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Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.

Control of neural stem cell (NSC) self-renewal and differentiationis of great importance to improve its therapeutic efficacy in the treatment of neurodegenerative diseases. Neurosphere culture for NSC expansion under undifferentiation condition determines the self-renewal capacity and differentiation propensity of NSCs. In this study, we examined the effects of controlled crosstalk between endothelial cells (ECs) and NSC neurospheres on self-renewal, differentiation, and functions of NSCs. Cultures of human fetal NSCs (hfNSCs) or human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs) in a microwell array with 500-μm well diameter facilitated cell–cell interaction and self-renewal ability, leading to increased neuronal differentiation and improved electrophysiological functions. Incorporation of ECs into size-controlled hfNSC neurospheres further promoted cell–cell interaction and self-renewal capacity. The decrease in EC density in hfNSC neurospheres effectively promoted cell–cell interaction and self-renewal. Under spontaneous differentiation condition, EC-containing hfNSC neurospheres differentiated into astrocytes rather than neuronal lineages. Therefore, we suggest the engineering of NSCs at neurosphere stage using microwell culture to control neurosphere size and that EC co-culture for vascularization may regulate behaviors, phenotypes, and functions of NSCs, leading to modulation of their therapeutic and regenerative potentials.

Blood plasma is a source of biomarkers in blood and a simple, fast, and easy extraction method is highly required for point-of-care testing (POCT) applications. This paper proposes a membrane filter integrated microfluidic device to extract blood plasma from whole blood, without any external instrumentation. A commercially available membrane filter was integrated with a newly designed dual-cover microfluidic device to avoid leakage of the extracted plasma and remaining blood cells. Nano-interstices installed on both sides of the microfluidic channels actively draw the extracted plasma from the membrane. The developed device successfully supplied 20 μL of extracted plasma with a high extraction yield (~45%) in 16 min.

Quantitative microfluidic point-of-care testing has been translated into clinical applications to support a prompt decision on patient treatment. A nanointerstice-driven filling technique has been developed to realize the fast and robust filling of microfluidic channels with liquid samples, but it has failed to provide a consistent filling time owing to the wide variation in liquid viscosity, resulting in an increase in quantification errors. There is a strong demand for simple and quick flow control to ensure accurate quantification, without a serious increase in system complexity. A new control mechanism employing two-beam refraction and one solenoid valve was developed and found to successfully generate digitized filling flow, completely free from errors due to changes in viscosity. The validity of digitized filling flow was evaluated by the immunoassay, using liquids with a wide range of viscosity. This digitized microfluidic filling flow is a novel approach that could be applied in conventional microfluidic point-of-care testing.

Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.

Engineering physiologically-relevant in vitro models of human organs remains a fundamental challenge. Despite significant strides made within the field, many promising organ-on-a-chip models fall short in recapitulating cellular interactions with neighboring cell types, surrounding extracellular matrix (ECM), and exposure to soluble cues due, in part, to the formation of artificial structures that obstruct >50% of the surface area of the ECM. Here, we report a three-dimensional (3D) cell culture platform based upon hydrophobic patterning of hydrogels that is capable of precisely generating a 3D ECM within a microfluidic channel with an interaction area >95%. In this study, for demonstrative purposes, type I collagen (COL1), Matrigel (MAT), COL1/MAT mixture, hyaluronic acid (HA), and cell-laden MAT were formed in the device. We demonstrate three potential applications, including creating a 3D endothelium model, studying the interstitial migration of cancer cells, and analyzing stem cell differentiation in a 3D environment. Our hydrophobic patterned-based 3D cell culture device provides the ease-of-fabrication and flexibility necessary for broad potential applications in organ-on-a-chip platforms.

The mechanical properties of cells change with their differentiation, chronological age, and malignant progression. Consequently, these properties may be useful label-free biomarkers of various functional or clinically relevant cell states. Here, we demonstrate mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell-analysis method that utilizes a four-terminal measurement of the current across a microfluidic channel to quantify simultaneously cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We define a new parameter, the whole-cell deformability index (wCDI), which provides a quantitative mechanical metric of the resistance to compressive deformation that can be used to discriminate among different cell types. The wCDI and the transverse deformation under constant strain show malignant MCF-7 and A549 cell lines are mechanically distinct from non-malignant, MCF-10A and BEAS-2B cell lines, and distinguishes between cells treated or untreated with cytoskeleton-perturbing small molecules. We categorize cell recovery time, ΔTr, as instantaneous (ΔTr~0 ms), transient (ΔTr⩽40 ms), or prolonged (ΔTr>40 ms), and show that the composition of recovery types, which is a consequence of changes in cytoskeletal organization, correlates with cellular transformation. Through the wCDI and cell-recovery time, mechano-NPS discriminates between sub-lineages of normal primary human mammary epithelial cells with accuracy comparable to flow cytometry, but without antibody labeling. Mechano-NPS identifies mechanical phenotypes that distinguishes lineage, chronological age, and stage of malignant progression in human epithelial cells.

The nanointerstices (NIs) at the sidewalls of a microfluidic channel impose a strong driving force on the main flow and render the flow less dependent on the surface contact angle of the microfluidic channel. NIs facilitate rapid and stable filling of aqueous solutions in plastic microfluidic channels and have been widely applied to many commercial applications. However, the air–liquid interface in an NI-driven microflow can be disrupted by small defects in the NIs, and bubbles can form around the defects. Here, we analyze the effects of the heterogeneous contact angle and the structural interrupts on the NI-driven microflow. We also formulated a mathematical model that precisely calculated the interrupted NI-driven microflow profile. From this study, the NI-driven microflow is anticipated to further facilitate commercial applications by providing reliable microfluidic channels and offering robust NI-driven microflow with perfect initial filling, a symmetric front meniscus, and the avoidance of bubble generation.

Vasculogenesis is an important morphogenetic event for vascular tissue engineering and ischemic disease treatment. Stem and progenitor cells can contribute to vasculogenesis via endothelial differentiation and direct participation in blood vessel formation. In this study, we developed an implantable microfluidic device to facilitate formation of three-dimensional (3D) vascular structures by human endothelial progenitor cells (hEPCs). The microfluidic device was made of biodegradable poly(lactic-co-glycolic acid) (PLGA) using a microchannel patterned silicon wafer made by soft lithography. A collagen type I (Col I) hydrogel containing hEPCs filled the microfluidic channels to reconstitute a 3D microenvironment for facilitating vascular structure formation by hEPCs. The device seeded with hEPCs was implanted into the subcutaneous space of athymic mice and retrieved one and four weeks after implantation. Histology and immunohistochemistry revealed that hEPCs formed a 3D capillary network expressing endothelial cell-specific proteins in the channel of the PLGA microfluidic device. This result indicates that a 3D microscale extracellular matrix reconstituted in the microchannel can promote the endothelial differentiation of hEPCs and in turn hEPC-mediated vasculogenesis. The PLGA microfluidic device reported herein may be useful as an implantable tissue-engineering scaffold for vascularized tissue reconstruction and therapeutic angiogenesis.

Neural stem cells (NSCs) reside in a vascular microenvironment termed the "NSC niche." Blood vessels in the NSC niche play an important role in maintaining an appropriate balance between NSC self-renewal and differentiation that serves to maintain homeostasis. Understanding the role of brain vessels in the NSC niche will facilitate basic research in neurogenesis and vasculogenesis as well as aid the development of potential therapies for degenerative disorders. Here, an in vitro-reconstituted NSC-vascular niche consisting of a 3D brain vasculature and extracellular matrix (ECM) microenvironment that allows NSCs to adopt physiologically relevant phenotypes through the combined effects of ECM components, chemical gradients, and signaling effectors from the brain vasculature is described. The reconstituted niche can provide precise spatiotemporal control of the NSC niche, regulating self-renewal, proliferation and colony formation of NSCs, and suppressing neuronal generation but promoting NSC differentiation into astrocytes and oligodendrocytes. It is proved that Notch effectors regulate both the astrocyte differentiation and NSC self-renewal, but the astrocyte differentiation is more active in NSCs in close proximity to brain vasculature. A potential role of the other vascular microenvironmental factor of pigment epithelium-derived factor from brain vasculature in the regulation of NSC self-renewal is also proved.

Plasticity and reciprocity of breast cancer cells to various extracellular matrice (ECMs) are three-dimensionally analyzed in quantitative way in a novel and powerful microfluidic in vitro platform. This successfully demonstrates the metastatic potential of cancer cells and their effective strategies of ECM proteolytic remodeling and morphological change, while interacting with other cells and invading into heterogeneous ECMs., International Research & Development Program (Grant 2009-00631), National Research Foundation of Korea (Basic Science Research Program Grant 2012-022481)

Micro-hoodoo (inverse trapezoidal) structures fabricated by silicon etching, soft-lithography, or photolithography have attracted great interest due to their improved hydrophobicity and self-cleaning properties. We present here a simple single-step UV diffraction lithography technique for fabricating micro-hoodoo structures with or without adjustable interconnecting bridges using a negative SU-8 photoresist. The theoretical calculations and the fabrication results revealed that the sizes and sidewall profiles of the micro-hoodoo structures and interconnecting bridges could be precisely controlled by the fabrication conditions, including the pattern-to-pattern spacing, exposure dose, and gap between the mask and the SU-8 surfaces. The theoretical calculations were conducted using an integrated model based on a Fresnel diffraction model for estimating the hoodoo size and an exponent decay model for estimating the sidewall profile. The integrated model agreed well with the fabricated hoodoo sizes and sidewall profiles, and the model provided an explanation for the structural instabilities observed during formation of the interconnecting bridges between hoodoos. The interconnecting bridges made the hydrophobic hoodoo structures sticky toward water, with a water contact angle hysteresis of up to 86.6°. The directional bridge interconnections produced directionally sticky hydrophobic surfaces that successfully mimicked function in butterfly wings to enable directional water removal.

In this work, the spreading and evaporation of 2–70 pL droplet (17–50 μm diameter) of water and ethylene glycol jetted by drop-on-demand piezo-driven jetting head on the heated substrate are studied. According to the experimental results, the interfacial oscillation phenomena of water droplet whose Ohnesorge number ( Oh ) is about 10 −2 is similar to that in inviscid impact driven region, while that of ethylene glycol droplet ( Oh ≈10 −1 ) is similar to that in highly viscous impact driven region followed by capillary driven extra spreading. In addition, various time scales used for nano/micro-liter droplets agree well with the times for interfacial oscillation, viscous damping, extra wetting, and evaporation in pico-liter droplets. In the case of water droplet, the spreading processes end before the evaporation becomes significant. However, in the case of highly viscous ethylene glycol droplet, the extra wetting overlaps the evaporation at high temperature.

The construction of well-controllable in vitro models of physiological and pathological vascular endothelium remains a fundamental challenge in tissue engineering and drug development. Here, we present an approach for forming a synthetic endothelial extracellular matrix (ECM) that closely resembles that of the native structure by locally depositing basement membrane materials onto type 1 collagen nanofibers only in a region adjacent to the endothelial cell (EC) monolayer. Culturing the EC monolayer on this synthetic endothelial ECM remarkably enhanced its physiological properties, reducing its vascular permeability, and promoting a stabilized, quiescent phenotype. We demonstrated that the EC monolayer on the synthetic endothelial ECM neither creates non-physiological barriers to cell-cell or cell-ECM interactions, nor hinders molecular diffusion of growth factors and other molecules. The synthetic endothelial ECM and vascular endothelium on it may help us enter in a new phase of research in which various models of the biological barrier behavior can be tested experimentally., Korea (South). Ministry of Science, ICT and Future Planning (National Research Foundation of Korea. Pioneer Research Center Program (NRF-2015M3C1A3002152), Korea (South). Ministry of Science, ICT and Future Planning (BioNano Health-Guard Research Center Global Frontier Project H-GUARD_2014M3A6B2060524)

Aptamers have recently emerged as reliable and promising targeting agents in the field of biology. However, their therapeutic potential has yet to be completely assessed due to their poor pharmacokinetics for systemic administration. Here, we describe a novel aptamer-antibody complex, designated an "oligobody" (oligomer+antibody) that may overcome the therapeutic limitations of aptamers. To provide proof-of-principle study, we investigated the druggability of oligobody in vivo using cotinine conjugated t44-OMe aptamer, which is specific for the sequence of pegaptanib, and an anti-cotinine antibody. The antibody part of oligobody resulted in extended in vivo pharmacokinetics of the aptamer without influencing its binding affinity. Moreover, the aptamer of oligobody penetrated deeply into the tumor tissues whereas the anti-VEGF antibody did not. Finally, the systemic administration of this oligobody reduced the tumor burden in a xenograft mouse model. Together, these results suggested that our oligobody strategy may represent a novel platform for rapid, low-cost and high-throughput cancer therapy.

Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix.

Pre-concentration methods are essential for detecting low concentrations of influenza virus in biological samples from patients. Here, we describe a new method for draining buffer from solution in the reservoir of a microfluidic device to increase the concentration of virus in the reservoir. Viruses were captured in the reservoir by an ion depletion barrier from connected ion selective microfluidic channels. 75 μl of buffer was successfully drained from a 100 μl sample, resulting in a 4-fold increase in influenza hemagglutinin concentration in the reservoir. The volume of the final concentrated sample was suitable for detection of influenza hemagglutinin by the enzyme-linked immunosorbent assay, demonstrating the usefulness of the developed platform for enhanced sensitivity of virus detection in a conventional analysis.

We report a microfluidic array for investigating and quantitatively analyzing human neural stem cell (hNSC) self-renewal and differentiation in an in vivo-like microenvironment. NSC niche conditions, including three-dimensional (3D) extracellular matrices and low oxygen tension, were effectively reconstituted in the microfluidic array in a combinatorial manner. The array device was fabricated to be detachable, rendering it compatible with quantitative real-time polymerase chain reaction for quantifying the effects of the biomimetic conditions on hNSC self-renewal and differentiation. We show that throughput of 3D cell culture and quantitative analysis can be increased. We also show that 3D hypoxic microenvironments maintain hNSC self-renewal capacity and direct neuronal commitment during hNSC differentiation.

In aquatic environments, microorganisms tend to form biofilms on surfaces to protect them from harsh conditions. The biofilms then accumulate into multilayered mat-like structures. In this study, we evaluated the effects of the hydrodynamic conditions on the ecology of biofilms produced by Pseudomonas aeruginosa (PA14). In microfluidic channels, we found that the development of biofilms was regulated by hydrodynamic conditions, but the developed biofilms also changed flow velocity by narrowing flow width. The coupled growing conditions were simplified by a new concept of consequent variables, and the dimensionless biofilm development (Ab/h(2)Ab/w(cs)(2)) was successfully expressed by the Reynolds number (Re) and the dimension of the channel (r). At low Re, higher flow rates encouraged growth of biofilms, while higher flow rates with high Re suppressed growth of biofilms. These results provide a simple model as a theoretical basis for understanding development of biofilms in microfluidic channels.

Here, we report a unique method to quantify the effects of in vivo-like extracellular matrix (ECM) for guiding differentiation of neural stem cells (NSCs) in three-dimensional (3D) microenvironments using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully monitored and quantified differentiation of NSCs in small volume ECMs and found that differentiation of NSCs, especially those differentiating towards neuronal and oligodendrocytic lineages, is significantly enhanced by 3D microenvironments reconstituted in the microfluidic channels.

Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.

Ablation of metal nanoparticle film using frequency doubled Nd:YAG nanosecond laser is explored to apply for trimming drop on demand (DOD) inkjet printed electrical micro-conductor for flexible electronics. While elevated rim structure due to expulsion of molten pool is observed in sintered nanoparticle film, the ablation of unsintered nanoparticle film results in a Gaussian-shaped ablation profile, so that a clean precise patterning is possible. In addition, the ablation fluence threshold of unsintered metal nanoparticle film is at least ten times lower than that of a corresponding metal film. Therefore, by using nanosecond laser ablation, inkjet printed metal nanoparticles compatible for flexible polymer can be patterned efficiently with a high resolution.

Nanosecond laser ablation of polyvinylpyrrolidone (PVP) protected silver nanoparticle (20 nm diameter) film is studied using a frequency doubled Nd:YAG nanosecond laser (532 nm wavelength, 6 ns full width half maximum pulse width). In the sintered silver nanoparticle film, absorbed light energy conducts well through the sintered porous structure, resulting in ablation craters of a porous dome shape or crown shape depending on the irradiation fluence due to the sudden vaporization of the PVP. In the unsintered silver nanoparticle film, the ablation crater with a clean edge profile is formed and many coalesced nanoparticles of 50 to 100 nm in size are observed inside the ablation crater. These results and an order of magnitude analysis indicate that the absorbed thermal energy is confined within the nanoparticles, causing melting of nanoparticles and their coalescence to larger agglomerates, which are removed following melting and subsequent partial vaporization.

Breast cancer doesn't contain only one colony of tumor cells. It appears to have heterogeneous cell composition of characteristically different subtypes with distinct molecular and gene expression signatures. Due to the tumor heterogeneity, which determines the risk of metastatic progression and drug resistance, it is difficult to predict progression patterns of cancer and provide the most suitable treatment to patients. Most research has focused on molecular definition and genetic association for the cancer heterogeneity studies. However, lack of in vitro models to precisely mimic the heterogeneous conditions of tumors has made it difficult to understand the role of heterogeneity in metastasis, especially invasion. So, to precisely regulate heterogeneous microenvironment of tumors and evaluate the role of the heterogeneity on invasion & metastasis, we developed a microfluidic in vitro assay incorporating 3D ECM scaffolds that emulate in vivo tumor microenvironment in 3D. We cultured highly invasive breast cancer cell, MDA-MB-231, and non-invasive breast cancer cell, MCF-7, together in a mixed population and let the cells interact with other cells and ECMs. As a result, we found that heterogeneity in cancer cell population promotes metastatic potential. Highly invasive breast cancer cells, MDA-MB-231, made microtracks in ECM and leaded collective migration of non-invasive breast cancer cells, MCF-7, into the microtracks. When MCF-7 were cultured alone, MCF-7 cells didn't invade into ECM. It has been widely announced that only stromal cells such as fibroblast remodel ECM and enhance the invasion potential of cancer cells. However, from the result of our experiment, cancer cells that have different characteristics in single tumor can promote the metastatic potential, result from that ECM remodeling (generating microtracks) by highly invasive cancer cells. It was precisely investigated by IF, rt-PCR and TEM analysis in microfluidic environments. Understanding about not only genetic and molecular definition of cancer heterogeneity, but also the role of heterogeneous cancer cell population in metastatic dissemination, could help new drugs and therapeutic strategies development. Citation Format: Yoojin Shin, Hyo Eun Jeong, Sewoon Han, Jihee Won, Seok Chung. The role of cancer heterogeneity in metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C13.

Spatiotemporal analysis of the inflammatory response has been limited by the difficulties of in vivo imaging and reconstitution of inflammation in vitro. Here, we present a novel method for establishing in vivo-like inflammatory models in a microfluidic device and quantitatively measuring the three-dimensional transmigration of neutrophils during the inflammatory process. This enabled us to concurrently characterize transendothelial migration behaviors of neutrophils under the influence of various inflammatory stimuli.

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.