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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Musculoskeletal Tissue Engineering Study Section (MTE)
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Moro, Manuel H
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Cell and Tissue Systems, Inc.
North Charleston
United States
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