Development of Cryoplates for Cytotoxicity Testing

用于细胞毒性测试的冷冻板的开发

• 批准号: 6633044
• 负责人: LIA H CAMPBELL
• 金额: $ 30.38万
• 依托单位: ORGAN RECOVERY SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633044
• 关键词: apoptosis; cell proliferation; collagen; corneal endothelium; cryopreservation; cytotoxicity; enzyme activity; extracellular matrix; fibronectins; high throughput technology; superoxide dismutase; technology /technique development; tissue /cell culture; transforming growth factors

DESCRIPTION (provided by applicant): Advancements in high throughput screening, and mandates all over the world to reduce animal testing, have driven the development of a variety of assays to evaluate potentially new drugs and their side effects. Environmental monitoring of potentially harmful chemicals and by-products has also driven the development of new assays. Convenient cell based systems for testing a wide variety of products with a variety of cell types has become a great need. In Phase I, we proposed the development of a system providing ready to use cells on microtiter plates for any desirable assay. We demonstrated that bovine corneal endothelial cells (BCE) attached to tissue culture plastic or an extracellular matrix can be cryopreserved and be viable after thawing. We have also shown that we are able to uniformly cool and warm microtiter plates and improve overall retention of cells to their substrate. The goal of the Phase II proposal, is to further define and optimize the parameters that will provide successful cryopreservation of BCE cells attached to microtiter plates. It was shown that cells attached to an extracellular matrix demonstrated better viability after cryopreservation than cells attached to tissue culture plastic. Further examination of extracellular matrix components will be performed to determine if adjustments to the composition of the matrix can improve viability and attachment. In addition other cryobiological variables will be evaluated including optimization of cooling, the choice of cryoprotectant vehicle solution and its influence on cell survival as well as long term storage of cells after cryopreservation on microtiter plates. All these experiments are designed to further optimize the system and provide cells on plates that are ready to use for a variety of applications. Validation of cryopreserved BCE cells will be provided by a battery of functional assays including, but not limited to, responses to toxicants, extracellular matrix formation, barrier function and nitric oxide production. It is anticipated that optimization of procedures for corneal endothelial cells will provide a technology platform that can be applied, with further development, to other cell types.

描述（由申请人提供）：高吞吐量筛查的进步，并要求全世界减少动物测试，并驱动了各种测定法的开发，以评估潜在的新药及其副作用。对潜在有害化学物质和副产品的环境监测也驱动了新测定的开发。基于各种细胞类型的各种产品的方便基于细胞的系统已成为巨大的需求。在第一阶段，我们提出了一个系统的开发，该系统可以准备在微量滴定板上使用细胞进行任何理想的测定。我们证明了附着在组织培养塑料或细胞外基质上的牛角膜内皮细胞（BCE）可以冷冻保存并在解冻后可行。我们还表明，我们能够统一冷却和温暖的微量滴定板并改善细胞对底物的总体保留。 II期建议的目的是进一步定义和优化将为微滴定板附着的BCE单元提供成功的冷冻保存的参数。结果表明，在冷冻保存后，附着在细胞外基质上的细胞比附着在组织培养的塑料上的细胞表现出更好的生存能力。将对细胞外基质组件进行进一步检查，以确定对基质组成的调整是否可以改善生存力和附着。另外，还将评估其他冷冻生物学变量，包括冷却的优化，冷冻保护剂溶液的选择及其对细胞存活的影响以及在微滴定板上冷冻保存后的细胞的长期存储。所有这些实验旨在进一步优化系统，并在准备用于各种应用的板上提供单元格。冷冻保存的BCE细胞的验证将由一系列功能分析提供，包括但不限于对毒物的反应，细胞外基质形成，屏障功能和一氧化氮产生。可以预料，角膜内皮细胞的程序的优化将提供一个技术平台，可以进一步开发到其他细胞类型。