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，允许在细胞培养底物上快速形成尺寸从50纳米到毫米的用户定义的蛋白质图案。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，它们可以用来研究真核细胞如何与细胞外环境中的分子相互作用。然而，制造图案化衬底的技术仍然局限于相对较少的实验室，而对于更大的细胞生物学研究界来说，它是不可用的。智能基质公司拥有一项基于蛋白质功能的局部激光灭活的图案化技术的专利申请权利。这项技术非常灵活，将成为一项服务的一部分，允许用户在没有特殊培训的情况下订购定制设计的图案基板，将在1-2天内交付。这项技术已经被证明可以在几微米的尺寸下工作。然而，细胞生物学应用的相关长度尺度是~10纳米或更大。在这里，我们建议推进ISI的基于激光的技术，以产生尺寸小至50纳米的特征图案。解决这个问题的方法是首先建立一个UV准分子激光构图系统，并确定两个细胞外基质蛋白最佳失活的剂量和激光强度。因为有一个失活的强度阈值，所以可以通过有效地削减活性基质蛋白质模式的边界来制造&lt；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。这些图案的功能将通过在其上培养几个不同的细胞系来测试，并通过评估细胞扩散和局部黏附形成来测试。这项研究的成功将使ISI能够提供定制的图案化服务，允许科学家订购特征小至100纳米的图案化组织培养基质。这将使在广泛领域进行重要的生物医学研究成为可能。细胞培养方法和技术开创于20世纪上半叶，使公共卫生领域取得了重大进展，如脊髓灰质炎疫苗的发现，是现代生物医学科学的主力军。这里提出的这项研究描述了一种基于激光的使能技术，允许科学家控制细胞功能，创造具有10纳米大小的特征的活性蛋白质图案。智能基质公司计划将这种方法商业化，并根据生物医学研究社区的需要生产定制图案。

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.