A user-friendly scalable microfluidic platform for enhanced neuron-cell culture

描述（由申请人提供）：神经元细胞培养广泛用于基础研究、药物发现和毒性测试。由于神经元的广泛和随意生长，传统的随机培养允许有限地进入亚细胞区室（轴突、树突、突触）。我们的长期目标是提供强大的，用户友好的，具有成本效益的培养平台，可以光学，流体和生化访问神经元及其亚细胞区室。通过销售由我们团队开发并获得专利的原型平台获得的数据显示了巨大且不断增长的需求。客户反馈还表明，技术改进将使我们的平台更易于访问和用户友好。这些改进包括确保设备的更大均匀性，减少最终用户组装程序，以及增强神经元通常需要的长期培养期的活力。因此，我们的第一个目标集中于通过以下方式解决这些问题：（1）将细胞加载端口并入平台中以减少细胞加载错误，（2）通过开发高分辨率、耐用的金属模具来改善特征均匀性，（3）开发减少损害神经元活力的蒸发损失的方法，（4）通过表面改性来增加装置材料的润湿性和生物相容性，和（5）将细胞外基质蛋白共价连接到玻璃上以使最终用户组装最小化。这一目标的成功完成将导致一个一致的，具有成本效益的，随时可用的神经元细胞培养平台。我们的下一个目标是开发新的基于细胞的工具来研究突触，这是神经科学的基石。对分离突触的方法存在相当大的需求，并且现有技术存在局限性。在目标2中，我们将开发用户友好的突触隔离工具，扩展目标1的核心技术进步。目标2将涉及（1）开发稳定的人工突触珠靶点，允许对突触前区室进行新的研究，以及（2）开发三室突触隔离平台，该平台利用装置几何形状和突触珠来促进隔离突触区室内的突触形成。这一目标的成功完成将导致一个用户友好的，可访问的和创新的基于细胞的工具，以光学和生物化学探针突触。这项工作的意义在于提高研究科学家可视化、操作和测量培养神经元的能力，从而更好地了解神经系统疾病的根本原因。这项研究是创新的，因为我们寻求通过开发新的基于细胞的工具来分离在没有胞体或神经胶质的情况下保持完整细胞形态的突触，从而改变当前的研究范式。

DESCRIPTION (provided by applicant): Neuron-cell culture is widely used for studies in basic research, drug discovery, and toxicity testing. Traditional random cultures allow limited access to subcellular compartments (axons, dendrites, synapses) due to extensive and haphazard growth of neurons. Our long-term goal is to provide robust, user-friendly, and cost effective culture platforms that can optically, fluidically, and biochemically access neurons and their subcellular compartments. Data acquired through sales of prototype platforms developed and patented by our team show a large and increasing demand. Customer feedback also indicates that technological improvements would make our platforms more accessible and user-friendly. Such improvements include ensuring greater uniformity of the device, reducing end-user assembly procedures, and enhancing viability for the long-term culturing period that is often needed for neurons. Thus, our first aim focuses on addressing these issues by (1) incorporating cell loading ports into the platform to reduce cell loading errors, (2) improving feature uniformit through the development of high-resolution, durable metal molds, (3) developing methods to reduce evaporative losses that impair neuron viability, (4) increasing the wettability and biocompatibility of the device material through surface modification, and (5) covalently linking extracellular matrix proteins onto glass to minimize end-user assembly. The successful completion of this aim will result in a consistent, cost-effective, and ready-to-use neuron-cell culture platform. Our next aim focuses on the development of novel cell-based tools to study synapses, the cornerstone of neuroscience. There is considerable demand for methods to isolate synapses and there are limitations in existing techniques. In Aim 2 we will develop user-friendly synapse isolation tools, expanding on core technological advancements from Aim 1. Aim 2 will involve (1) development of stable, artificial synaptic bead targets allowing novel investigations of the presynaptic compartment and (2) development of a three-compartment synapse isolation platform that exploits device geometry and synaptic beads to encourage synapse formation within an isolated synaptic compartment. The successful completion of this aim will result in a user-friendly, accessible, and innovative cell-based tool to optically and biochemically probe synapses. The significance of the proposed work is to improve research scientists' ability to visualize, manipulate, and measure cultured neurons leading to greater understanding of the underlying causes of neurological diseases. This research is innovative because we seek to shift current research paradigms through the development of novel cell-based tools to isolate synapses that maintain intact cell morphology in the absence of somata or glia.