Junghyo Yoon

Viscoelastic lithography for fabricating self-organizing soft micro-honeycomb structures with ultra-high aspect ratios.

High-aspect ratio micro- and nano-structures have been used for the production of a variety of applications. In this paper, we describe a simple and cost-effective approach to fabricate an arrayed microarchitecture with an ultra-high aspect ratio using soft materials. The shapes and sizes of the honeycomb structure can be easily modulated by changing the dimensions and position of the base mould pattern and the pressure. The honeycomb structure is used to prepare a drug delivery patch and a microwell array to form cell spheroids without cell loss. The honeycomb structures prepared using natural ECM (collagen–Matrigel) materials are successfully fabricated. The hepatocytes and endothelial cells are seeded and co-cultured in the ECM-based micro-honeycomb to prepare a 3D liver model successfully mimicking an ultrastructure of liver and providing enhanced liver function.

Microfluidic in-reservoir pre-concentration using a buffer drain technique

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.

Ethanol-dispersed and antibody-conjugated polymer nanofibers for the selective capture and 3-dimensional culture of EpCAM-positive cells

Electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (NFs) were used as a platform for the selective capture and three-dimensional culture of EpCAM-positive cells in cell culture medium and whole blood. The NFs were treated with streptavidin to facilitate bond formation between the amino groups of streptavidin and the maleic anhydride groups of the NFs. A biotinylated anti-EpCAM monoclonal antibody (mAb) was attached to the streptavidin-conjugated NFs via the selective binding of streptavidin and biotin. Upon simple mixing and shaking with EpCAM-positive cancer cells in a wide concentration range from 10 to 1000,000 cells per 10mL, the mAb-attached NFs (mAb-NFs) captured the Ep-CAM positive cells in an efficiency of 59%-67% depending on initial cell concentrations, with minor mechanical capture of 14%-36%. Captured cells were directly cultured, forming cell aggregates, in the NF matrix, which ensures the cell proliferation and follow-up analysis. Furthermore, the capture capacity of mAb-NFs was assessed in the presence of whole blood and blood lysates, indicating cluster formation that captured target cells. It is anticipated that the antibody-attached NFs can be employed for the capture and analysis of very rare EpCAM positive circulating cancer cells.

Ion concentration polarization for pre-concentration of biological samples without pH change

In this paper, a method was developed for pre-concentrating large-volume biological samples for subsequent analysis. We previously developed another pre-concentration device, but it unfortunately altered the pH of the sample when an electric field was applied to the sample reservoir. Changes in the pH are not suitable for subsequent antibody-antigen reactions because of the stability issues that arise based on the target molecules isoelectric point (pI). Here, this problem was overcome using ion concentration polarization (ICP) with a cation selective membrane (Nafion). Phosphate buffered saline was used as a test solution for the sample. The sample was contained in a reservoir that was not affected by the electric field, and an ICP barrier was formed in front of the reservoir. This device could concentrate microliter-scale samples without changing the pH because the biomolecules were blocked from passing through the ICP barrier while the sample (phosphate buffered saline) was drained. A 40 μL sample was successfully pre-concentrated to 20 μL in a single channel device and 10 μL in a dual channel device, resulting in 2.1-fold and 3.3-fold increases, respectively, in influenza hemagglutinin concentrations. These changes in the concentration were confirmed by ELISA.

Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

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.

Enhanced oxygen permeability in membrane-bottomed concave microwells for the formation of pancreatic islet spheroids

Oxygen availability is a critical factor in regulating cell viability that ultimately contributes to the normal morphogenesis and functionality of human tissues. Among various cell culture platforms, construction of 3D multicellular spheroids based on microwell arrays has been extensively applied to reconstitute in vitro human tissue models due to its precise control of tissue culture conditions as well as simple fabrication processes. However, an adequate supply of oxygen into the spheroidal cellular aggregation still remains one of the main challenges to producing healthy in vitro spheroidal tissue models. Here, we present a novel design for controlling the oxygen distribution in concave microwell arrays. We show that oxygen permeability into the microwell is tightly regulated by varying the poly-dimethylsiloxane (PDMS) bottom thickness of the concave microwells. Moreover, we validate the enhanced performance of the engineered microwell arrays by culturing non-proliferated primary rat pancreatic islet spheroids on varying bottom thickness from 10 μm to 1050 μm. Morphological and functional analyses performed on the pancreatic islet spheroids grown for 14 days prove the long-term stability, enhanced viability, and increased hormone secretion under the sufficient oxygen delivery conditions. We expect our results could provide knowledge on oxygen distribution in 3-dimensional spheroidal cell structures and critical design concept for tissue engineering applications. STATEMENT OF SIGNIFICANCE In this study, we present a noble design to control the oxygen distribution in concave microwell arrays for the formation of highly functional pancreatic islet spheroids by engineering the bottom of the microwells. Our new platform significantly enhanced oxygen permeability that turned out to improve cell viability and spheroidal functionality compared to the conventional thick-bottomed 3-D culture system. Therefore, we believe that this could be a promising medical biotechnology platform to further develop high-throughput tissue screening system as well as in vivo-mimicking customised 3-D tissue culture systems.

Fabrication of type I collagen microcarrier using a microfluidic 3D T-junction device and its application for the quantitative analysis of cell-ECM interactions.

We presented a new quantitative analysis for cell and extracellular matrix (ECM) interactions, using cell-coated ECM hydrogel microbeads (hydrobeads) made of type I collagen. The hydrobeads can carry cells as three-dimensional spheroidal forms with an ECM inside, facilitating a direct interaction between the cells and ECM. The cells on hydrobeads do not have a hypoxic core, which opens the possibility for using as a cell microcarrier for bottom-up tissue reconstitution. This technique can utilize various types of cells, even MDA-MB-231 cells, which have weak cell-cell interactions and do not form spheroids in conventional spheroid culture methods. Morphological indices of the cell-coated hydrobead visually present cell-ECM interactions in a quantitative manner.

Tunable sheathless microfluidic focusing using ion concentration polarization

In this study, we developed a tunable sheathless focusing method for focusing micrometer- and nanometer-sized particles, using ion concentration polarization (ICP) in an ion-selective, resin-coated channel. The particle movement was regulated using an electric field, and by varying the flow rate and ionic strength of the liquid solution; various phenomena were observed, depending on the particle properties. Here, we provide insights into the physical basis of the ICP-focusing phenomena, and a statistical approach for analyzing the particle movement. This ICP-focusing technology is an approach that could be applied for the separation and sorting of various particles, including cells, proteins, and bacteria.

Generation of digitized microfluidic filling flow by vent control

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.

On-Chip Lipid Extraction Using Superabsorbent Polymers for Mass Spectrometry

Pretreatment of samples is one of the most important steps in analytical methods for efficient and accurate results. Typically, an extraction method used for lipid analysis with mass spectrometry is accompanied by complex liquid-liquid extraction. We have devised a simple, rapid, and efficient lipid extraction method using superabsorbent polymers (SAPs) and developed a high-throughput lipid extraction platform based on a microfluidic system. Since SAPs can rapidly absorb an aqueous solution from a raw sample and convert it into the gel, the lipid extraction process can be remarkably simplified. The hydrophobic lipid components were captured into the fibrous SAP gel and then solubilized and eluted directly into the organic solvent without significant interference by this polymer. The small-scale lipid extraction process minimizes the liquid handling and unnecessary centrifugation steps, thereby enabling the implementation of a SAP-integrated microfluidic lipid extraction platform. The SAP method successfully induced reproducible extraction and high recovery rates (95-100%) compared to the conventional Folch method in several lipid classes. We also demonstrated the feasibility of the SAP method for the analysis of lipids in complex biological samples, such as the brain and liver, as well as Escherichia coli. This small-scale SAP method and its microfluidic platform will open up new possibilities in high-throughput lipidomic research for diagnosing diseases because this new technique saves time, labor, and cost.