular regents), as compared to the solute concentration in an applied solution. Consequently, biomolecular reagent access to in-gel scaffolded biological samples (e.g., encapsulated cells, microbial cultures, target analytes) is adversely impacted in hydrogels. Further, biomolecular reagents are typically introduced to the hydrogel via diffusion. This passive process requires long incubation periods compared to active biomolecular delivery techniques. Electrotransfer is an active technique used in Western blots and other gel-based immunoassays that overcomes limitations of size exclusion (increasing the total probe mass delivered into gel) and expedites probe delivery, even in millimeter-thick slab gels. While compatible with conventional slab gels, electrotransfer has not been adapted to thin gels (50-250 μm thick), which are of great interest as components of open microfluidic devices (vs enclosed microchannel-based devices). Mechanically delicate, thin gels are often mounted on rigid support substrates (glass, plastic) that are electrically insulating. Consequently, to adapt electrotransfer to thin-gel devices, we replace rigid insulating support substrates with novel, mechanically robust, yet electrically conductive nanoporous membranes. We describe grafting nanoporous membranes to thin-polyacrylamide-gel layers via silanization, characterize the electrical conductivity of silane-treated nanoporous membranes, and report the dependence of in-gel immunoprobe concentration on transfer duration for passive diffusion and active electrotransfer. Alternative microdevice component layers─including the mechanically robust, electrically conductive nanoporous membranes reported here─provide new functionality for integration into an increasing array of open microfluidic systems.

Low-cost rapid prototyping and assembly of an open microfluidic device for a 3D vascularized organ-on-a-chip.

Reconstruction of 3D vascularized microtissues within microfabricated devices has rapidly developed in biomedical engineering, which can better mimic the tissue microphysiological function and accurately model human diseases in vitro. However, the traditional PDMS-based microfluidic devices suffer from the microfabrication with complex processes and usage limitations of either material properties or microstructure design, which drive the demand for easy processing and more accessible devices with a user-friendly interface. Here, we present an open microfluidic device through a rapid prototyping method by laser cutting in a cost-effective manner with high flexibility and compatibility. This device allows highly efficient and robust hydrogel patterning under a liquid guiding rail by spontaneous capillary action without the need for surface treatment. Different vascularization mechanisms including vasculogenesis and angiogenesis were performed to construct a 3D perfusable microvasculature inside a tissue chamber with various shapes under different microenvironment factors. Furthermore, as a proof-of-concept we have created a vascularized spheroid by placing a monoculture spheroid into the central through-hole of this device, which formed angiogenesis between the spheroid and microvascular network. This open microfluidic device has great potential for mass customization without the need for complex microfabrication equipment in the cleanroom, which can facilitate studies requiring high-throughput and high-content screening.

Aspiration-mediated hydrogel micropatterning using rail-based open microfluidic devices for high-throughput 3D cell culture

Microfluidics offers promising methods for aligning cells in physiologically relevant configurations to recapitulate human organ functionality. Specifically, microstructures within microfluidic devices facilitate 3D cell culture by guiding hydrogel precursors containing cells. Conventional approaches utilize capillary forces of hydrogel precursors to guide fluid flow into desired areas of high wettability. These methods, however, require complicated fabrication processes and subtle loading protocols, thus limiting device throughput and experimental yield. Here, we present a swift and robust hydrogel patterning technique for 3D cell culture, where preloaded hydrogel solution in a microfluidic device is aspirated while only leaving a portion of the solution in desired channels. The device is designed such that differing critical capillary pressure conditions are established over the interfaces of the loaded hydrogel solution, which leads to controlled removal of the solution during aspiration. A proposed theoretical model of capillary pressure conditions provides physical insights to inform generalized design rules for device structures. We demonstrate formation of multiple, discontinuous hollow channels with a single aspiration. Then we test vasculogenic capacity of various cell types using a microfluidic device obtained by our technique to illustrate its capabilities as a viable micro-manufacturing scheme for high-throughput cellular co-culture.

Programmed Cell-Death Mechanism Analysis Using Same-Cell, Multimode DNA and Proteoform Electrophoresis.

Gaining insight into the timing of cell apoptosis events requires single-cell-resolution measurements of cell viability. We explore the supposition that mechanism-based scrutiny of programmed cell death would benefit from same-cell analysis of both the DNA state (intact vs fragmented) and the protein states, specifically the full-length vs cleaved state of the DNA-repair protein PARP1, which is cleaved by caspase-3 during caspase-dependent apoptosis. To make this same-cell, multimode measurement, we introduce the single-cell electrophoresis-based viability and protein (SEVAP) assay. Using SEVAP, we (1) isolate human breast cancer SKBR3 cells in microwells molded in thin polyacrylamide gels, (2) electrophoretically separate protein molecular states and DNA molecular states—using differences in electrophoretic mobility—from each single-cell lysate, and (3) perform in-gel DNA staining and PARP1 immunoprobing. Performed in an open microfluidic device, SEVAP scrutinized hundreds to thousands of individual SKBR3 cells. In each single-cell lysate separation, SEVAP baseline-resolved fragmented DNA from intact DNA (Rs = 5.17) as well as cleaved PARP1 from full-length PARP1 (Rs = 0.66). Comparing apoptotic and viable cells showed statistically similar profiles (expression, mobility, peak width) of housekeeping protein β-tubulin (Mann–Whitney U test). Clustering and cross-correlation analysis of DNA migration and PARP1 migration identified nonapoptotic vs apoptotic cells. Clustering analysis further suggested that cleaved PARP1 is a suitable apoptosis marker for this system. SEVAP is an efficient, multimode, end-point assay designed to elucidate cell-to-cell heterogeneity in mechanism-specific signaling during programmed cell death.

Enhanced Liquid Transport on a Highly Scalable, Cost‐Effective, and Flexible 3D Topological Liquid Capillary Diode

AbstractDirectional liquid‐transport surfaces have various applications, such as, open microfluidic devices, fog collection, oil–water separation, and surface lubrication. However, current liquid‐transport surfaces are expensive, complicated to manufacture, and lack scalability. Moreover, they exhibit low transport speeds and distances. In this study, a laser cutter is used to fabricate scalable, low‐cost unidirectional liquid‐transporting surfaces with enhanced transport speed and distance using polymeric materials. Cutting and engraving methods are used to create a liquid capillary diode comprising 3D wedge shapes, thereby obtaining an appropriate pressure gradient and liquid pinning. The developed liquid capillary diode exhibits the fastest transport speed (3–17.7 mm s−1) reported so far, and a large normalized distance (L/R: transport distance/radius of dispensed droplet). The transport distance increases with the square root of time under various contact angles and liquid viscosities, which agree well with the theoretical scaling results obtained using the modified Washburn model. Additionally, the flexible liquid capillary diode operates adequately even when bent with the maximum curvature of 0.1 mm−1. The results provide better design guidelines for 3D topological liquid‐transport surfaces for various applications.

Characterization of Nanoparticle Adsorption on Polydimethylsiloxane-Based Microchannels

Nanoparticles (NPs) are used in various medicinal applications. Exosomes, bio-derived NPs, are promising biomarkers obtained through separation and concentration from body fluids. Polydimethylsiloxane (PDMS)-based microchannels are well-suited for precise handling of NPs, offering benefits such as high gas permeability and low cytotoxicity. However, the large specific surface area of NPs may result in nonspecific adsorption on the device substrate and thus cause sample loss. Therefore, an understanding of NP adsorption on microchannels is important for the operation of microfluidic devices used for NP handling. Herein, we characterized NP adsorption on PDMS-based substrates and microchannels by atomic force microscopy to correlate NP adsorptivity with the electrostatic interactions associated with NP and dispersion medium properties. When polystyrene NP dispersions were introduced into PDMS-based microchannels at a constant flow rate, the number of adsorbed NPs decreased with decreasing NP and microchannel zeta potentials (i.e., with increasing pH), which suggested that the electrostatic interaction between the microchannel and NPs enhanced their repulsion. When exosome dispersions were introduced into PDMS-based microchannels with different wettabilities at constant flow rates, exosome adsorption was dominated by electrostatic interactions. The findings obtained should facilitate the preconcentration, separation, and sensing of NPs by PDMS-based microfluidic devices.

Open access tool and microfluidic devices for phenotypic quantification of heart function of intact fruit fly and zebrafish larvae

In this paper, the heartbeat parameters of small model organisms, i.e. Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish), were quantified in-vivo in intact larvae using microfluidics and a novel MATLAB-based software. Among different developmental stages of flies and zebrafish, the larval stage is privileged due to biological maturity, optical accessibility, and the myogenic nature of the heart. Conventional methods for parametric quantification of heart activities are complex and mostly done on dissected, irreversibly immobilized, or anesthetized larvae. Microfluidics has helped with reversible immobilization without the need for anesthesia, but heart monitoring is still done manually due to challenges associated with the movement of floating organs and cardiac interruptions. In our MATLAB software applied to videos recorded in microfluidic-based whole-organism assays, we have used image segmentation to automatically detect the heart and extract the heartbeat signal based on pixel intensity variations of the most contractile region of the heart tube. The smoothness priors approach (SPA) was applied to remove the undesired low-frequency noises caused by environmental light changes or heart movement. Heart rate and arrhythmicity were automatically measured from the detrended heartbeat signal while other parameters including end-diastolic and end-systolic diameters, shortening distance, shortening time, fractional shortening, and shortening velocity were quantified for the first time in intact larvae, using M-mode images under bright field microscopy. The software was able to detect more than 94% of the heartbeats and the cardiac arrests in intact Drosophila larvae. Our user-friendly software enables in-vivo quantification of D. melanogaster and D. rerio larval heart functions in microfluidic devices, with the potential to be applied to other biological models and used for automatic screening of drugs and alleles that affect their heart.

Comparison of photoactivatable crosslinkers for in-gel immunoassays.

While fluorescence readout is a key detection modality for hydrogel-based immunoassays, background fluorescence due to autofluorescence or non-specific antibody interactions impairs the lower limit of detection of fluorescence immunoassays. Chemical modifications to the hydrogel structure impact autofluorescence and non-specific interactions. Benzophenone is a common photoactivatable molecule, and benzophenone methacrylamide (BPMA) has been used for cross-linking protein in polyacrylamide (PA) hydrogels. However, previous studies have suggested that the aromatic structure of benzophenone can contribute to increased autofluorescence and non-specific hydrophobic interactions with unbound fluorescent probes. Here, we synthesize diazirine methacrylamide (DZMA) as an alternative photoactivatable molecule to crosslink into PA hydrogels for in-gel protein capture for in-gel immunoassays. We hypothesize that the less hydrophobic structure of diazirine (based on previously reported predicted and experimental log P values) exhibits both reduced autofluorescence and non-specific hydrophobic interactions. We find that while equal concentrations of DZMA and BPMA result in lower protein target photocapture in the diazirine configuration, increasing the DZMA concentration up to 12 mM improves in-gel protein capture to be on par with previously reported and characterized 3 mM BPMA hydrogels. Furthermore, despite the higher concentration of diazirine, we observe negligible autofluorescence signal and a 50% reduction in immunoassay fluorescence background signal in diazirine gels compared to BPMA gels resulting in comparable signal-to-noise ratios (SNR) of the probed protein target. Finally, we test the utility of DZMA for single-cell immunoblotting in an open microfluidic device and find that protein migrates ∼1.3× faster in DZMA hydrogels than in BPMA hydrogels. However, in DZMA hydrogels we detect only 15% of the protein signal compared to BPMA hydrogels suggesting that the diazirine chemistry results in greater protein losses following electrophoretic separations. We establish that while diazirine has lower background fluorescence signal, which may potentially improve immunoassay performance, the lower capture efficiency of diazirine reduces its utility in open microfluidic systems susceptible to sample losses.

Open Space Diffusive Filter for Simultaneous Species Retrieval and Separation.

We present a novel method for the local retrieval of surface bound species and their rapid in-line separation using an open space microfluidic device. Separation can be performed in less than 30 s using the difference in diffusivities within parallel microfluidic flows. As a proof-of-principle, we report the rapid and efficient filtration of polystyrene beads from small molecules and surface bound red blood cells from dimethyl sulfoxide for antigen typing.

Integrating Microstructured Electrospun Scaffolds in an Open Microfluidic System for in Vitro Studies of Human Patient-Derived Primary Cells.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

MP51-15 PERIPROSTATIC FAT INDUCES CENTROSOMAL AMPLIFICATION IN THE PROSTATE CANCER MICROENVIRONMENT: A MICROFLUIDIC APPROACH

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-15 PERIPROSTATIC FAT INDUCES CENTROSOMAL AMPLIFICATION IN THE PROSTATE CANCER MICROENVIRONMENT: A MICROFLUIDIC APPROACH Max Greenberg, Victoria Gil, Philip Fitchev, Francesca Nardi, Evanston, Deepak Daroach, Omar Franco, Simon Hayward, Renee Vickman, Xiaojing Su, Tristan Nicholson, Ashleigh Theberge, and Susan Crawford* Max GreenbergMax Greenberg More articles by this author , Victoria GilVictoria Gil More articles by this author , Philip FitchevPhilip Fitchev More articles by this author , Francesca Nardi, EvanstonFrancesca Nardi, Evanston More articles by this author , Deepak DaroachDeepak Daroach More articles by this author , Omar FrancoOmar Franco More articles by this author , Simon HaywardSimon Hayward More articles by this author , Renee VickmanRenee Vickman More articles by this author , Xiaojing SuXiaojing Su More articles by this author , Tristan NicholsonTristan Nicholson More articles by this author , Ashleigh ThebergeAshleigh Theberge More articles by this author , and Susan Crawford*Susan Crawford* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Periprostatic fat (PPF) encases the prostate and is an understudied visceral fat depot and a stromal component of the prostate tumor microenvironment (TME). Visceral obesity is associated with ectopic lipid accumulation in non-adipocytes and tumor progression; however, the mechanisms responsible for aberrant adipocyte-tumor cell crosstalk are not clear. The PPF stores neutral lipid in the form of lipid droplets (LDs) and secretes a wide array of adipokines and cytokines. In human cancer-associated fibroblasts (CAFs), we recently found that increased lipid content promoted centrosomal and microtubule-organizing center (MTOC) amplification as well as a proliferative phenotype. Studying the TME has been hampered by limited experimental systems. We used an innovative multicellular open microfluidic platform, the Monorail, to study metabolic reprogramming and LD trafficking in tri-cultured human adipocytes, prostate tumor cells, and primary cancer-associated fibroblasts (CAFs). METHODS: Primary PPF derived from surgical prostatectomy specimens were co-cultured with prostate cancer and 3T3-L1 cell lines, and primary CAFs. The cells were cultured in a polystyrene open microfluidic device where only 22µL of medium is required per well. Low temperature-sensitive agarose gel was used to partition cells into various wells. The cells were analyzed using immunofluorescence stains (pericentrin and metalloproteinases), BODIPY and Oil-Red-O was used for neutral lipid. LD velocity was analyzed using live cell imaging. RESULTS: We found that the co-culture of prostate cancer cells with adipocytes (3T3-L1 or primary PPF) resulted in an increased LD area, and the addition of CAFs elevated both LD density and area in the tumor cells. Immunofluorescent studies for MTOCs revealed exposure of CAFs or adipocyte cells to cancer cells in the Monorail promoted amplification in centrosomes/MTOCs to two or more per cell. Live-cell imaging demonstrated that exposure to adipocytes caused the LD velocity to increase in cancer cells, and metalloproteinases were located on the surface of the LDs. CONCLUSIONS: The Monorail is an open microfluidic platform that simulates the complex TME. The device allows testing of primary human samples where cell number is limited. Adipocytes promote lipid reprogramming and MTOC amplification in both prostate cancer cells and CAFs. In the setting of obesity, higher intracytoplasmic lipid accumulation and centrosomal amplification are potential mechanisms responsible for accelerated prostate tumor growth and progression. Source of Funding: This work has been supported by the Rob Brooks Fund for Precision Prostate Cancer Care © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e769-e770 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Max Greenberg More articles by this author Victoria Gil More articles by this author Philip Fitchev More articles by this author Francesca Nardi, Evanston More articles by this author Deepak Daroach More articles by this author Omar Franco More articles by this author Simon Hayward More articles by this author Renee Vickman More articles by this author Xiaojing Su More articles by this author Tristan Nicholson More articles by this author Ashleigh Theberge More articles by this author Susan Crawford* More articles by this author Expand All Advertisement PDF downloadLoading ...

Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device.

In an open microfluidic device, we investigate protein polyacrylamide gel electrophoresis (PAGE) separation performance on single-cell lysate. Single-cell protein electrophoresis is performed in a thin layer of polyacrylamide (PA) gel into which microwells are molded. Individual cells are isolated in these open microwells, then lysed on-chip with a dual lysis and electrophoresis sodium dodecyl sulfate (SDS) buffer. We scrutinize the effect of sieving gel composition on electromigration of protein targets, using a wide range of cellular protein standards (36 kDa to 289 kDa). We find that as PA concentration increases, protein electromigration deviates from the empirical log-linear relationship predicted between migration distance and molecular mass. We perform Ferguson analysis to calculate retardation coefficients and free solution mobilities of nine cellular protein standards and observe that the largest-molecular-mass protein, mTOR (289 kDa), does not behave as predicted by established linear-fit models for SDS-denatured proteins, indicating that mTOR is beyond the linear range of this assay. Lastly, we performed in-gel immunoprobing on the single-cell electrophoretic separations and observed that smaller pore-size gels (higher gel concentration) reduce protein diffusion out of the gel, which does not notably impact the measured immunoprobed protein expression. Compared to larger pore-size gels, the smaller pore-size gels lead to higher local concentrations of the target protein in each protein band, resulting in an increase in the signal-to-noise ratio (SNR) for each protein. Understanding the separation and immunoprobing performance at different gel concentrations improves assay design and optimization for target proteins.

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

Profiling Cell-Matrix Adhesion Using Digitalized Acoustic Streaming.

Profiling the kinetics of cell-matrix adhesion is of great importance to understand many physiological and pathological processes such as morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Here, we developed a novel digital acoustofluidic device for parallel profiling cell-matrix adhesion at single-cell level. By introduction of localized and uniform acoustic streaming into an open chamber microfluidic device, the adherent cells within the open chamber can be detached by the streaming-induced Stokes drag force. By digital regulation of pulsed acoustic power from a low level to high levels, the hundreds of adherent cells can be ruptured from the fibronectin-coated substrate accordingly, and their adhesive forces (from several pN to several nN) and kinetics can be determined by the applied power and cell incubation time. As a proof-of-concept application for studying cancer metastasis, we applied this technique to measure the adhesion strength and kinetics of human breast cancer cells to extracellular matrix such as fibronectin and compared their metastatic potentials by measuring the rupture force of cancer cells representing malignant (MCF-7 cells and MDA-MB-231 cells) and nonmalignant (MCF-10A cells) states. Our acoustofluidic device is simple, easy to operate, and capable of measuring, in parallel, hundreds of individual cells' adhesion forces with a resolution at the pN level. Thus, we expect this device could be widely used for both fundamental cell biology research as well as development of cancer diagnostics and tissue engineering technologies.

Asymmetric micro-ratchets regulated drop dispensing on bamboo mimetic surface

Asymmetric micro-ratchet-regulated drop dispensing on a bamboo mimetic surface performs well for controlling the drop emission in open microfluidic devices.

Controlling Dispersion during Single-Cell Polyacrylamide-Gel Electrophoresis in Open Microfluidic Devices.

New tools for measuring protein expression in individual cells complement single-cell genomics and transcriptomics. To characterize a population of individual mammalian cells, hundreds to thousands of microwells are arrayed on a polyacrylamide-gel-coated glass microscope slide. In this "open" fluidic device format, we explore the feasibility of mitigating diffusional losses during lysis and polyacrylamide-gel electrophoresis (PAGE) through spatial control of the pore-size of the gel layer. To reduce in-plane diffusion-driven dilution of each single-cell lysate during in-microwell chemical lysis, we photopattern and characterize microwells with small-pore-size sidewalls ringing the microwell except at the injection region. To reduce out-of-plane-diffusion-driven-dilution-caused signal loss during both lysis and single-cell PAGE, we scrutinize a selectively permeable agarose lid layer. To reduce injection dispersion, we photopattern and study a stacking-gel feature at the head of each <1 mm separation axis. Lastly, we explore a semienclosed device design that reduces the cross-sectional area of the chip, thus reducing Joule-heating-induced dispersion during single-cell PAGE. As a result, we observed a 3-fold increase in separation resolution during a 30 s separation and a >2-fold enhancement of the signal-to-noise ratio. We present well-integrated strategies for enhancing overall single-cell-PAGE performance.