Jaewon Park

Microfluidic compartmentalized co-culture platform for CNS axon myelination research

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16–18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.

Micropatterning of poly(dimethylsiloxane) using a photoresist lift-off technique for selective electrical insulation of microelectrode arrays.

A poly(dimethylsiloxane) (PDMS) patterning method based on a photoresist lift-off technique to make an electrical insulation layer with selective openings is presented. The method enables creating PDMS patterns with small features and various thicknesses without any limitation in the designs and without the need for complicated processes or expensive equipments. Patterned PDMS layers were created by spin-coating liquid phase PDMS on top of a substrate having sacrificial photoresist patterns, followed by a photoresist lift-off process. The thickness of the patterned PDMS layers could be accurately controlled (6.5-24 µm) by adjusting processing parameters such as PDMS spin-coating speeds, PDMS dilution ratios, and sacrificial photoresist thicknesses. PDMS features as small as 15 µm were successfully patterned and the effects of each processing parameter on the final patterns were investigated. Electrical resistance tests between adjacent electrodes with and without the insulation layer showed that the patterned PDMS layer functions properly as an electrical insulation layer. Biocompatibility of the patterned PDMS layer was confirmed by culturing primary neuron cells on top of the layer for up to two weeks. An extensive neuronal network was successfully formed, showing that this PDMS patterning method can be applied to various biosensing microdevices. The utility of this fabrication method was further demonstrated by successfully creating a patterned electrical insulation layer on flexible substrates containing multi-electrode arrays.

A Microchip for Quantitative Analysis of CNS Axon Growth under Localized Biomolecular Treatments

Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration.

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability. The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput. Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 microm) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master. Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

A magnetic resonance (MR) microscopy system using a microfluidically cryo-cooled planar coil

We present the development of a microfluidically cryo-cooled planar coil for magnetic resonance (MR) microscopy. Cryogenically cooling radiofrequency (RF) coils for magnetic resonance imaging (MRI) can improve the signal to noise ratio (SNR) of the experiment. Conventional cryostats typically use a vacuum gap to keep samples to be imaged, especially biological samples, at or near room temperature during cryo-cooling. This limits how close a cryo-cooled coil can be placed to the sample. At the same time, a small coil-to-sample distance significantly improves the MR imaging capability due to the limited imaging depth of planar MR microcoils. These two conflicting requirements pose challenges to the use of cryo-cooling in MR microcoils. The use of a microfluidic based cryostat for localized cryo-cooling of MR microcoils is a step towards eliminating these constraints. The system presented here consists of planar receive-only coils with integrated cryo-cooling microfluidic channels underneath, and an imaging surface on top of the planar coils separated by a thin nitrogen gas gap. Polymer microfluidic channel structures fabricated through soft lithography processes were used to flow liquid nitrogen under the coils in order to cryo-cool the planar coils to liquid nitrogen temperature (-196 °C). Two unique features of the cryo-cooling system minimize the distance between the coil and the sample: (1) the small dimension of the polymer microfluidic channel enables localized cooling of the planar coils, while minimizing thermal effects on the nearby imaging surface. (2) The imaging surface is separated from the cryo-cooled planar coil by a thin gap through which nitrogen gas flows to thermally insulate the imaging surface, keeping it above 0 °C and preventing potential damage to biological samples. The localized cooling effect was validated by simulations, bench testing, and MR imaging experiments. Using this cryo-cooled planar coil system inside a 4.7 Tesla MR system resulted in an average image SNR enhancement of 1.47 ± 0.11 times relative to similar room-temperature coils.

A Microchip for High-Throughput Axon Growth Drug Screening

It has been recently known that not only the presence of inhibitory molecules associated with myelin but also the reduced growth capability of the axons limit mature central nervous system (CNS) axonal regeneration after injury. Conventional axon growth studies are typically conducted using multi-well cell culture plates that are very difficult to use for investigating localized effects of drugs and limited to low throughput. Unfortunately, there is currently no other in vitro tool that allows investigating localized axonal responses to biomolecules in high-throughput for screening potential drugs that might promote axonal growth. We have developed a compartmentalized neuron culture platform enabling localized biomolecular treatments in parallel to axons that are physically and fluidically isolated from their neuronal somata. The 24 axon compartments in the developed platform are designed to perform four sets of six different localized biomolecular treatments simultaneously on a single device. In addition, the novel microfluidic configuration allows culture medium of 24 axon compartments to be replenished altogether by a single aspiration process, making high-throughput drug screening a reality.

Hybrid Open Drain Method and Fully Current-Based Characterization of Asymmetric Resistance Components in a Single MOSFET

Separate extraction of source (RS) from drain resistance (RD) is important in the systematic modeling of electrical characteristics and investigation of physical mechanism related to the performance and reliability in MOSFETs and their integrated circuits. We report a hybrid open drain method (ODM), as a fully current-based characterization technique, for a comprehensive separation of asymmetric source and drain resistance components in a single MOSFET. In the hybrid ODM, the ODM through the parasitic bipolar transistor is combined with the dual-sweep combinational transconductance technique, the channel resistance method, and the parasitic junction current method. We fully considered the asymmetry in the source and the drain possibly caused by the layout, process, and degradation under bias. We successfully extracted the resistance components with RSe = 6.66-7.35 Q, RDe = 7.64-8.34 Q, RSo = 0.78-8.07 Q, RDo = 1.11-10.08 Q, and RSUB = 6.29-9.17 Q in the n-channel MOSFETs. RSe (RDe) is the VGS-independent external source (drain) resistance. RSo (RDo) is the VGS-independent external spreading source (drain) resistance and RSi (RDi) is the VGS-dependent intrinsic source (drain) resistance, respectively. RSUB is the substrate resistance. The hybrid ODM is expected to be useful in the characterization of parasitic resistances in each MOSFET with asymmetry caused by the layout, process, and degradation without using multiple devices with different channel length (L) and width (W) for measurement.

Lateral-flow particle filtration and separation with multilayer microfluidic channels

Separating particles from a suspension and sorting particles into different size ranges are important to many chemical, biological, and bioengineering applications. In this article, a novel lateral-flow particle separation device is presented for continuous particle fractionation from suspensions. This device is based on three-dimensional multilayer poly(dimethylsiloxane) microchannels, which can be fabricated by high-yield and low-cost molding and transfer-bonding techniques. By varying the dimensions of the microchannels in each layer, particles in a suspension can be fractionated into specific layers based on their sizes. Particle separation is successfully achieved in sorting polystyrene microbeads of 1, 10, and 45μm in diameter into different layers. The yield and selectivity of particle separation can be controlled by device geometries such as channel width and length. This novel continuous-flow particle filtration and separation device is expected to find applications in micrototal analysis systems...

Microfluidic systems for axonal growth and regeneration research

Damage to the adult mammalian central nervous system (CNS) often results in persistent neurological deficits with limited recovery of functions. The past decade has seen increasing research efforts in neural regeneration research with the ultimate goal of achieving functional recovery. Many studies have focused on prevention of further neural damage and restoration of functional connections that are compromised after injury or pathological damage. Compared to the peripheral nervous system, the failure of the adult CNS to regenerate is largely attributed to two basic aspects: inhibitory environmental influences and decreased growth capabilities of adult CNS neurons. Since early demonstration of successful growth of injured CNS axons into grafted peripheral nerve (David and Aguayo, 1981), multiple CNS axonal growth inhibitory factors have been identified and are mainly associated with degenerating CNS myelin (such as Nogo, MAG, OMgp) and with glial scar (such as chondroitin sulfate proteoglycans, CSPGs) (Yiu and He, 2006). However, blockade of these extracellular inhibitory signals alone is often insufficient for the majority of injured axons to achieve long-distance regeneration, as intrinsic regenerative capacity of mature CNS neurons is also a critical determinant for axon re-growth(Sun et al., 2011). Combinatory strategies that enhance neuronal growth and in the meantime overcome environmental inhibitory cues appear to confer better axonal regeneration and neural repair (Wang et al., 2012). While animal models are instrumental and indispensable to our understanding of CNS responses to injury and investigation of intervention strategies and functional regeneration, in vitro models have been designed to address specific and unique questions due to their accessibilities to experimental manipulations and relatively low cost. For instance, early findings from in vitro culture experiments formed the basis for the concept of CNS myelin-associated inhibitory molecules such as Nogo (Schwab and Thoenen, 1985; Chen et al., 2000; GrandPre et al., 2000). Attempts to identify compounds that overcome the inhibition of CNS myelin and CSPGs on neurite outgrowth in culture have revealed key neuronal signaling components that mediate the inhibitory effects of myelin and CSPGs (Sivasankaran et al., 2004). However, conventional culture system often has to use neuronal cultures at low cell density and for only a short period of time due to technical difficulties in monitoring and quantifying axonal growth. To develop effective CNS regenerative strategies, fast and reliable assessment of axonal growth would be critical not only to identify and select potentially interesting candidate molecules that promote axon extension over inhibitory molecules, but also to rule out poor ones. Equally important are considerations that neurons are highly polarized cells and that damaged axon could be extending far away from the cell body and encountering drastically different microenvironment than that of the soma. As such, signaling events elicited by extrinsic factors are most likely spatially regulated and may have different functional outputs. Over the years, compartmentalized neuronal culture systems have been developed. Now the emergence of microfluidics technology offers many advantages and versatilities for axonal growth and regeneration studies (Figure 1). Figure 1 Schematic illustrations of compartmentalized neuron culture platforms for axon isolation.

Liquid-Phase Capillary Etching of Poly(Dimethylsiloxane) Microchannels With Tetra-n-Butylammonium Fluoride

Easy fabricability, along with other unique properties, has made poly(dimethylsiloxane) (PDMS) one of the most commonly used materials for microfluidics-based microdevices. However, unlike other polymer materials commonly used for microdevices, the PDMS cannot be easily etched or further changed upon polymerization, and therefore, fabrication method has been rather limited to replication molding. Here, we demonstrated liquid-phase capillary etching of the PDMS microchannels with tetra-n-butylammonium fluoride (TBAF) and characterized its etching profiles depending on the initial microchannel widths, TBAF flow rates, TBAF concentrations, and substrates used for the microfluidic channel sealing. The characterization showed that the microchannel circumference etch rate was linearly proportional to the TBAF flow rate and was faster at higher TBAF concentration. On the other hand, influence of the TBAF concentration on the horizontal and vertical etch rates was vastly different, with the vertical etch rate being much more affected by the concentration change. Potential applications of the liquid-phase capillary etching method were demonstrated by fabricating the PDMS microchannels with round-shaped cross sections, a microdevice with embedded 3-D metal electrodes with the electrodes directly exposed to the microfluidic channel, and a glass-PDMS-glass sandwich microchannel for high-clarity optical detection. We believe that the characterization of the liquid-phase capillary etching along with the applications demonstrated here will provide new capabilities for fabricating the PDMS-based microdevices that could not be easily fabricated by conventional replication molding processes.