由共轭聚合物活细胞电活动的光控制
Summary
A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.
Abstract
Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.
Introduction
能够操作与精确的空间和时间分辨率的细胞活性的可能性表示在神经科学研究和在神经和精神障碍的治疗的关键策略1的传统方法是基于使用电极细胞电刺激定位成接近或接触目标系统，2可以是不同的复杂性（单电池，蜂窝网络，脑切片， 在体内脑组织）。在过去的一个世纪中，利用膜片钳，金属基板集成电极所提供的生理学和神经网络的运作机制的单个神经元和病理生理学的详细图片。然而，电刺激患有重要的限制。第一个涉及一种普遍较差空间分辨率由于电极和它们的固定几何形状的的物理尺寸，这是不能容易地适应像生物组织复杂的组织系统。另外，有关的刺激和记录系统之间的串扰的电极阻抗和问题可能恶化的测量值的最终信噪比3另一方面，利用光的刺激可能有助于克服许多局限性电方式。首先，它提供了前所未有的空间（<1微米）和时间分辨率（<1毫秒），使得能够针对特定的细胞类型或甚至亚细胞区室。此外，它是高度非侵入性，因为它避免了与感兴趣的组织的任何物理接触和disentangles刺激从记录。而且，无论光的强度和波长，可以精确地调节，从而多样化刺激方案都可以应用。3,4-
然而，动物细胞中的绝大多数不呈现给光任何特定的灵敏度。几种策略用于光学stimulation的因而被提出，或者利用附近或在细胞内的感光分子介质，或使用光敏器件外部设置的，靠近小区。前一类是指内生机制，例如通过可见光或红外光（IR）的刺激，以及利用或者光异构化/光解的化合物或光敏分子致动器（光遗传学）基因的表达。后一类包括技术实现了与使用无机纳米/微米颗粒或光电的硅衬底的外源性刺激。5然而，所有这些系统都具有光明的两侧和缺点。具体地，细胞在可见光范围内源性的吸收弱，不可靠，以及伴随的产生活性氧的物质可以是有害的细胞。在一般情况下，红外用于诱导局部热加热因吸水，但水的消光系数小，因此要求圣荣红外光（从几十到几百瓦/毫米2），它是难以通过标准显微镜光学器件以提供并可能造成安全问题为在体内的应用程序。另一方面，光笼蔽切换化合物具有时间限制的动作，往往需要UV光，是很难传递由于有限的组织穿透。此外，他们还从激活的化合物后，光解照射范围外扩散问题的困扰。最后，光遗传学工具使得科学家能够针对特定的细胞亚群和副舱室，并迅速成为的关键技术在神经科学研究之一。然而，通过病毒载体中插入的外源DNA区段提出了重要的安全问题，特别是考虑通过对人类患者。5,6-出于这些原因，新材料和设备能够细胞的光学操纵研究是一个非常热门的话题。
最近，一种新型基于使用的光敏共轭聚合物，能够有效地转导的光刺激成细胞电活动的调制手段，已经提出了。由聚合物光激发的细胞刺激（CSPP）技术利用许多键支持特性的典型的有机半导体：它们在本质上是敏感的光在可见光范围内; 7它们是生物相容的，柔软和适形和它们的机械灵活性允许与组织亲密的接口无论是在体外 和体内8-10。除此之外，他们可以很容易地官能化，以更好地适应与活细胞的界面，并启用特定激励，探测和感测能力。11,12而且，它们支持电子以及离子迁移，从而使它们适合的组合电子广告生物学13,14有趣的是，他们可以在光伏模式下工作，避免了需要应用外部偏置˚F或有效的细胞光刺激。15
CSPP技术的可靠性已在几个系统先前已经证实，包括原代神经元，星形胶质细胞15,16，17二次电池线 18和取出的视网膜组织16在这项工作中，所有必要的步骤，以制造光敏感的生物聚合物的接口 19 在体外系统的光学刺激进行详细说明。作为研究的情况下，区域规则性聚（3-己基噻吩）的一个典型的有机光生伏打共混物（RR-P3HT），用作电子给体，和苯基C61-丁酸 – 甲基酯（PCBM），充当电子受体采用。作为生物系统，人胚肾（HEK-293）细胞中被使用。设置有细胞的活性通过电生理学测量的相对记录一个光刺激协议的一个例子。
所描述的平台然而一般有效性，并且它可以很容易地扩展到使用其它共轭聚合物（通过适当地调节溶液的制备方法和淀积参数），不同类型的细胞（通过适当改变细胞培养协议，电镀过程和时间请求细胞接种和增殖）和不同的刺激方案（光的波长，刺激频率和持续时间，光激发密度）。
Protocol
Representative Results
Discussion
关键步骤所报告的协议为体外细胞光学刺激主要涉及光敏聚合物的选择，热灭菌参数，强度和光刺激的持续时间。一个P3HT：PCBM薄膜在这里被选中，因为它保证了良好的时间和电化学稳定性。然而，人们应该注意到，并非所有的感光聚合物可提供模拟表演，22更具体在照明。此外，在这种情况下，选定的加热杀菌参数不导致的聚合物光电特性相当大的退化;通知然而，在情况下，灭菌…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The work was supported by EU through project FP7-PEOPLE-212-ITN 316832-OLIMPIA,
Telethon – Italy (grants GGP12033 and GGP14022), Fondazione Cariplo (grant ID 2013-0738).
Materials
| rr-P3HT | Sigma Aldrich | 698989-5G | |
| ITO-coated substrates | Nano-CS | IT10300100 | |
| Fibronectin | Sigma Aldrich | F1141 | |
| chlorobenzene | Sigma Aldrich | 319996 | |
| PCBM | Nano-C | Nano-CPCBM-BF | |
| acetone | Sigma Aldrich | 270725 | |
| isopropyl alcohol | Sigma Aldrich | 563935 | |
| HEK cells | LGC standards srl | ATCC-CRL-1573 | |
| HEPES | Sigma Aldrich | H0887 | |
| PBS | Sigma Aldrich | P5244 | |
| E-MEM | LGC standards srl | ATCC-30-2003 | |
| EDTA | Sigma Aldrich | E8008- | |
| FBS | LGC standards srl | ATCC-30-2020 |
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