Cryopreservation of Human Epidermal Skin Models

描述（由申请人提供）：为了减少毒性试验中使用动物，公司开发了体外皮肤模型或等效模型，以取代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）验证了两种皮肤模型，以取代Draize测试。这些工程组织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，这些结构是按订单制造的，在使用之前需要几周的交货时间。冷冻保存的产品将在需要时可用，消除了制造产品所需的前置时间。此外，可以对准备装运的库存进行质量控制检查，并且可以制造大批量，为供应商节省成本。冷冻保存的皮肤模型还将为最终用户提供更大的灵活性，以安排实验而无需担心构建体的可用性，并且可以从同一批次的产品订购，以便更好地控制研究中的均匀性。本建议旨在确定冷冻保存这些皮肤等同物的可行性。拟定的研究将开始，建立组织培养板和孔插入物中角质形成细胞的冷冻保存条件。然后将最佳保存条件用于冷冻保存在孔插入物上建立的含有角质形成细胞的市售表皮皮肤模型。预计由于组织和递送系统（由多孔板中的膜插入物组成）的复杂性，将需要重大创新。在II期研究中，这些条件将应用于市售的其他表皮模型和包含一种以上细胞类型的更复杂模型。 公共卫生相关性：几家公司已经开发了人表皮皮肤模型，其中两种已被验证可用作体内皮肤刺激试验的体外替代品。用于这些皮肤模型的保存方法将确保这些构造在需要时容易获得，从而提供较便宜、一致、质量受控的产品。该项目的成功将有助于减少用于研究的动物数量，并增加人类测试模型的可用性，这些模型可能比目前的活体动物模型更能预测人类的毒性和刺激性。

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.