Seog Woo Rhee

A microfluidic culture platform for CNS axonal injury, regeneration and transport.

Investigation of axonal biology in the central nervous system (CNS) is hindered by a lack of an appropriate in vitro method to probe axons independently from cell bodies. Here we describe a microfluidic culture platform that polarizes the growth of CNS axons into a fluidically isolated environment without the use of targeting neurotrophins. In addition to its compatibility with live cell imaging, the platform can be used to (i) isolate CNS axons without somata or dendrites, facilitating biochemical analyses of pure axonal fractions and (ii) localize physical and chemical treatments to axons or somata. We report the first evidence that presynaptic (Syp) but not postsynaptic (Camk2a) mRNA is localized to developing rat cortical and hippocampal axons. The platform also serves as a straightforward, reproducible method to model CNS axonal injury and regeneration. The results presented here demonstrate several experimental paradigms using the microfluidic platform, which can greatly facilitate future studies in axonal biology.

Microfluidic culture platform for neuroscience research

This protocol describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) and peripheral nervous system neurons for neuroscience applications. This method uses replica-molded transparent polymer parts to create miniature multi-compartment cell culture platforms. The compartments are made of tiny channels with dimensions of tens to hundreds of micrometers that are large enough to culture a few thousand cells in well-controlled microenvironments. The compartments for axon and somata are separated by a physical partition that has a number of embedded micrometer-sized grooves. After 3–4 days in vitro (DIV), cells that are plated into the somal compartment have axons that extend across the barrier through the microgrooves. The culture platform is compatible with microscopy methods such as phase contrast, differential interference microscopy, fluorescence and confocal microscopy. Cells can be cultured for 2–3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration. This protocol can be completed in 1–2 days.

Enhanced upconversion luminescence in NaGdF4:Yb,Er nanocrystals by Fe3+ doping and their application in bioimaging

The visible green and red upconversion emissions in Er(3+)/Yb(3+) doped β-NaGdF4 nanoparticles were enhanced by tridoping with Fe(3+) ions (0-40 mol%). XRD, XPS, ICP-AES and EDS data demonstrated successful incorporation of Fe(3+) ions in NaGdF4:Yb(3+)/Er(3+) nanoparticles. The effect of Fe(3+) tridoping on the upconversion luminescence in NaGdF4:Yb(3+)/Er(3+) NPs was investigated in detail. The green and red emission intensities were enhanced by 34 and 30 times, respectively. The maximum emission was observed in a sample containing 30 mol% Fe(3+) ions. A possible mechanism for the enhanced upconversion emission is proposed. In addition, a layer of silica was coated onto the surface of UCNPs to improve the biocompatibility. Folic acid was covalently linked to the silica coated UCNPs to form UCNP@SiO2-FA nanoprobes, which have been successfully applied to the fluorescent imaging HeLa cells.

Assessment of cytocompatibility of surface-modified CdSe/ZnSe quantum dots for BALB/3T3 fibroblast cells

With the widespread use of quantum dots (QDs), the likelihood of exposure to QDs has been assumed to have increased substantially. Recently, QDs have been employed in numerous biological and medical applications. However, there is a lack of toxicological data pertaining to QDs. In this study, we aimed to investigate the cytocompatibility of surface-modified CdSe/ZnSe QDs for BALB/3T3 fibroblast cells. The ligands used for surface modification are mercaptopropionic acid (MPA) and Gum arabic (GA)/tri-n-octylphosphine oxide (TOPO). Cells were exposed to different concentrations of QDs followed by illustrative cytotoxicity analyses. Furthermore, we used a confocal microscope to assess intracellular uptake of QDs. Confocal images showed that MPA-coated QDs were distributed inside the cytoplasmic region of cells. In contrast, GA/TOPO-coated QDs were not found inside cells. MPA-coated QDs were highly cytocompatible, whereas GA/TOPO-coated QDs were toxic to the cells. Cells treated with GA/TOPO-coated QDs showed altered morphology, decreased viability, significant concentrations of intracellular free cadmium, detectable reactive oxygen species (ROS) formation, depolymerized cytoskeleton, and irregular-shaped nuclei. This study suggests that surface modification by ligands plays a significant role in the prevention of cytotoxicity of QDs.

Microfluidic Chambers for Cell Migration and Neuroscience Research

This chapter describes the fabrication and use microfluidic chambers for cell migration and neuroscience research. Both microfluidic chambers are made using soft lithography and replica molding. The main advantages of using soft lithography to create microfluidic chambers are reproducibility, ease of use, and straightforward fabrication procedures. The devices can be fabricated in biology and chemistry laboratories with minimal access to clean-room facilities. First, a microfluidic chemotaxis chamber, which has been used in investigating chemotaxis of neutrophils, human breast cancer cells, and other cell types, is described. Precise and stable gradients of chemoattractants with arbitrary shapes can be generated for different applications. Second, a multicompartment culture chamber that can fluidically isolate neuronal processes from cell bodies is described. The design of this chamber is such that only neurites grow through a series of microgrooves embedded in a physical barrier. Both devices are compatible with phase, differential interference contrast, and fluorescence microscopy.

A new perspective on in vitro assessment method for evaluating quantum dot toxicity by using microfluidics technology.

In this study, we demonstrate a new perspective on in vitro assessment method for evaluating quantum dot (QD) toxicity by using microfluidics technology. A new biomimetic approach, based on the flow exposure condition, was applied in order to characterize the cytotoxic potential of QD. In addition, the outcomes obtained from the flow exposure condition were compared to those of the static exposure condition. An in vitro cell array system was established that used an integrated multicompartmented microfluidic device to develop a sensitive flow exposure condition. QDs modified with cetyltrimethyl ammonium bromide∕trioctylphosphine oxide were used for the cytotoxicity assessment. The results suggested noticeable differences in the number of detached and deformed cells and the viability percentages between two different exposure conditions. The intracellular production of reactive oxygen species and release of cadmium were found to be the possible causes of QD-induced cytotoxicity, irrespective of the types of exposure condition. In contrast to the static exposure, the flow exposure apparently avoided the gravitational settling of particles and probably assisted in the homogeneous distribution of nanoparticles in the culture medium during exposure time. Moreover, the flow exposure condition resembled in vivo physiological conditions very closely, and thus, the flow exposure condition can offer potential advantages for nanotoxicity research.

Integrated Microfluidics Platforms for Investigating Injury and Regeneration of CNS Axons

We describe the development of experimental platforms to quantify the regeneration of injured central nervous system (CNS) neurons by combining engineering technologies and primary neuronal cultures. Although the regeneration of CNS neurons is an important area of research, there are no currently available methods to screen for drugs. Conventional tissue culture based on Petri dish does not provide controlled microenvironment for the neurons and only provide qualitative information. In this review, we introduced the recent advances to generate in vitro model system that is capable of mimicking the niche of CNS injury and regeneration and also of testing candidate drugs. We reconstructed the microenvironment of the regeneration of CNS neurons after injury to provide as in vivo like model system where the soluble and surface bounded inhibitors for regeneration are presented in physiologically relevant manner using microfluidics and surface patterning methods. The ability to control factors and also to monitor them using live cell imaging allowed us to develop quantitative assays that can be used to compare various drug candidates and also to understand the basic mechanism behind nerve regeneration after injury.

Surface Selective Deposition of PMMA on Layered Double Hydroxide Nanocrystals Immobilized on Solid Substrates

2009 WILEY-VCH Verlag Gm Assembly of nanometer-sized particles on various solid substrates has been the focus of intense interest in the development of new integrated functional materials. Layered double hydroxides (LDHs), known as anionic or hydrotalcite-like clays, have been investigated as a multifunctional inorganic material, for example, host materials, catalysts, sorbents, and bioinorganic and polymer–inorganic composites. To date in this area, most of the work performed has been on powder samples in colloidal solutions, where the bulk properties of the randomly assembled nanocrystals predominate over the contribution of the individual ones. LDH particles in the form of powders are considered one of the strongly correlated systems in the field of strong interparticle interactions involving electrostatic forces as well as hydrogen bonding. These hydrophilic ensembles of LDH particles are expected to be less reactive toward incoming reactants, especially organic anions such as carboxylates. In this context, we recently introduced a novel method of controlling the face-to-face assembly of [Mg4Al2(OH)12]CO3 nH2O (MgAl-LDH) nanocrystals on Si substrates in closely packed arrays with a highly-ordered orientation, which can be used for solvothermal anion exchange to give a drastic anisotropic size change that can be observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Multilayer LDH nanocrystals on solid substrates not only make chemical reactions much more reliable, but also open up a new platform of other useful chemical interfaces difficult to achieve in a bulk system. Motivated by the need to assemble functional nanomaterials based on hybrid thin films, we demonstrate in the present study that the LDH nanocrystal organization method can provide a tunable reactive inorganic interface, depending on the liquid media and the surface characteristics of the applied substrates. We were able to precisely modify the surface potentials of the LDH nanocrystals in colloid solutions by changing the solvents, leading to the well-oriented LDH monolayer films acting as a reactive inorganic interface for the fabrication of polymer–LDH hybrid films. The hydroxide groups of LDH provide a facile route to produce the additional surface modification required to develop nanoscale inorganic composite thin films, such as superhydrophobic and polymer–inorganic hybrid films. Among the existing synthetic approaches to the preparation of polymer–inorganic hybrid nanocomposites, surface-initiated polymerization (SIP) allows for the high affinity of the graft polymer by employing the surface modification of the initiator on the surface of layered inorganic compounds. Graft polymers generated on clay surfaces by SIP have especially attracted considerable interest because of their practical applications involving improved mechanical, thermal, and barrier properties. The density of the grafting surfaces could be adjusted by employing different initiators. However, in most systems based on silicate materials reported to date it has been proved that the grafting polymer films have low polymer densities because of the stepwise generation of the initiator molecules to form a monolayer, the difficulty in introducing initiators and monomers into the clay surfaces, and the occurrence of unnecessary side reactions. Herein, we present a precise control method to increase the grafting polymer density on hydroxyl-rich LDH surfaces by using self-assembled monolayers (SAMs) to create a uniform initiator monolayer, in which we were able to change the area coverage of the assembled LDH nanocrystals on the substrates. To the best of our knowledge, this is the first example of the graft density control of polymer films by adjusting the area coverage of an immobilized LDH monolayer with a highly oriented structure on oxide, metal, and polymer substrates. Specifically, the incorporation of poly(methyl methacrylate) (PMMA) on the immobilized LDH surface provides us with new polymer–LDH hybrid films as well as a nanoscale reaction platform, which is extremely difficult in bulk systems. We investigated the orientation and area coverage of MgAl-LDH depending upon the applied substrates and solvents. Figure 1 shows that the tile-like LDH crystals were bound in parallel to the substrate planes on Si. Protic solvents gave a monolayer of MgAl-LDH with higher coverage of at least 50%, whereas nonprotic solvents such as toluene resulted in double and triple layers in some parts with a lower coverage of about 20%. Additional ultrasonic treatment in clean organic solvents for 30min produced no distinguishable changes in the particle assembly, implying that the adhesion is strong enough to resist the ultrasound-induced vibration. Figure 2a presents the surface coverage ratios, namely the percentage areas covered by the MgAl-LDH nanocrystals with respect to the whole Si surface, which are governed by the degree of attraction of MgAl-LDH to the substrates. Alcohols gave higher values of the lateral packing among the solvents. Most of the alcohols, denoted as Group I, gave ratios of around 70% to 90%.

Microfluidic platforms for advanced risk assessments of nanomaterials

Abstract In the past few years, promising efforts to utilize microfabrication-based technologies have laid the foundation for developing advanced, and importantly more physiologically-realistic, microfluidic methods for risk assessment of engineered nanomaterials (ENMs). In the present review, we discuss the wave of recent developments using microfluidic-based in vitro models and platforms for nanotoxicological assays, such as determination of cell viability, cellular dose, oxidative stress and nuclear damage. Here, we specifically highlight the tangible advantages of microfluidic devices in providing promising tools to tackle many of the current and ongoing challenges faced with traditional toxicology assays. Most importantly, microfluidic technology not only allows to recreate physiologically-relevant in vitro models for nanotoxicity examinations, but also provides platforms that deliver an attractive strategy towards improved control over applied ENM doses. In a final step, we present examples of state-of-the-art microfluidic platforms for in vitro assessment of potential adverse ENM effects.

Multicompartmented microfluidic device for characterization of dose-dependent cadmium cytotoxicity in BALB/3T3 fibroblast cells

This paper describes the development of a miniaturized multicompartmented microfluidic device for high-throughput cell cytotoxicity assays and its applicability to the investigation of cadmium-induced cytotoxicity. A steady gradient of cadmium was generated inside the compartments to study the effects of cadmium ion on BALB/3T3 fibroblast cells in a dose-dependent fashion. The device allowed the performance of multiplexed assays to probe the dosage effect of cadmium, morphological alterations of live cells, regulation of proliferation and viability of cells, determination of reactive oxygen species, mechanisms of cell death, i.e. apoptosis and/or necrosis, and immunocytochemical staining of cells in parallel and/or serially, or on a single population simultaneously. The outcomes of all the microfluidic assays were compared to conventional plates-based cytotoxicity assays. The results indicated that the cells cultured in this device were morphologically healthy with greater than 90% viability. They further suggested that the basic mode of cell death behind cadmium-induced cytotoxicity was apoptosis, which was regulated by intracellular oxidative stress via cytoskeleton disorganization and nuclear condensation. Such microenvironments resemble the in vivo physiological conditions very closely and thus offer a unique platform for more accurate observations of cytotoxicity assays and more precise estimation of the IC50 value in comparison to conventional analytical assays.