Laboratory of Biosensors and Bioelectronics, ETH Zurich , Gloriastrasse 35, 8092 Zurich, Switzerland.
SuSoS AG, Lagerstrasse 14, 8600 Dübendorf, Switzerland.
Langmuir. 2017 Sep 5;33(35):8594-8605. doi: 10.1021/acs.langmuir.7b01437. Epub 2017 Aug 9.
Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer's ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.
通过表面修饰将培养细胞排列成图案是生物学家用来以特定和受控的方式回答问题的工具。在过去的十年中，自下而上的神经科学作为一个新的应用出现，其目的是通过逆向工程和分析体外基本电路来更好地理解大脑。构建明确定义的神经网络是最终目标。抗污涂层通常用于控制神经突的生长。由于错误的连接会改变整个拓扑结构和微电路的功能，因此要求很高。目前最先进的涂层解决方案，如广泛使用的聚（L-赖氨酸）-g-聚（乙二醇）（PLL-g-PEG），无法防止原代神经元在长期培养中产生不需要的连接。在这项研究中，开发了一种具有大大增强的抗污性能的新型共聚物，并对其可靠性、稳定性和多功能性进行了表征和评估。为此，将以下成分接枝到聚丙烯酰胺（PAcrAm）骨架上：己胺，以支持在缓冲水溶液中的自发静电吸附，以及丙基二甲基乙氧基硅烷，以通过共价键合到羟基化的培养表面和抗污聚合物聚（2-甲基-2-恶唑啉）（PMOXA）来提高耐久性。在用于神经连接控制的测定中，将新共聚物有效防止不需要的神经突生长的能力与金标准 PLL-g-PEG 进行了比较。此外，还在聚苯乙烯、玻璃和聚二甲基硅氧烷上使用原代海马和皮质大鼠神经元以及 C2C12 成肌细胞和人成纤维细胞对其多功能性进行了评估。PAcrAm-g-（PMOXA、NH、Si）在所有测试的培养表面和细胞类型上都始终优于 PLL-g-PEG，并且它是第一种能够可靠地防止排列的原代神经元节点在长期内形成不需要的连接的表面涂层。虽然目前的工作重点是新抗污涂层成功和可持续地防止不需要的连接的概念验证，但它是体外神经科学的一个重要里程碑，使后续研究能够构建神经相关网络。此外，由于 PAcrAm-g-（PMOXA、NH、Si）可以快速应用于各种表面和细胞类型，因此它是体外生物学和生物医学工程工具包的一个有吸引力的扩展。
