Cryopreservation of Human Epidermal Skin Models

描述（由申请人提供）：为了减少动物毒性试验的使用，公司已经开发出体外皮肤模型或等同物来取代Draize皮肤刺激试验。欧盟的一项法规进一步推动了这一发展，该法规将从2009年开始禁止使用动物收集化妆品成分的毒理学数据。最近，欧洲替代方法验证中心（ECVAM）已经验证了两种皮肤模型来取代Draize测试。这些工程组织的皮肤等效物的供应是一个重要的问题，可以通过开发有效的冷冻保存方法来解决。目前，这些结构是定制的，需要几个星期的交货时间才能使用。冷冻保存的产品将在需要时提供，从而消除生产产品所需的前置时间。此外，可以对准备发货的库存进行质量控制检查，并且可以批量生产，从而为供应商节省成本。低温保存的皮肤模型还将为最终用户提供更大的灵活性来安排实验，而不必担心结构的可用性，并且可以从同一批次的产品中订购，以便更好地控制研究中的一致性。这项提议是为了确定冷冻保存这些皮肤等价物的可行性。提出的研究将首先建立组织培养板和孔插入物中角质形成细胞的低温保存条件。然后将最佳保存条件用于商业上可获得的表皮皮肤模型的低温保存，该模型含有建立在孔插入物上的角质形成细胞。由于由多孔板中的膜插入组成的组织和输送系统的复杂性，预计将需要进行重大创新。在第二阶段的研究中，这些条件将应用于其他商业化的表皮模型，以及包含多种细胞类型的更复杂的模型。

DESCRIPTION (provided by applicant): In an effort to reduce the use of animals for toxicity testing, companies have developed in vitro skin models or equivalents to replace the Draize skin irritation test. This development has been further pushed by a European Union regulation that will prohibit the use of animals for collecting toxicological data on cosmetic ingredients beginning in 2009. Recently, the European Centre for the Validation of Alternative Methods (ECVAM) has validated two skin models to replace the Draize test. Supply of these engineered tissues skin equivalents is a significant issue that can be solved by development of effective cryopreservation methods. Presently, these constructs are made to order and require a lead time of several weeks before they can be used. A cryopreserved product would be available when needed eliminating lead time required to manufacture the product. Additionally, performance of quality control checks for stock that is ready to be shipped could be performed and large batches could be manufactured providing a cost savings to suppliers. A cryopreserved skin model would also provide the end-user greater flexibility for scheduling of experiments without concern regarding availability of the constructs and it would be possible to order from the same batch of product in order to have better control of uniformity within research studies. This proposal is to determine the feasibility of cryopreserving these skin equivalents. The proposed studies will begin by establishing conditions for cryopreservation of keratinocytes in tissue culture plates and well inserts. The optimum preservation conditions would then be used for cryopreservation of a commercially available epidermal skin model containing keratinocytes established on well inserts. It is anticipated that major innovation will be required due to the complexity of both the tissue and delivery system consisting of membrane inserts in multiwell plates. In Phase II studies, these conditions will be applied to other epidermal models that are commercially available and to more complex models which contain more than one cell type. PUBLIC HEALTH RELEVANCE: Human epidermal skin models have been developed by several companies and two have been validated for use as in vitro replacements for the in vivo skin irritation test. A preservation method for these skin models will insure that these constructs are readily available when needed, providing a less expensive, consistent, quality controlled product. Success in this project will help reduce the number of animals employed for research and increase availability of human test models that may be more predictive of toxicity and irritation in humans than current live animal models.