Development of Cryoplates for Cytotoxicity Testing

描述（由申请人提供）：高通量筛选的进步，以及世界各地减少动物试验的要求，推动了各种试验的发展，以评估潜在的新药及其副作用。对潜在有害化学品和副产品的环境监测也推动了新检测方法的发展。用于测试具有各种细胞类型的各种产品的方便的基于细胞的系统已经成为极大的需求。在第一阶段，我们提出了一个系统的发展，提供随时使用的细胞在微量滴定板上的任何理想的测定。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，并在解冻后存活。我们还表明，我们能够均匀地冷却和加热微量滴定板，并改善细胞对其底物的整体保留。II期提案的目标是进一步定义和优化参数，这些参数将为附着在微量滴定板上的BCE细胞提供成功的冷冻保存。结果表明，与附着于组织培养塑料的细胞相比，附着于细胞外基质的细胞在冷冻保存后表现出更好的活力。将对细胞外基质组分进行进一步检查，以确定对基质组成的调整是否可以改善活力和附着。此外，还将评价其他低温生物学变量，包括冷却的优化、冷冻保护剂溶剂溶液的选择及其对细胞存活的影响以及在微量滴定板上冷冻保存后细胞的长期储存。所有这些实验旨在进一步优化系统，并在板上提供可用于各种应用的细胞。将通过一系列功能试验对冻存BCE细胞进行验证，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。预计角膜内皮细胞程序的优化将提供一个技术平台，该平台可以进一步开发，应用于其他细胞类型。

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.