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Networked neural spheroid by neuro-bundle mimicking nervous system created by topology effect.

Jeong GS, Chang JY, Park JS, Lee SA, Park D, Woo J, An H, Lee CJ, Lee SH - Mol Brain (2015)

Bottom Line: During neural-network formation, neural progenitor cells successfully differentiated into glial and neuronal cells.These cells secreted laminin, forming an extracellular matrix around the host and satellite spheroids.Electrical stimuli were transmitted between networked neurospheroids in the resulting nerve-like neural bundle, as detected by imaging Ca(2+) signals in responding cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Health Science, Korea University, Seoul, 136-100, South Korea. arysu94@gmail.com.

ABSTRACT
In most animals, the nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS), the latter of which connects the CNS to all parts of the body. Damage and/or malfunction of the nervous system causes serious pathologies, including neurodegenerative disorders, spinal cord injury, and Alzheimer's disease. Thus, not surprising, considerable research effort, both in vivo and in vitro, has been devoted to studying the nervous system and signal transmission through it. However, conventional in vitro cell culture systems do not enable control over diverse aspects of the neural microenvironment. Moreover, formation of certain nervous system growth patterns in vitro remains a challenge. In this study, we developed a deep hemispherical, microchannel-networked, concave array system and applied it to generate three-dimensional nerve-like neural bundles. The deep hemicylindrical channel network was easily fabricated by exploiting the meniscus induced by the surface tension of a liquid poly(dimethylsiloxane) (PDMS) prepolymer. Neurospheroids spontaneously aggregated in each deep concave microwell and were networked to neighboring spheroids through the deep hemicylindrical channel. Notably, two types of satellite spheroids also formed in deep hemispherical microchannels through self-aggregation and acted as an anchoring point to enhance formation of nerve-like networks with neighboring spheroids. During neural-network formation, neural progenitor cells successfully differentiated into glial and neuronal cells. These cells secreted laminin, forming an extracellular matrix around the host and satellite spheroids. Electrical stimuli were transmitted between networked neurospheroids in the resulting nerve-like neural bundle, as detected by imaging Ca(2+) signals in responding cells.

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Characterization of neural bundle-spheroid network function. (a) Fluorescence image of neuronal spheroids. Green and blue indicate neurites (β-tubulin) and nuclei (DAPI), respectively. (b) Bundle formation in neurospheroid networks; each neural bundle is composed of several neurite fibers. (c) Schematic depiction of the method used to assess signal transmission within a neurospheroid network. One neuronal spheroid was electrically stimulated and the electrical signal was detected by imaging Ca2+ in another spheroid. Red rectangle indicates the region of interest (ROI). (d) An optical microscope image of an electric stimulator on the nerve-like bundle and the ROI at which Ca2+ signals were recorded. Black arrowheads indicate a neural bundle. (e) A fluorescence microscope image of Ca2+ signals (dotted circles) detected in the ROI shown in (d). The white and black arrowheads indicate satellite spheroid and bundle formation in deep HCWN (f) Electrical transmission through the nerve-like bundle without (left) and with (right) TTX. Signals were detected at all sites prior to TTX treatment, and all signals were eliminated by TTX.
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Fig5: Characterization of neural bundle-spheroid network function. (a) Fluorescence image of neuronal spheroids. Green and blue indicate neurites (β-tubulin) and nuclei (DAPI), respectively. (b) Bundle formation in neurospheroid networks; each neural bundle is composed of several neurite fibers. (c) Schematic depiction of the method used to assess signal transmission within a neurospheroid network. One neuronal spheroid was electrically stimulated and the electrical signal was detected by imaging Ca2+ in another spheroid. Red rectangle indicates the region of interest (ROI). (d) An optical microscope image of an electric stimulator on the nerve-like bundle and the ROI at which Ca2+ signals were recorded. Black arrowheads indicate a neural bundle. (e) A fluorescence microscope image of Ca2+ signals (dotted circles) detected in the ROI shown in (d). The white and black arrowheads indicate satellite spheroid and bundle formation in deep HCWN (f) Electrical transmission through the nerve-like bundle without (left) and with (right) TTX. Signals were detected at all sites prior to TTX treatment, and all signals were eliminated by TTX.

Mentions: Neural cells were cultured in deep HCWN plates for 7 days to allow the development of neural networks connected through nerve-like bundles (Figure 5a and b). Satellite spheroids (Figure 5b, white arrowheads) in the deep hemicylindrical channel were anchored at the center of the channel and were connected to the host spheroids (Figure 5a and b, orange arrowheads), showing that satellite spheroids act as intermediaries to enhance structural networking between host spheroids. To determine whether this nerve-like network supports signal transmission functions, we applied an electrical stimulus to one spheroid (Figure 5c) and recorded Ca2+ responses of fura-2-loaded cells in a connected spheroid. As shown in Figure 5d, electrical stimulation at one point produced simultaneous Ca2+ fluorescence signals in responding cells located at a distance of ~1 mm (Figure 5d, white dotted rectangle) from the stimulator, which is indicative of transmission of a Ca2+ signal through a neural bundle (Figure 5e, white dotted circles) and confirming that spheroids are functionally networked. The stimulation-evoked Ca2+ response was transient and was abolished by the voltage-gated Na+ channel inhibitor tetrodotoxin (TTX; 0.5 μM), indicating that a stimulation-induced action potential could be propagated through the networked bundles, leading to a postsynaptic Ca2+ response at the recording site (Figure 5f). These results show that neural network bundles consist of several neuronal cells and connect spheroid to spheroid.Figure 5


Networked neural spheroid by neuro-bundle mimicking nervous system created by topology effect.

Jeong GS, Chang JY, Park JS, Lee SA, Park D, Woo J, An H, Lee CJ, Lee SH - Mol Brain (2015)

Characterization of neural bundle-spheroid network function. (a) Fluorescence image of neuronal spheroids. Green and blue indicate neurites (β-tubulin) and nuclei (DAPI), respectively. (b) Bundle formation in neurospheroid networks; each neural bundle is composed of several neurite fibers. (c) Schematic depiction of the method used to assess signal transmission within a neurospheroid network. One neuronal spheroid was electrically stimulated and the electrical signal was detected by imaging Ca2+ in another spheroid. Red rectangle indicates the region of interest (ROI). (d) An optical microscope image of an electric stimulator on the nerve-like bundle and the ROI at which Ca2+ signals were recorded. Black arrowheads indicate a neural bundle. (e) A fluorescence microscope image of Ca2+ signals (dotted circles) detected in the ROI shown in (d). The white and black arrowheads indicate satellite spheroid and bundle formation in deep HCWN (f) Electrical transmission through the nerve-like bundle without (left) and with (right) TTX. Signals were detected at all sites prior to TTX treatment, and all signals were eliminated by TTX.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4379946&req=5

Fig5: Characterization of neural bundle-spheroid network function. (a) Fluorescence image of neuronal spheroids. Green and blue indicate neurites (β-tubulin) and nuclei (DAPI), respectively. (b) Bundle formation in neurospheroid networks; each neural bundle is composed of several neurite fibers. (c) Schematic depiction of the method used to assess signal transmission within a neurospheroid network. One neuronal spheroid was electrically stimulated and the electrical signal was detected by imaging Ca2+ in another spheroid. Red rectangle indicates the region of interest (ROI). (d) An optical microscope image of an electric stimulator on the nerve-like bundle and the ROI at which Ca2+ signals were recorded. Black arrowheads indicate a neural bundle. (e) A fluorescence microscope image of Ca2+ signals (dotted circles) detected in the ROI shown in (d). The white and black arrowheads indicate satellite spheroid and bundle formation in deep HCWN (f) Electrical transmission through the nerve-like bundle without (left) and with (right) TTX. Signals were detected at all sites prior to TTX treatment, and all signals were eliminated by TTX.
Mentions: Neural cells were cultured in deep HCWN plates for 7 days to allow the development of neural networks connected through nerve-like bundles (Figure 5a and b). Satellite spheroids (Figure 5b, white arrowheads) in the deep hemicylindrical channel were anchored at the center of the channel and were connected to the host spheroids (Figure 5a and b, orange arrowheads), showing that satellite spheroids act as intermediaries to enhance structural networking between host spheroids. To determine whether this nerve-like network supports signal transmission functions, we applied an electrical stimulus to one spheroid (Figure 5c) and recorded Ca2+ responses of fura-2-loaded cells in a connected spheroid. As shown in Figure 5d, electrical stimulation at one point produced simultaneous Ca2+ fluorescence signals in responding cells located at a distance of ~1 mm (Figure 5d, white dotted rectangle) from the stimulator, which is indicative of transmission of a Ca2+ signal through a neural bundle (Figure 5e, white dotted circles) and confirming that spheroids are functionally networked. The stimulation-evoked Ca2+ response was transient and was abolished by the voltage-gated Na+ channel inhibitor tetrodotoxin (TTX; 0.5 μM), indicating that a stimulation-induced action potential could be propagated through the networked bundles, leading to a postsynaptic Ca2+ response at the recording site (Figure 5f). These results show that neural network bundles consist of several neuronal cells and connect spheroid to spheroid.Figure 5

Bottom Line: During neural-network formation, neural progenitor cells successfully differentiated into glial and neuronal cells.These cells secreted laminin, forming an extracellular matrix around the host and satellite spheroids.Electrical stimuli were transmitted between networked neurospheroids in the resulting nerve-like neural bundle, as detected by imaging Ca(2+) signals in responding cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Health Science, Korea University, Seoul, 136-100, South Korea. arysu94@gmail.com.

ABSTRACT
In most animals, the nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS), the latter of which connects the CNS to all parts of the body. Damage and/or malfunction of the nervous system causes serious pathologies, including neurodegenerative disorders, spinal cord injury, and Alzheimer's disease. Thus, not surprising, considerable research effort, both in vivo and in vitro, has been devoted to studying the nervous system and signal transmission through it. However, conventional in vitro cell culture systems do not enable control over diverse aspects of the neural microenvironment. Moreover, formation of certain nervous system growth patterns in vitro remains a challenge. In this study, we developed a deep hemispherical, microchannel-networked, concave array system and applied it to generate three-dimensional nerve-like neural bundles. The deep hemicylindrical channel network was easily fabricated by exploiting the meniscus induced by the surface tension of a liquid poly(dimethylsiloxane) (PDMS) prepolymer. Neurospheroids spontaneously aggregated in each deep concave microwell and were networked to neighboring spheroids through the deep hemicylindrical channel. Notably, two types of satellite spheroids also formed in deep hemispherical microchannels through self-aggregation and acted as an anchoring point to enhance formation of nerve-like networks with neighboring spheroids. During neural-network formation, neural progenitor cells successfully differentiated into glial and neuronal cells. These cells secreted laminin, forming an extracellular matrix around the host and satellite spheroids. Electrical stimuli were transmitted between networked neurospheroids in the resulting nerve-like neural bundle, as detected by imaging Ca(2+) signals in responding cells.

Show MeSH
Related in: MedlinePlus