Nanometer-scale protein patterns for cell culture by laser inactivation

描述（由申请人提供）：该项目的长期目标是开发新技术，该技术将允许在50纳米至毫米的尺寸范围内在细胞培养基质上快速用户定义蛋白质图案。图案化细胞培养基质正在成为一种强大的生物医学研究工具。例如，它们可用于研究真核细胞如何与细胞外环境中的分子相互作用。然而，制造图案化基底的技术仍然限于相对少量的实验室，并且不适用于更大的细胞生物学研究社区。Intelligent Substrates，Inc.拥有一种基于局部激光灭活蛋白质功能的图案化技术的专利权。这项技术是高度灵活的，并将成为一项服务的一部分，允许用户没有特殊的培训，以订购定制设计的图案基板，将在1-2天内交付。该技术已被证明可以在几微米的尺寸上工作。然而，用于细胞生物学应用的相关长度尺度为~10纳米和更大。在这里，我们建议推进ISI的基于激光的技术，以产生具有尺寸小至50 nm的特征的图案。解决这个问题的方法是首先建立一个紫外准分子激光图案化系统，并建立最佳的剂量和激光强度的两个细胞外基质蛋白的灭活。由于存在灭活的强度阈值，因此可以通过有效地削减活性基质蛋白质图案的边界来制作<100纳米的特征。这些底物将使用免疫荧光显微镜和原子力显微镜进行表征。将通过在其上培养几种不同的细胞系并通过评价细胞铺展和粘着斑形成来测试图案的功能性。这项研究的成功将使ISI能够提供定制的图案化服务，使科学家能够订购具有小于100纳米特征的图案化组织培养基质。这将使重要的生物医学研究在广泛的领域。世纪上半叶开创的细胞培养方法和技术使公共卫生领域取得了重大进展，例如发现了脊髓灰质炎疫苗，并且是现代生物医学科学的主力。这里提出的研究描述了一种基于激光的技术，使科学家能够控制细胞功能，创造具有10纳米大小特征的活性蛋白质模式。Intelligent Substrates，Inc.计划将这种方法商业化，并根据生物医学研究界的需求生产定制模式。

DESCRIPTION (provided by applicant): The long term goal of this project is to develop new technology that will allow rapid user- defined patterning of proteins on cell culture substrates at dimensions ranging from 50 nanometers to millimeters. Patterned cell culture substrates are emerging as a powerful biomedical research tool. For example, they can be used to study how eukaryotic cells interact with molecules in the extracellular environment. However, the technology for making patterned substrates is still limited to a relatively small number of laboratories, and it is not available to the larger cell biology research community. Intelligent Substrates, Inc. has patent-pending rights to a patterning technology that is based on local laser inactivation of protein function. This technology is highly flexible, and will become part of a service that allows a user with no special training to order custom designed patterned substrates that will be delivered in 1-2 days. The technology has been demonstrated to work at dimensions of a few micrometers. However, the relevant length scale for the cell biological applications is ~10 nanometers and greater. Here we propose to advance ISI's laser-based technology to produce patterns with features with dimensions as small as 50 nm. The approach to this problem is first to build a UV excimer laser patterning system, and to establish the dose and laser intensity for optimal inactivation of two extracellular matrix proteins. Because there is an intensity threshold for inactivation, features of <100 nanometers can be made by effectively whittling the boundaries of the patterns of active matrix proteins. These substrates will be characterized using immunofluorescence microscopy and atomic force microscopy. The functionality of the patterns will be tested by culturing several different cell lines on them and by evaluating cell spreading and focal adhesion formation. Success of this research will enable ISI to offer a custom patterning service that permits scientists to order patterned tissue culture substrates with features as small as <100 nanometers. This will enable important biomedical research in a wide range of areas. Cell culture methods and technology, pioneered in the first half of the 20th century, have enabled the significant advances in public health, such as the discovery of the polio vaccine, and are the work horse of modern biomedical sciences. The research proposed here describes an enabling laser-based technology that allows scientists to control cell function creating patterns of active proteins with features the size of 10s of nanometers. Intelligent Substrates, Inc. plans to commercialize this method and produce custom patterns on demand for the biomedical research community.