DEVELOPMENT OF AN IMPROVED INVITRO METASTATIC ASSAY

改进的体外转移测定的开发

• 批准号: 3493174
• 负责人: DAVID L CLAPPER
• 金额: $ 5万
• 依托单位: SURMODICS, INC.
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3493174
• 关键词: artificial membranes; basement membrane; bioassay; cell adhesion; cell migration; chemoattractants; clone cells; collagen; heparin; membrane reconstitution /synthesis; metastasis; method development; neoplasm /cancer invasiveness; neoplastic cell; photochemistry; tritium

There are currently no rapid and reliable in vitro assays that measure the metastatic activity of cells. In this SBIR project, we propose to develop an improved Boyden chamber chemoinvasion assay, which will utilize Transwell (TM) membranes with a defined basement membrane overlayer that will allow in vitro assays for metastatic cells to be conducted with greater speed, reproducability, and sensitivity than existing assays. The chemoinvasion assays evaluate the invasive activity of metastatic cells by quantitating their ability to respond to a chemoattractant by penetrating a layer of reconstituted basement membrane (RBM). Several recent papers which utilize Matrigel (TM) as an RBM preparation have shown that this assay can be conducted in 6 hours and clearly discriminates between invasive and non-invasive cells. However, there is considerable assay variability, which is caused by: l) variability in the Matrigel overlayer and 2) poor adherence of Matrigel to the polycarbonate membrane (resulting in gaps through which non-metastatic cells can migrate). During Phase I, patented BSI photochemistry will be used to covalently couple type IV collagen and heparin onto the polycarbonate membranes with the expectation of greatly improving the adherence of RBM to the membrane. Also, initial experiments will be conducted to develop a defined RBM preparation to replace Matrigel. Modified polycarbonate membranes overlaid with Matrigel and defined RBM compositions will be evaluated with metastatic and non-metastatic cell lines. Phase II studies will complete the development of a defined RBM preparation.

目前尚无快速可靠的体外测定法来测量 细胞的转移活性。在这个SBIR项目中，我们建议开发 改进的Boyden室化学侵入测定法，将利用 带有定义的地下膜覆盖层的Transwell（TM）膜 将允许对转移细胞进行体外测定 比现有测定法更高的速度，可重复性和灵敏度。这 化学鉴定测定评估转移细胞的侵入性活性 通过穿透来量化其对化学吸引剂反应的能力 一层重构的基底膜（RBM）。最近的几篇论文 哪个利用Matrigel（TM）作为RBM准备，这表明这一点 可以在6小时内进行测定，并明确区分 侵入性和非侵入性细胞。但是，有很多测定法 可变性，是由以下原因引起的：l）Matrigel叠加仪中的可变性 和2）母质凝胶对聚碳酸酯膜的依从性不佳（由此产生 在非转移细胞可以迁移的间隙中）。 在第一阶段，获得专利的BSI光化学将用于共价 夫妇与IV胶原蛋白和肝素类型 人们对RBM对膜的依从性的期望。 此外，将进行初始实验以开发定义的RBM 准备更换Matrigel的准备。改良的聚碳酸酯膜覆盖 使用Matrigel和定义的RBM组合物将通过 转移和非转移细胞系。第二阶段研究将完成 定义的RBM制备的开发。