DEVELOPMENT OF CRYOPLATES FOR CYTOTOXICITY TESTING

寻找替代方法以减少生物医学研究和毒理学测试中使用的动物数量的研究集中在培养细胞的体外方法上。我们的概念是开发一种现成的冷冻微量滴定板（“Cryoplate”），它将提供一种易于获得的细胞递送系统，用于细胞毒性的体外测定。 我们建议检查的一般假设，细胞分配在组织培养板，并连接到一个天然或合成的基板可以承受冷冻和解冻的严酷条件，提供一个细胞输送系统的体外细胞毒性试验。 最初的研究将利用培养的牛角膜细胞，其允许方便地检查对冷冻保存应力的耐受性，作为直接附着于组织培养板的合成基质的单个细胞或作为附着于由这些细胞在培养中形成的天然细胞外基质（ECM）的细胞层。 据设想，低温生物学变量的优化将使冷冻板概念的发展，用于各种各样的毒理学和诊断实验室。该产品不仅消除了用于体外测试目的的平板接种和扩增细胞的需要，而且可能需要处理比传统方法更少的细胞。 这项研究中开发的基本技术可能最终导致将细胞冷冻保存在其他基底上，例如携带传感器或其他诊断工具的小型化合成芯片。拟定商业应用：预计冷冻保存的细胞递送系统将商业化用于化学测试和诊断测定。所述方法可适用于开发用于多种细胞类型和目的的体外细胞测定试剂盒。 将与行业合作伙伴寻求许可证机会，以获得替代应用。

The search for alternative methodologies to reduce the number of animals used in biomedical research and toxicology testing has focused on in vitro methods of cultured cells. Our concept is to develop an off-the-shelf frozen microtiter plate ("Cryoplate") which will provide a readily available cell delivery system for in vitro assaying of cytotoxicity. We propose to examine the general hypothesis that cells dispensed in a tissue culture plate and attached to either a natural or synthetic substrate can withstand the rigors of freezing and thawing to provide a cell delivery system for in vitro cytotoxicity assays. Initial studies will utilize cultured bovine corneal cells which permit convenient examination of tolerance to the stresses of cryopreservation, either as individual cells attached directly to the synthetic substrate of the tissue culture plate or as a layer of cells attached to the natural extracellular matrix (ECM) laid down by these cells in culture. It is envisaged that optimization of cryobiological variables will enable development of the Cryoplate concept for use in a wide variety of toxicology and diagnostic laboratories. This product will not only eliminate the need for plating and expanding cells for in vitro testing purposes, but will possibly require handling fewer cells than conventional methods. The basic technology developed in this research may eventually lead to the cryopreservation of cells on other substrates such as miniaturized synthetic chips carrying sensors or other diagnostic tools. PROPOSED COMMERCIAL APPLICATION: It is anticipated that cryopreserved cell delivery systems will be commercialized for chemical testing and diagnostic assays. The methods may be applicable to the development of in vitro cell assay kits for a variety of cell types and purposes. Licensing opportunities will be sought with industry partners for alternative applications.