저자는 매우 도움이 토론 오퍼 펜어맨, 프레드 울프, 메나헴 시걸, 안드레아스 니프과 에이 탄 Reuveny 감사합니다. 저자는 기술의 초기 버전을 개발 일란 브레스킨와 조르디 소리아노 감사합니다. 저자는 이론적 인 개념에 도움을 트비 틀러스티와 장 피에르 에크 만 감사합니다. 이 연구는 미네르바 재단, 과학 기술, 이스라엘의 교육부와 이스라엘 과학 재단 보조금 1320년에서 1309년까지과 양성 국립 과학 재단 (National Science Foundation) 교부금 2,008,331에 의해 지원되었다.

<ol>
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저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

1D 패턴은 다양한 애플리케이션에 사용될 수있는 중요한 도구입니다. 예를 들어, 우리는 신경 세포의 배양 물 (29)로부터 논리 게이트를 생성하고 최근 래트 해마 뉴런 5 Chronaxie 및 Rheobase를 측정하고, 상기 비교 다운 증후군 해마 뉴런 소성 활동 신호 전파 속도의 감속을위한 1 차원 패턴을 사용한 야생형 (WT) 해마의 뉴런 (27). 1 차원 패터닝 제안...

신경 세포와 외부 전기장 사이의 상호 작용은 근본적인 의미뿐만 아니라 실제 사람이있다. 이 외부인가 전계 조직을 자극 할 수있는 볼타 시대부터 알려져 있지만, 뉴런에서 얻어진 활동 전위의 제조를 담당하는 기전은 최근 4 3 2 1 풀어되기 시작한다. 이 전기장 2, 5에 응답하는 신경 세포의 막전위의 탈분극, 막 특성 및 이온 채널의 역할, 심지어 지역을 유발하는 메커니즘에 관한 질문에 대한 답을 찾는 포함되어 있습니다. 신경 자극 6, 7, 8, 9, 치료 <sup10 가지 방법론은이 정보에 특히 의존하며, 이는 고통받는 지역을 대상으로하고 치료 결과를 이해하는 데 중요 할 수 있습니다. 이러한 이해는 또한 치료 프로토콜과 뇌의 다른 영역을 자극하기위한 새로운 접근법을 개발하는 데 도움이 될 수 있습니다. 생체 내 뇌 에서 의 상호 작용을 측정하는 것은 이러한 이해에 중요한 요소를 추가하지만, 두개골 내 측정의 부정확성과 낮은 제어 가능성으로 인해 방해 받는다. 대조적으로, 배양 물에서의 측정은 고정밀, 우수한 신호 대 잡음 성능 및 고도의 재현성 및 제어로 대량으로 용이하게 수행 될 수있다. 문화를 사용하여 집단 네트워크 행동의 다양한 연결 특성을 밝혀 낼 수 있습니다 11 , 12 , 13 , 14 , </sup&gt; 15, 16. 마찬가지로,이 또한 제어 시스템 optogenetically 활성 뉴런 17, 18의 광 자극 동안의 채널 개구부 (19)가 활동 전위를 생성 할 책임이 방법, 예를 들면 다른 자극 방법이 작동하는 메커니즘을 해명에 매우 효율적일 것으로 예상된다. 여기에 초점을 효율적으로 외부 전기장을 통해 신경 세포를 자극 수있는 도구의 개발과 이해를 설명에 있습니다. 본 연구에서 우리는 하나 차원 배양 상이한 구성 및 목욕 전극에 의해 직접인가 전계의 방향 및 2 차원의 마지막 자극을 이용하여 자극, 2 차원 일차원 패터닝 해마 배양의 제조를 설명하고 패터닝 시변 전기장을 유도하는 자기장을5, 20, 21.

<table><tbody><tr><td>APV</td><td>Sigma-Aldrich</td><td>A8054</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>B27 supp</td><td>Gibco</td><td>17504-044</td><td>Plating medium. Mentioned in Section 1.1.1</td></tr><tr><td>bicuculline</td><td>Sigma-Aldrich</td><td>14343</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>Borax (sodium tetraborate decahydrate)</td><td>Sigma-Aldrich</td><td>S9640</td><td>Borate buffer. Mentioned in Section 1.1.2</td></tr><tr><td>Boric acid</td><td>Frutarom LTD</td><td>5550710</td><td>Borate buffer. Mentioned in Section 1.1.2</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; , 1 M</td><td>Fluka&amp;nbsp;</td><td>21098</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>CNQX</td><td>Sigma-Aldrich</td><td>C239</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>COMSOL</td><td>COMSOL Inc</td><td>Multiphysics 3.5</td><td>Numerical simulation. Mentioned in Section 3.5.2</td></tr><tr><td>D-(+)-Glucose, 1 M</td><td>Sigma-Aldrich</td><td>65146</td><td>Plating medium, Extracellular recording solution. Mentioned in Sections 1.1.1 and 1.5.2</td></tr><tr><td>D-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>Cell Cultures. Mentioned in Sections 1.2.4 and 1.2.6</td></tr><tr><td>FCS (FBS)</td><td>Gibco</td><td>12657-029</td><td>Plating medium. Mentioned in Section 1.1.1</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td>Bio Coating. Mentioned in Section 1.2.6</td></tr><tr><td>Fluo4AM</td><td>Life technologies</td><td>F14201</td><td>Imaging of spontaneous or evoked activity. Mentioned in Sections 1.5.1, 1.5.3, and 1.5.5</td></tr><tr><td>FUDR</td><td>Sigma-Aldrich</td><td>F0503</td><td>Changing medium. Mentioned in Section 1.4.1</td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1272</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td></tr><tr><td>GlutaMAX 100x</td><td>Gibco</td><td>35050-038</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td></tr><tr><td>Hepes, 1 M</td><td>Sigma-Aldrich</td><td>H0887</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>HI HS</td><td>BI</td><td>04-124-1A</td><td>Plating medium, Changing medium, Final medium. Mentioned in Sections 1.1.1, 1.4.1, and 1.4.2</td></tr><tr><td>KCl,&amp;nbsp; 3 M</td><td>Merck</td><td>1049361000</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Laminin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L2020</td><td>Bio Coating. Mentioned in Section 1.2.6</td></tr><tr><td>MEM x 1</td><td>Gibco</td><td>21090-022</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&amp;nbsp;&amp;nbsp;&amp;nbsp; 1.4.2</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; , 1 M</td><td>Sigma-Aldrich</td><td>M1028</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>NaCl, 4 M</td><td>Bio-Lab</td><td>19030591</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Octadecanethiol</td><td>Sigma-Aldrich</td><td>01858</td><td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td></tr><tr><td>Pluracare F108 NF Prill</td><td>BASF Corparation&amp;nbsp;</td><td>50475278</td><td>Bio-Rejection Coating, Bio Coating. Mentioned in Sections 1.2.4 and 1.2.6</td></tr><tr><td>Poly-L-lysine 0.01% solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;P47075</td><td>Promote cell division. Mentioned in Section 1.1.4</td></tr><tr><td>Sucrose, 1 M</td><td>Sigma-Aldrich</td><td>S1888</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Thiol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>1858</td><td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td></tr><tr><td>URIDINE</td><td>Sigma-Aldrich</td><td>U3750</td><td>Changing medium. Mentioned in Section 1.4.1</td></tr><tr><td>Sputtering machine</td><td>AJA International, Inc</td><td>ATC Orion-5Series&amp;nbsp;</td><td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td></tr><tr><td>Pen plotter&amp;nbsp;</td><td>Hewlett Packard&amp;nbsp;</td><td>HP 7475A</td><td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td></tr><tr><td>Electrodes wires&amp;nbsp;</td><td>A-M Systems, Carlsborg WA</td><td>767000</td><td>Electric stimulation of neuronal cultures. Mentioned in Sections 2.1, 2.2, 2.3, and 2.4.5</td></tr><tr><td>Signal generator</td><td>BKPrecision</td><td>4079</td><td>Shaping of the electric signal. Mentioned in Section 2.3</td></tr><tr><td>Amplifier</td><td>Homemade</td><td></td><td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td></tr><tr><td>Power supply</td><td>Matrix&amp;nbsp;</td><td>MPS-3005 LK-3&amp;nbsp;</td><td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td></tr><tr><td>Transcranial magnetic stimulation</td><td>Magstim, Spring Gardens, UK</td><td>Rapid 2</td><td>Magnetic stimulation of neuronal culture. Mentioned in Sections 3.1, 3.3, and 3.4</td></tr><tr><td>Epoxy</td><td>Cognis</td><td>Versamid 140</td><td>Casting of homemade coils. Mentioned in Section 3.4</td></tr><tr><td>Epoxy</td><td>Shell</td><td>EPON 815&amp;nbsp;</td><td>Casting of homemade coils. Mentioned in Section 3.4</td></tr><tr><td>Platinum wires 0.005&#039;&#039; thick; A-M Systems,&amp;nbsp;</td><td>&amp;nbsp;Carlsborg WA</td><td>&amp;nbsp;767000</td><td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td></tr><tr><td>Circular magnetic coil</td><td>Homemade</td><td></td><td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td></tr><tr><td>WaveXpress SW</td><td>B&amp;amp;K Precision&amp;nbsp;</td><td></td><td>Waveform editing software. Mentioned in Section 2.1.32</td></tr><tr><td>Xion Ultra 897</td><td>Andor</td><td></td><td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td></tr></tbody></table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

뉴런은 멤브레인 전위가 특정 임계 값을 초과 할 때 활동 전위를 발화시킵니다. 뇌의 전형적인 활동에서, 이것은 시냅스에 대한 화학 물질 투입의 결과로서 발생합니다. 그러나 뉴런은 부과 된 전기장에 의해 흥분 될 수도 있습니다. 특히 최근의 임상 응용은 외부에서 전기장을 만들어 뉴런을 활성화시킵니다. 따라서 신경 세포가 외부 장에 어떻게 반응하는지 그리고 활동 잠재력을 유발하는 원인을 조사하는 것이 중요합니다. 다행히도, 외부 전기장의 정밀하고 제어 된 적용은 배양에서 절제되고, 해리되고 성장되는 배아 신경 세포에 대해 가능하다. 이를 통해 매우 재현 가능한 시스템에서 이러한 질문을 조사 할 수 있습니다. 이 논문에서는 신경 세포 배양에 외부 전계의 제어 된 적용을 위해 사용되는 기술의 일부가 재검토된다. 네트워크는 1 차원 일 수 있으며, 즉 선형으로 패턴 화 될 수 있습니다R 양식 또는 상기 기판의 전체면 상에 성장시키고, 따라서 이차원. 또한, 음원은 유체 (욕 전극) 또는 자기 펄스 원격 생성을 이용하여 전계를 유도하여 침지 전극을 통하여 전계를 직접 애플리케이션에 의해 생성 될 수있다.

<html> 제시된 프로토콜은의 연결 문화의 쉬운 patterning 수 있습니다. 우리가 자극을 위해 개발 한 여러 가지 방법과 결합되면 Chronaxie 및 Rheobase 5 와 같은 일부 고유 신경 세포 특성을 측정하고 건강한 사람과 질병이있는 뉴런의 특성을 비교하고 문화를 자극하는 최적의 방법을 찾기 위해 사용할 수 있습니다. 그들의 구조와 더 많은 새로운 접근 방식을 제공...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

신경 세포의 문화는 하나의 신경 세포에 대한 효과 나 신경 세포의 인구를 통해 새로운 뇌 자극 기술을 연구하는데 좋은 모델입니다. 목욕 전극에 의해 직접 생산 또는 시변 자기장에 의해 유도되는 전계에 의해 패터닝 신경 배양 자극하는 다른 방법들이 여기에 제시 하였다.

전기 단일 및 두 개 차원 문화에 자기장을 사용하여 신경 세포의 외부인가 전압

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

external-excitation-neurons-using-electric-magnetic-fields-one-two

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13.2K 조회수.  The Salk Institute for Biological Studies.  신경 세포의 문화는 하나의 신경 세포에 대한 효과 나 신경 세포의 인구를 통해 새로운 뇌 자극 기술을 연구하는데 좋은 모델입니다. 목욕 전극에 의해 직접 생산 또는 시변 자기장에 의해 유도되는 전계에 의해 패터닝 신경 배양 자극하는 다른 방법들이 여기에 제시 하였다. 

비디오: External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

연구

생체공학

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

외부 응용 직류 전기 분야에 신경 전구체 세포 마이그레이션 속도론 분석을위한 Galvanotaxis 분석

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

신경과학

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

다중 전극 배열에 나선 신경절 신경 이식편 문화 및 전기 생리학

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

미세 전극 배열에서 신경 세포 배양의 자극에 대한 네트워크 반응의 시간 의존적 증가

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

실리콘 나노 와이어 및 세포 간 전기 커플링의 조사를위한 광학 자극

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

미세 유체 장치에서 전기장 유발 신경 전구체 세포 분화

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

생물학적 조직의 자극을 위한 전기 및 자기장 장치

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

External Excitation of One-Dimensional Patterned Neuronal Cultures

Source:Stern, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for stimulating line-patterned neuronal cultures with a uniform, unidirectional electric field.

1차원 패턴화된 신경 세포 배양의 외부 흥분(External Excitation of one-dimensional patterned neuronal cultures)

1. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.

	Prepare a solution of 50 &#181;g calcium sensitive fluorescent ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Source: Stern, S., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

자기장에 의한 패턴화된 신경 배양의 자극(Stimulation of patterned neuronal cultures by a magnetic field)

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

Source: Tedesco, M., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53080">Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model.</a> J. Vis. Exp. (2015)
This video demonstrates the development of a 3D neuronal culture coupled to a planar microelectrode array with hippocampal neurons. The isolated neurons form a 2D network on the microelectrode array&#39;s active area and adhere to glass microbeads in a multi-well plate with a membrane insert. Upon transferring these neuron-coated microbeads to the electrode, they organize into an interconnected 3D neural network.