3D living neural networks

项目总结/摘要 精神疾病，包括精神分裂症，抑郁症和自闭症谱系障碍， 虽然很明显，它们主要代表皮质疾病。大脑皮层是 更高的精神功能，然而，尽管进行了广泛的研究，仍然没有统一的理论来解释大脑皮层是如何工作的。 这部分是由于神经科学家对神经元的精确可重复研究所需的工具有限。 神经回路的形成和控制可塑性和修复机制的研究。大多数研究 迄今为止完成的研究依赖于2D细胞培养或活脑研究。控制细胞分布的能力 已经通过使细胞外蛋白质（例如聚赖氨酸）图案化， 直接神经细胞附着。然而，随后将细胞洗涤到基底上。而细胞 通常粘附在图案化的表面上，没有控制细胞分布的机制， 单细胞分辨率。其他技术已经研究了具有"笼"的平面电极阵列的网格的使用。 每个笼子里只能放一个细胞这提高了明确映射 检测信号到特定的神经元，并限制刺激单细胞。然而，这种方法不是 容易扩展到3D环境，金属基底不能准确地模拟细胞的自然状态， 环境，这可以改变细胞的行为。或者，使用光刺激和 钙成像避免了许多这些问题。然而，这种方法呈现出令人生畏的水平， 复杂性使得神经科学家们很难解开大脑的功能。 我们的方法为神经科学研究提供了几个关键好处。通过利用最近 在钙成像和光刺激的进步，我们消除了对电极的需要，在整个我们的神经 网络来刺激和探测连通性。因此，纯水凝胶支架可以用作微球。 支持结构，并作为渠道的来源，直接神经生长。立体光刻使用户能够 为了快速确定聚合物网络的形状，分步重复方法能够实现任意的结构， 在x和y方向上的尺寸，并且附加分层使得能够实现大尺度轴向尺寸。此外，通过合并 具有光学捕获的立体光刻，聚合物结构内细胞位置的微米级控制 实现了。我们的商用光学捕获系统能够操纵数百个物体 同时，以高速，并具有亚细胞分辨率。当光学捕获系统与 与立体光刻，完整的解决方案将允许科学家研究生物过程， 前所未有的速度、分辨率和可重复性。 博尔德非线性系统公司和科罗拉多大学提议将他们的专业知识联合收割机用于建筑 SLM和SLM显微镜在一个两阶段的项目，最终目标是使动态3D组织 支架制造在神经科学和临床研究中具有实际意义。在第一阶段，我们计划建立一个 紧凑、廉价、用户友好的倒置显微镜，带有光学捕获模块，以及 立体光刻该设备将是自对准的，并与适当的软件集成，以便它可以 使用，开箱即用，在几个神经生物学项目中的应用，包括研究机制， 可塑性和修复，药物和毒素筛选，化学和生物传感，生物相容性测试， 修复装置与人体之间的接口，以及神经连接再生的研究 治疗脊髓损伤在第二阶段，我们将增加制造系统的吞吐量，并扩大 系统的自动化。最终目标是设计一种能够制造大规模神经元的工具， 网络和组织支架具有微米分辨率，无需用户控制。此外，在第二阶段，BNS将 与奥林巴斯合作，设计"螺栓"光学捕获和立体光刻模块， 奥林巴斯显微镜。这将为拟议的研究提供一个既定的分销渠道， 允许用户利用专用于其个体研究的现有成像模态。

Project Summary/Abstract Mental disease, including schizophrenia, depression and autism spectrum disorders, are still poorly understood, although it is clear that they mostly represent cortical disorders. The cortex is the primary site of higher mental functions, yet despite extensive research there is still no unified theory of how the cortex works. This is partly due to the fact that neuroscientists have limited tools required for precise repeatable studies of neural circuit formation and for studies of the mechanisms that control plasticity and repair. Most research completed to date relies on 2D cell cultures or studies of live brains. Some ability to control cellular distribution within 2D cultures has been demonstrated by patterning an extra-cellular protein, for example polylysine, to direct neuronal cell attachment. However, cells are subsequently washed onto the substrate. While cells generally adhere to the patterned surfaces, there is no mechanism for controlling cellular distribution with single cell resolution. Other techniques have investigated use of grids of planar electrode arrays with "cages" that only allow a single cell to be deposited in each cage. This improved the ability to unambiguously map detected signals to specific neurons, and to confine stimulation to single cells. However, this approach is not easily scalable to 3D environments, and metallic substrates do not accurately mimic a cells natural environment, which can alter cell behavior. Alternatively, studies of brain slices using photostimulation and calcium imaging circumvent many of these problems. However, this approach presents a daunting level of complexity making it challenging for neuroscientists to unravel function of the brain. Our approach offers several key benefits to neuroscience research. By taking advantage of recent advances in calcium imaging and photostimulation we remove the need for electrodes throughout our neural network to stimulate and probe connectivity. As a result a purely hydrogel scaffold can be used as the supporting structure, and as the source of channels to direct neural growth. Stereolithography enables the user to rapidly define the shape of the polymer network, step and repeat methods enable structures of arbitrary dimensions in x and y, and additive layering enables large scale axial dimensions. Furthermore, by merging stereolithography with optical trapping, micron scale control of the position of cells within the polymer structure is realized. Our commercially available optical trapping system is capable of manipulating hundreds of objects simultaneously, at high speed, and with sub-cellular resolution. When the optical trapping system is combined with stereolithography, the complete solution will allow scientists to study biological processes with unprecedented speed, resolution, and repeatability. Boulder Nonlinear Systems and the University of Colorado propose to combine their expertise in building SLMs and in SLM microscopy in a two-phase project with the ultimate goal of making dynamic 3D tissue scaffold fabrication a practical reality in neuroscience and clinical research. In the first phase we plan to build a compact, inexpensive, user-friendly inverted microscope with modules for optical trapping, and stereolithography. The device will be self-aligning and integrated with appropriate software so that it can be used, out of the box, for applications in several neurobiological projects including studies of mechanisms for plasticity and repair, drug and toxin screening, chemical and biological sensing, biocompatibility tests at the interface between a prosthetic device and human body, and research into regeneration of nerve connections for spinal cord injuries. In Phase II we will increase the throughput of the fabrication system, and extend the automation of the system. The ultimate goal is to design a tool capable of fabricating large scale neural networks and tissue scaffolds with micron resolution free of user control. Additionally in the Phase II BNS will collaborate with Olympus to design "bolt-on" optical trapping and stereolithography modules for existing Olympus microscopes. This will provide an established distribution channel for the proposed research and will allow users to utilize existing imaging modalities specialized to their individual studies.