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.
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.
In accordance with the World Health Organization, 360 million people worldwide suffer from hearing loss with profound consequences on professional and private life. Hearing aids can restore sensory function in moderate forms of hearing loss; however, for the most severe cases, the most effective treatment option is a prosthetic device called a cochlear implant (CI). CIs contain a linear electrode array of up to 24 electrodes, which is surgically inserted into the scala tympani of the cochlea. The electrodes directly stimulate the spiral ganglion neurons, forming the auditory nerve 1.
With more than 300,000 devices implanted worldwide, CIs are very successful medical implants and rank among the most cost-effective procedure ever reported. Despite its success the cochlear implant still has limitations such as reduced frequency resolution compared to physiological hearing. This can lead to deficits in effective communication in groups or noisy environments, and the ability to decipher very complex sounds such as music. This reduced frequency resolution is likely due to the gap between the CI electrodes and the spiral ganglion neurons, leading to stimulation of large groups of neurons. This gap is in the range of hundreds of micrometers 2,3. Elimination of this gap would facilitate the stimulation of smaller groups of neurons per electrode, thereby increasing frequency resolution and overall performance of the device 4.
To study the influence of the gap between the electrode and the neuron and the effect of various optimized stimulation protocols, we have developed an in vitro bioassay based on a non-invasive electrophysiological characterization of SGNs on multi electrode arrays (MEAs) 5. Additionally, MEAs can be easily modified to vary electrode shape, size, material and surface roughness, to optimize the neuron-electrode interface. The following is a step-by-step protocol to reproducibly obtain recordings from murine spiral ganglion neuron cultures and assess the dependency on the above-mentioned parameters.
这里描述的协议显示了如何在文化MEA SGN外植体和细胞外无创记录评估SGN活动。这个平台,我们最近开发5允许对新刺激方案和电极材料从而减少能源需求的识别,激活SGN,为进一步实施人工耳蜗等神经修复术的潜在兴趣的协议。在介绍过程一些预防措施是准确的和可重复的实验成就的根本。
螺旋神经节和主要组织进一步的处理小心拨开需要特别谨慎。这些实验已经使用图5和7天前之间的C57 / BL6小鼠中进行。在相同的年龄范围使用Wistar大鼠获得类似的结果(未显示数据)。我们相信这是精细解剖的最佳年龄为耳蜗骨仍是软连接ough容易去除由镊子,和科尔蒂的螺旋神经节和器官可以在不破裂树枝状进程或SGN胞体容易地分离。在较早的年龄的组织过于柔软,机会翻录SGN胞体连同柯蒂氏器是高,而在后期阶段,耳蜗骨胶囊的硬化增加损伤组织而解剖的危险。对SG的适当隔离以及外植体的仔细切割是至关重要的。这些应在范围200〜500微米的大小,以附着最大化到MEA表面,并有在所述外植体,从该突起将再生足够数目的SG胞体的。以增加神经营养支持在培养的最初几天,新鲜的BDNF每天加入柯蒂氏器被放置在共培养。
夹层的速度也很重要。所有步骤应以最小化组织劣化迅速,使用冰冷的溶液来进行。该安乐死和放置外植体在MEA之间的时间应该是10-15分钟之间,并且是成功的培养物是至关重要的。让所有的工具准备好,以避免在文化镀延误。
所有步骤,包括精细解剖,将培养的维护和制备介质和设备都是在无菌条件下进行的。当与ECM溶液涂布在MEA解冻它在冰上并在较高温度下使用冰冷枪头和冰冷介质作为ECM的混合物的凝胶是非常重要的。当将植上的多边环境协定,第一介质添加到电极区域,随后放置外植体。如果介质在第二步骤中加入,外植体趋于分离从MEA由于剪切应力。因为介质的小体积在第5天施加到培养,培养基的蒸发,必须最小化。因此,强烈建议创建使用含PBS密切Proxim的小培养皿加湿室性的多边环境协定。
这里所描述的实验,利用市售的MEA的电极与特定的电极尺寸,涂层材料和帧内电极的距离(如在点2所述,注)进行。培养条件在这里已经进行了优化,以最大化特定电极网格设计的神经元的覆盖。这是可能的的电极间距,几何形状和表面其它配置可能需要不同的涂层或细胞密度。这些步骤可能要求初始故障处理,以达到高密度培养物。
关于电生理记录,在开始录像的培养是从培养基中在37℃下在RT转移到细胞外溶液中之前。为了避免不稳定的录音,等待约10分钟,使文化保持稳定。当刺激的文化,特别应谨慎在选择刺激使用作为大振幅(> 3 V)和持续时间可能会导致在培养的损坏。如对刺激脉冲的形状和持续时间的参考见参考文献5。
用于数据采集和处理自制硬件(记录室中,到放大器和放大器连接)被用来与被选择的特定分析程序(见材料表 ,点7)。然而,其他商业MEA设置和其他软件包适合以及对这些操作。之前我们已经评估我们的文化的答复在3个不同的时间点,且随时间延长培养时间5活性增加。在这里,我们建议18天体外录音。 MEA系统允许连续记录在无菌,加湿室,可以促进对每个培养类型的最佳的时间点的识别。
这种生物测定的主要缺点是在T高变异他数的每培养电极,显示对刺激的反应。的刺激响应这个速率主要取决于四个因素:在培养物中的神经突的密度,突起和电极,所述突起或突起束的直径,并且电极的阻抗之间的联系。关于突起的密度,只有生长在至少两个电极突起可以用于刺激实验。突起和电极之间的接触依赖于周围组织,可以一方面隔离电极的突起,因而恶化了接触,或者,另一方面,隔离突起和从周围浴的电极,从而改善接触。组织密度,以及突起的大小,由所使用的外植体和培养条件限定。解离的神经元培养物也已成功地进行测试,但在这些培养的神经元密度低得多,从而导致RECO的数量减少录制电极。因此,细胞培养物应适应要处理的具体的科学问题。
最后,该电极的阻抗主要由大小和电极的表面上给出。材料,形成一个大的表面,如铂黑, 如图3,降低了电极的阻抗也可提高电极和突起7-9之间的耦合。
因此策略提高刺激的成功率包括的培养条件的优化,总电极或电极密度的数量的增加与电极表面10的调制。
截至目前,已经利用膜片钳技术进行SG神经元的电生理特性。这使得动作电位的细胞内记录和细胞内的离子电流的详细分析单个神经元。这里,我们提出的体外生物测定可用于通过同时分析自发活动或响应许多神经元的细胞外的刺激来研究螺旋神经节神经元的活性谱。此外,电极和螺旋神经节神经元之间的相互作用,可以研究和修改的或新的材料的应用进行优化。最后,即使这里没有示出,该平台可组合使用的外部电极,安装在显微最近由我们的组5所示,在为了研究刺激电极和文化活动的距离之间的关系。所有这些新颖的方面允许的耳蜗植入电极的主要功能的模仿可能导致新的假体装置的设计。
我们的模型是一种在体外工具非常有用的研究策略,提高听神经元,并进一步运算的刺激功效timize CI技术。一旦技术被掌握,人们可以设想筛选一些变量的修改:一个)的不同神经元群体,二)不同电极材料/尺寸/阻抗三)执行慢性实验来检验材料毒性或电极刺激诱导的毒性,这可以对体内电极阵列更安全,更有效的刺激方案线索。
The authors have nothing to disclose.
作者感谢露丝Rubli在瑞士伯尔尼大学的生理学系与实验宝贵的技术帮助。这项工作是由欧盟FP7-NMP程序(。 – www.nanoci.org项目NANOCI赠款协议没有281056)的部分资助。
culture medium | |||
Neurobasal medium | Invitrogen | 21103-049 | 24 ml (for 25 ml) |
HEPES | Invitrogen | 15630-080 | 250 μl (for 25 ml) |
Glutamax | Invitrogen | 35050-061 | 250 μl (for 25 ml) |
B27 | Invitrogen | 17504-044 | 500 μl (for 25 ml) |
FBS | GIBCO | 10099-141 | 10% (for 25 ml) |
BDNF | R&D Systems | 248-BD-025/CF | final 5 ng/ml (for 25 ml) |
Name | Company | Catalog Number | Comments |
extracellular solution (pH 7.4) | |||
NaCl | 145mM | ||
KCl | 4mM | ||
MgCl2 | 1mM | ||
CaCl2 | 2mM | ||
HEPES | 5mM | ||
Na-pyruvate | 2mM | ||
Glucose | 5mM | ||
Name | Company | Catalog Number | Comments |
blocking solution | |||
PBS | Invitrogen | 10010023 | |
BSA | Sigma | A4503-50G | 2% |
Triton X-100 | Sigma | X100 | 0.01% |
Name | Company | Catalog Number | Comments |
Immunostaining solutions | |||
TuJ | R&D Systems | MAB1195 | dil 1:200 |
DAPI | Sigma | D9542 | |
Paraformaldehyde | Sigma | 158127 | 4% |
Fluoreshild | Sigma | F6057 | |
Name | Company | Catalog Number | Comments |
plastic/tools | |||
petri dish 35 mm | Huberlab | 7.627 102 | |
petri dish 94 mm | Huberlab | 7.633 180 | |
Dumont #5 tweezer | WPI | 14098 | |
Dumont #55 tweezer | WPI | 14099 | |
Name | Company | Catalog Number | Comments |
Materials | |||
Enzymatic solution: Terg-a-Zyme | Sigma | Z273287-11KG | |
Extracellular Matrix (ECM) mix: Matrigel TM | Corning | 356230 | |
MEA electrodes | Qwane Biosciences | (Lausanne, Switzerland) | |
Name | Company | Catalog Number | Comments |
Software | |||
Labview | National Instruments | Switzerland | |
IgorPro | WaveMetrics | Lake Oswega, USA |