Wonhye Lee

Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.

We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing.

Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture

Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs promotes the migration and proliferation of embedded cells, which is an important factor for designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF (vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural tissue. We examined the morphological changes of the printed C17.2 cells embedded in the collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%) after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within 1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their morphology. The cells printed in this range also migrated toward the fibrin gel, with the total migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of the GF in the collagen scaffold. The presented method can be gainfully used in the development of three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications.

On-Demand Three-Dimensional Freeform Fabrication of Multi-Layered Hydrogel Scaffold With Fluidic Channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186.

Three-dimensional bioprinting of rat embryonic neural cells

We present a direct cell printing technique to pattern neural cells in a three-dimensional (3D) multilayered collagen gel. A layer of collagen precursor was printed to provide a scaffold for the cells, and the rat embryonic neurons and astrocytes were subsequently printed on the layer. A solution of sodium bicarbonate was applied to the cell containing collagen layer as nebulized aerosols, which allowed the gelation of the collagen. This process was repeated layer-by-layer to construct the 3D cell–hydrogel composites. Upon characterizing the relationship between printing resolutions and the growth of printed neural cells, single/multiple layers of neural cell–hydrogel composites were constructed and cultured. The on-demand capability to print neural cells in a multilayered hydrogel scaffold offers flexibility in generating artificial 3D neural tissue composites.

Image-Guided Transcranial Focused Ultrasound Stimulates Human Primary Somatosensory Cortex

Focused ultrasound (FUS) has recently been investigated as a new mode of non-invasive brain stimulation, which offers exquisite spatial resolution and depth control. We report on the elicitation of explicit somatosensory sensations as well as accompanying evoked electroencephalographic (EEG) potentials induced by FUS stimulation of the human somatosensory cortex. As guided by individual-specific neuroimage data, FUS was transcranially delivered to the hand somatosensory cortex among healthy volunteers. The sonication elicited transient tactile sensations on the hand area contralateral to the sonicated hemisphere, with anatomical specificity of up to a finger, while EEG recordings revealed the elicitation of sonication-specific evoked potentials. Retrospective numerical simulation of the acoustic propagation through the skull showed that a threshold of acoustic intensity may exist for successful cortical stimulation. The neurological and neuroradiological assessment before and after the sonication, along with strict safety considerations through the individual-specific estimation of effective acoustic intensity in situ and thermal effects, showed promising initial safety profile; however, equal/more rigorous precautionary procedures are advised for future studies. The transient and localized stimulation of the brain using image-guided transcranial FUS may serve as a novel tool for the non-invasive assessment and modification of region-specific brain function.

Linear and non-linear EEG analysis of adolescents with attention-deficit/ hyperactivity disorder during a cognitive task

OBJECTIVE We aimed to investigate whether electroencephalograph (EEG) dynamics differ in adolescents with attention-deficit/hyperactivity disorder (ADHD) compared with healthy subjects during the performance of a cognitive task. METHODS We recorded EEGs from 19 scalp electrodes in 11 adolescent boys with ADHD and 12 age-matched healthy boys while the subjects were at rest and during a continuous performance test (CPT). The approximate entropy (ApEn), a non-linear information-theoretic measure, was calculated to quantify the complexity of the EEGs. RESULTS The mean ApEn of the ADHD patients was significantly lower than the healthy subjects over the right frontal regions (Fp2 and F8) during the performance of the cognitive task, but not at rest. The spectral analysis showed significant differences between the two groups in the P3 and T4 regions at rest and the Fp2 and F8 regions during task performance. CONCLUSIONS The differences in EEG complexity between the two groups suggest that cortical information processing is altered in ADHD adolescents, and thus their levels of cortical activation may be insufficient to meet the cognitive requirements of attention-demanding tasks. SIGNIFICANCE This study suggests that a non-linear measure such as ApEn is useful for investigating neural dysfunctions in adolescents with ADHD.

Transcranial focused ultrasound stimulation of human primary visual cortex

Transcranial focused ultrasound (FUS) is making progress as a new non-invasive mode of regional brain stimulation. Current evidence of FUS-mediated neurostimulation for humans has been limited to the observation of subjective sensory manifestations and electrophysiological responses, thus warranting the identification of stimulated brain regions. Here, we report FUS sonication of the primary visual cortex (V1) in humans, resulting in elicited activation not only from the sonicated brain area, but also from the network of regions involved in visual and higher-order cognitive processes (as revealed by simultaneous acquisition of blood-oxygenation-level-dependent functional magnetic resonance imaging). Accompanying phosphene perception was also reported. The electroencephalo graphic (EEG) responses showed distinct peaks associated with the stimulation. None of the participants showed any adverse effects from the sonication based on neuroimaging and neurological examinations. Retrospective numerical simulation of the acoustic profile showed the presence of individual variability in terms of the location and intensity of the acoustic focus. With exquisite spatial selectivity and capability for depth penetration, FUS may confer a unique utility in providing non-invasive stimulation of region-specific brain circuits for neuroscientific and therapeutic applications.

Transcranial focused ultrasound to the thalamus is associated with reduced extracellular GABA levels in rats.

Objective: Transcranial focused ultrasound (FUS), with its ability to non-invasively modulate the excitability of region-specific brain areas, is gaining attention as a potential neurotherapeutic modality. The aim of this study was to examine whether or not FUS administered to the brain could alter the extracellular levels of glutamate and γ-aminobutyric acid (GABA), which are representative excitatory and inhibitory amino acid neurotransmitters, respectively. Methods: FUS, delivered in the form of a train of pulses, was applied to the thalamus of Sprague-Dawley rats transcranially. Glutamate and GABA were directly sampled from the frontal lobe of the rat brain via a direct microdialysis technique before, during, and after the sonication. The dialysate concentrations were determined by high-performance liquid chromatography. Results: The individual levels of the neurotransmitters sampled were normalized to the baseline level for each rat. In terms of the changes in extracellular glutamate levels, there was no difference between the FUS-treated group and the unsonicated control group. However, extracellular GABA levels started to decrease upon sonication and remained reduced (approximately 20% below baseline; repeated-measures ANOVA, p < 0.05, adjusted for multiple comparisons) compared to the control group. Conclusion: The ability to modulate region-specific brain activity, along with the present evidence of the ability to modulate neurotransmission, demonstrates the potential utility of FUS as a completely new non-invasive therapeutic modality.

Microvalve-Assisted Patterning Platform for Measuring Cellular Dynamics Based on 3D Cell Culture

A microfluidic platform to satisfy both 3D cell culture and cell‐based assay is required for credible assay results and improved assay concept in drug discovery. In this article, we demonstrate a microvalve‐assisted patterning (MAP) platform to provide a new method for investigating cellular dynamics by generating a linear concentration gradient of a drug as well as to realize 3D cell culture in a microenvironment. The MAP platform was fabricated by multilayer soft lithography and several microvalves made it possible to pattern a cell–matrix (scaffold) and to exchange media solutions without breaking cell–matrix structure in a microchannel. This approach provides not only exact fluids control, bubble removal, and stable solution exchange in a microchannel, but also reliable scaffold fabrication and 3D cell culture. In this study, hepatotoxicity tests with human hepatocellular liver carcinoma cells (HepG2) were also performed in real‐time monitoring where cell morphologies exposed to different drug concentrations were observed at a time. Compared to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay, the MAP platform could be used to reduce drug amount and assay time for cell‐based assays as much as 10 and 3 times, respectively. Biotechnol. Bioeng. 2008;101: 1005–1013.

Suppression of EEG visual-evoked potentials in rats through neuromodulatory focused ultrasound.

We investigated the use of pulsed low-intensity focused ultrasound (FUS) to suppress the visual neural response induced by light stimulation in rodents. FUS was administered transcranially to the rat visual cortex using different acoustic intensities and pulsing duty cycles. The visual-evoked potentials (VEPs) generated by an external strobe light stimulation were measured three times before, once during, and five times after the sonication. The VEP magnitude was suppressed during the sonication using a 5% duty cycle (pulse-repetition frequency of 100 Hz) and a spatial-peak pulse-average acoustic intensity of 3 W/cm2; however, this suppressive effect was not present when a lower acoustic intensity and duty cycle were used. The application of a higher intensity and duty cycle resulted in a slight elevation in VEP magnitude, which suggested excitatory neuromodulation. Our findings demonstrate that the application of pulsed FUS to the region-specific brain area not only suppresses its excitability, but can also enhance the excitability depending on the acoustic intensity and the rate of energy deposition. This bimodal feature of FUS-mediated neuromodulation, which has been predicted by numerical models on neural membrane capacitance change by the external acoustic pressure waves, suggests its versatility for neurotherapeutic applications.