Arrays for Cloning Growth Suppressed Cells

描述（由申请人提供）：为研究特定基因对细胞生理学的影响而创建的分子工程细胞是生物医学发现的基本工具。当代癌症研究的一个主要焦点是通过创建表达肿瘤抑制基因或癌基因敲除的细胞系来研究细胞存活和复制的分子机制。虽然对研究癌细胞不受控制的复制至关重要，但由于它们的生长劣势，这些细胞系极难产生和维持。基于微制造细胞阵列的新技术有可能为这些细胞工具的日常创建和维护提供一个有利的平台。在目前的拨款申请中，一个跨学科团队将开发可释放的细胞“筏”阵列，以克服在分离和维持生长不利的克隆菌落方面的技术挑战。易于实现和廉价的制造技术将用于制造阵列。将优化阵列的设计和材料，以便在阵列上转染和扩增细胞。将进行关键的生物控制以确认细胞健康、活力和克隆性。将根据标准显微镜成像开发阵列分析和克隆菌落选择方法。一个简单的机械系统将被优化，以释放目标细胞所在的单个筏。这项工作将包括硬件和软件的设计和测试，以实现半自动分析和细胞分离。肿瘤抑制生物学研究所需的细胞系将被创建和维护，该技术将通过与传统方法的比较得到验证。在这项研究中开发的基本创新将降低阵列制造的成本，并将产生一种灵活、可靠和易于使用的技术。这种细胞克隆新方法所产生的能力将导致对控制癌细胞生长和生存的新见解。

DESCRIPTION (provided by applicant): Molecularly engineered cells created to study the impact of specific genes on cell physiology are fundamental tools for biomedical discovery. A major focus of contemporary cancer research is the study of the molecular mechanisms underlying cell survival and replication by creation of cell lines that express tumor suppressor genes or have oncogene knockouts. While critical for the study of the uncontrolled replication that characterizes cancer cells, these cell lines are extremely difficult to generate and maintain due to their growth disadvantage. New technologies based on microfabricated cell arrays have the potential to provide an enabling platform for routine creation and maintenance of these cellular tools. In the current grant application, an interdisciplinary team will develop arrays of releasable cell "rafts" to overcome the technical challenges in isolation and maintenance of clonal colonies with a growth disadvantage. Easily implemented and inexpensive fabrication techniques will be used to manufacture the arrays. Array design and materials will be optimized for transfection and expansion of cells on the array. Critical biological controls will be performed to confirm cell health, viability, and clonality. Methods for array analysis and selection of clonal colonies will be developed based on standard microscopic imaging. A simple mechanical system will be optimized in order to release individual rafts on which target cells reside. This work will include the design and testing of hardware and software to enable semi-automated analysis and cell isolation. Cell lines needed for studies of tumor suppressor biology will be created and maintained, and the technology will be validated by comparison with conventional approaches. The fundamental innovations developed in this research will decrease the costs of array manufacture and will produce a flexible, reliable and simple to use technology. The capabilities engendered by this new method for cell cloning will lead to new insights into the control of cancer cell growth and survival.