Min Jee Jang

Directional neurite growth using carbon nanotube patterned substrates as a biomimetic cue.

Researchers have made extensive efforts to mimic or reverse-engineer in vivo neural circuits using micropatterning technology. Various surface chemical cues or topographical structures have been proposed to design neuronal networks in vitro. In this paper, we propose a carbon nanotube (CNT)-based network engineering method which naturally mimics the structure of extracellular matrix (ECM). On CNT patterned substrates, poly-L-lysine (PLL) was coated, and E18 rat hippocampal neurons were cultured. In the early developmental stage, soma adhesion and neurite extension occurred in disregard of the surface CNT patterns. However, later the majority of neurites selectively grew along CNT patterns and extended further than other neurites that originally did not follow the patterns. Long-term cultured neuronal networks had a strong resemblance to the in vivo neural circuit structures. The selective guidance is possibly attributed to higher PLL adsorption on CNT patterns and the nanomesh structure of the CNT patterns. The results showed that CNT patterned substrates can be used as novel neuronal patterning substrates for in vitro neural engineering.

Pitch‐Dependent Acceleration of Neurite Outgrowth on Nanostructured Anodized Aluminum Oxide Substrates

Nervous systems are composed of microstructured scaffolds with three-dimensional nanofeatured textures. These textures enable the systems to give nanometer-scaled physical cues to the overlying cells, along with biochemical cues. However, the topographical effects on the neurons are still an unexplored territory, although there have been many reports on the biochemical cues for neuronal behavior. It is practically very difficult to investigate the topographical environments in vivo in the biological systems and/or to mimic them precisely in vitro. There is much recent evidence that the cellular response is affected by the physical properties of artificial materials. Studies with such materials could therefore provide us with new insight into the developmental processes of the brain and enable elucidation of the unexplored nanotopographical effects on neuronal behavior. The responses of nerve cells to surface roughness have been studied on various substrates, such as porous silicon, thin titanium nitride films, carbon nanotubes, topographically molded poly(dimethylsiloxane), silicon pillar arrays, gallium phosphide nanowires, aligned nanofiber arrays, and silicon nanowires. Previous studies showed that nerve cells exhibit enhanced attachment and viability as well as the axonal guidance effect on rough surfaces, as opposed to topographically flat surfaces. However, there have been few reports on how nanometer-scaled features regulate neuronal behavior in terms of neurite development. To generate nanotopographical stimuli to neurons in a controllable and systematic manner, it is necessary to make reproducible, rigid structures with variable topographical features. Among the methods for creating topographies on surfaces at the nanometer scale, the fabrication of anodized aluminum oxide (AAO) is highly effective, straightforward,

Axon-First Neuritogenesis on Vertical Nanowires

In this work, we report that high-density, vertically grown silicon nanowires (vg-SiNWs) direct a new in vitro developmental pathway of primary hippocampal neurons. Neurons on vg-SiNWs formed a single, extremely elongated major neurite earlier than minor neurites, which led to accelerated polarization. Additionally, the development of lamellipodia, which generally occurs on 2D culture coverslips, was absent on vg-SiNWs. The results indicate that surface topography is an important factor that influences neuronal development and also provide implications for the role of topography in neuronal development in vivo.

Aqueous micro-contact printing of cell-adhesive biomolecules for patterning neuronal cell cultures

Micro-contact printing (μCP) technique has been widely used for generating micro-scale patterns of biomolecules for patterning live cells. The contact-printing process is carried out in air, while most of the biomolecules including proteins and antibodies should be handled in a solution to preserve their bioactivity. Here we attempted to print biomolecules under aqueous conditions by modifying certain steps that are known to be critical for the bioactivity. The proposed contact-printing process is as follows: After inking the stamp with biomolecule in a solution, the stamp was rinsed in ultra-sonication bath to remove excessive inked biomolecules on the stamp and the following contact-printing process (‘stamping’) was carried out in a buffered solution. By this way, inked biomolecules were consistently handled under a well-defined aqueous condition. Results showed that high-resolution micropatterns of biomolecules can be printed under the aqueous condition (aqueous micro-contact printing, aq-μCP) and it was readily applicable for patterning neuronal cell cultures. Using the modified process, we were able to print widely separated patterns (2 μm-wide lines with 400 μm spacing), which was not achievable with conventional μCP. Extracellular matrix proteins (laminin and fibronectin) were readily printed in a few micrometer scale patterns and their biological activities were confirmed by immunoassays and neuronal cell cultures. We also demonstrated that pH sensitive surface biofunctionalization scheme can be implemented with the proposed aq-μCP for patterning neuronal cell cultures. The aq-μCP improves the existing surface patterning strategy by extending printable patterns and proteins for neuronal cell chip design.

Function of Ezrin‐Radixin‐Moesin Proteins in Migration of Subventricular Zone‐Derived Neuroblasts Following Traumatic Brain Injury

Throughout life, newly generated neuroblasts from the subventricular zone migrate toward the olfactory bulb through the rostral migratory stream. Upon brain injury, these migrating neuroblasts change their route and begin to migrate toward injured regions, which is one of the regenerative responses after brain damage. This injury‐induced migration is triggered by stromal cell‐derived factor 1 (SDF1) released from microglia near the damaged site; however, it is still unclear how these cells transduce SDF1 signals and change their direction. In this study, we found that SDF1 promotes the phosphorylation of ezrin‐radixin‐moesin (ERM) proteins, which are key molecules in organizing cell membrane and linking signals from the extracellular environment to the intracellular actin cytoskeleton. Blockade of ERM activation by overexpressing dominant‐negative ERM (DN‐ERM) efficiently perturbed the migration of neuroblasts. Considering that DN‐ERM‐expressing neuroblasts failed to maintain proper migratory cell morphology, it appears that ERM‐dependent regulation of cell shape is required for the efficient migration of neuroblasts. These results suggest that ERM activation is an important step in the directional migration of neuroblasts in response to SDF1‐CXCR4 signaling following brain injury. STEM CELLS 2013;31:1696–1705

NeuroCa: integrated framework for systematic analysis of spatiotemporal neuronal activity patterns from large-scale optical recording data

Abstract. Optical recording facilitates monitoring the activity of a large neural network at the cellular scale, but the analysis and interpretation of the collected data remain challenging. Here, we present a MATLAB-based toolbox, named NeuroCa, for the automated processing and quantitative analysis of large-scale calcium imaging data. Our tool includes several computational algorithms to extract the calcium spike trains of individual neurons from the calcium imaging data in an automatic fashion. Two algorithms were developed to decompose the imaging data into the activity of individual cells and subsequently detect calcium spikes from each neuronal signal. Applying our method to dense networks in dissociated cultures, we were able to obtain the calcium spike trains of ∼1000 neurons in a few minutes. Further analyses using these data permitted the quantification of neuronal responses to chemical stimuli as well as functional mapping of spatiotemporal patterns in neuronal firing within the spontaneous, synchronous activity of a large network. These results demonstrate that our method not only automates time-consuming, labor-intensive tasks in the analysis of neural data obtained using optical recording techniques but also provides a systematic way to visualize and quantify the collective dynamics of a network in terms of its cellular elements.

Agarose-Assisted Micro-Contact Printing for High-Quality Biomolecular Micro-Patterns†

Micro-contact printing has been developed to print biomolecules, such as cell adhesive molecules, proteins, or DNAs, on a substrate, which can serve as experimental platforms for investigating biological issues and engineering biosensors. Despite the popularity of this method, it has been technically challenging to use a conventional stamp made of a hydrophobic polydimethoxysilane (PDMS) elastomer that often requires surface treatments to facilitate the inking and stamping of biomolecules. In this work, we proposed a new surface modification method for a PDMS stamp using agarose hydrogel and demonstrated the applications to the design of micro-patterned substrates with biomolecules. By using a simple bench-top dip-coating method with a commercial syringe pump to steadily pull out the stamp from boiled agarose solution, we coated an agarose layer on the stamp. It consequentially enhanced the transferability of ink molecules to the target substrate and the uniformity of printed patterns compared to the traditional methods for treating stamp surface such as surfactant coating and temporary oxidation with air plasma. In addition, this microstamping method was also used to produce patterns of proteins with the preservation of bioactivity, which could guide neuronal growth. Thus, we demonstrated the applicability to the interface designs of biochips and biosensors.

BrainFilm, a novel technique for physical compression of 3D brain slices for efficient image acquisition and post-processing

Tissue clearing enables us to observe thick tissue at a single cell resolution by reducing light scattering and refractive index matching. However, imaging of a large volume of tissue for 3D reconstruction requires a great deal of time, cost, and efforts. Few methods have been developed to transcend these limitations by mechanical compression or isotropic tissue shrinkage. Tissue shrinkage significantly lessens the imaging burden; however, there is an inevitable trade-off with image resolution. Here, we have developed the “BrainFilm” technique to compress cleared tissue at Z-axis by dehydration, without alteration of the XY-axis. The Z-axis compression was approximately 90%, and resulted in substantial reduction in image acquisition time and data size. The BrainFilm technique was successfully used to trace and characterize the morphology of thick biocytin-labelled neurons following electrophysiological recording and trace the GFP-labelled long nerve projections in irregular tissues such as the limb of mouse embryo. Thus, BrainFilm is a versatile tool that can be applied in diverse studies of 3D tissues in which spatial information of the Z-axis is dispensable.

Cell-Type Dependent Effect of Surface-Patterned Microdot Arrays on Neuronal Growth

Surface micropatterns have been widely used as chemical cues to control the microenvironment of cultured neurons, particularly for neurobiological assays and neurochip designs. However, the cell-type dependency on the interactions between neurons and underlying micropatterns has been rarely investigated despite the inherent differences in the morphology of neuronal types. In this study, we used surface-printed microdot arrays to investigate the effect of the same micropatterns on the growth of mouse spinal interneuron, mouse hippocampal neurons, and rat hippocampal neurons. While mouse hippocampal neurons showed no significantly different growth on control and patterned substrates, we found the microdot arrays had different effects on early neuronal growth depending on the cell type; spinal interneurons tended to grow faster in length, whereas hippocampal neurons tended to form more axon collateral branches in response to the microdot arrays. Although there was a similar trend in the neurite length and branch number of both neurons changed across the microdot arrays with the expanded range of size and spacing, the dominant responses of each neuron, neurite elongation of mouse spinal interneurons and branching augmentation of rat hippocampal neurons were still preserved. Therefore, our results demonstrate that the same design of micropatterns could cause different neuronal growth results, raising an intriguing issue of considering cell types in neural interface designs.

Optimizing tissue-clearing conditions based on analysis of the critical factors affecting tissue-clearing procedures

Tissue-clearing techniques have received great attention for volume imaging and for the potential to be applied in optical diagnosis. In principle, tissue clearing is achieved by reducing light scattering through a combination of lipid removal, size change, and matching of the refractive index (RI) between the imaging solution and the tissue. However, the contributions of these major factors in tissue clearing have not been systematically evaluated yet. In this study, we experimentally measured and mathematically calculated the contribution of these factors to the clearing of four organs (brain, liver, kidney, and lung). We found that these factors differentially influence the maximal clearing efficacy of tissues and the diffusivity of materials inside the tissue. We propose that these physical properties of organs can be utilized for the quality control (Q/C) process during tissue clearing, as well as for the monitoring of the pathological changes of tissues.