DEVELOPMENT OF CRYOPLATES FOR CYTOTOXICITY TESTING

寻找替代方法来减少生物医学研究和毒理学试验中使用的动物数量，主要集中在体外培养细胞的方法上。我们的概念是开发一种现成的冷冻微量滴度板（“Cryoplate”），它将为体外细胞毒性测定提供一种现成的细胞递送系统。我们建议检验一般假设，即细胞分布在组织培养板中并附着在天然或合成底物上，可以承受严格的冷冻和解冻，从而为体外细胞毒性试验提供细胞传递系统。最初的研究将利用培养的牛角膜细胞，它可以方便地检查对低温保存压力的耐受性，或者作为单个细胞直接附着在组织培养板的合成底物上，或者作为细胞层附着在这些细胞在培养中铺设的天然细胞外基质（ECM）上。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毒理学和诊断实验室。该产品不仅可以消除体外测试中对细胞的电镀和扩增的需要，而且可能比传统方法需要处理更少的细胞。在这项研究中开发的基本技术可能最终导致细胞在其他基质上的低温保存，例如携带传感器或其他诊断工具的微型合成芯片。拟议的商业应用：预计冷冻保存细胞递送系统将用于化学测试和诊断分析的商业化。所述方法可适用于开发用于各种细胞类型和目的的体外细胞测定试剂盒。将与工业伙伴寻求许可机会，以获得其他应用。

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.