Based on an accredited test method, the Fraunhofer IGB has developed an in vitro phototoxicity assay to measure the phototoxic potential of substances in medications and lotions used to protect the skin against environmental influences, which can become toxic when exposed to UV light. The assay uses human skin cells that have been grown into three-dimensional tissue as a human skin model.
The Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB) in Stuttgart has long-standing expertise in producing artificial skin. Fraunhofer IGB teams now have so much control over the processes that they are able to produce a variety of skin models consisting of dermis and epidermis at relatively low cost and consistent quality. The cooperation with the Fraunhofer Institute for Production Engineering and Automation (IPA) has made it possible to fully automate the production of skin models in a factory-like facility. The models are produced using human skin cells isolated from biopsies and grown into three-dimensional tissues in the IGB laboratories. The skin produced can be adapted to specific requirements. For example, the IGB researchers have developed a skin cancer model by adding skin tumour cells to their standard skin model. They can also integrate cells that form blood vessels, melanocytes, which are responsible for skin pigmentation, and even fat layers, which enable them to investigate the deposition and accumulation of certain substances.
In the search for applications for the skin model, Sibylle Thude came up with the idea of optimising the model for phototoxicity tests. Thude is a biologist who is in charge of the investigations into the accreditation of assays at the IGB and in the IGB’s Department for Cell and Tissue Engineering. “We decided to develop phototoxicity tests as such methods are not yet very common; there are as yet no ISO-certified methods that meet the required standards. We therefore believed that our in vitro models would have good market potential,” says Thude. “Phototox”, the researchers' name for the test method, was developed by Thude and another IGB researcher.
The phototoxicity test is performed on an in vitro skin model built from typical human epidermis cells, i.e. keratinocytes, the predominant skin cells on the outermost layer of the epidermis. Thude and her team isolated human primary keratinocytes from tissue cells, propagated and cultured them for two to three weeks to form an artificial epidermis with natural layers. The horny keratinocyte layer of the skin is crucial for realistic test scenarios, as it is an important barrier against environmental influences and dehydration. Although the researchers could have made the skin model even more realistic by adding additional skin cell types such as fibroblasts, they refrained from doing so as this would have made production of the model more expensive and time-consuming, as Thude explains.
The skin model undergoes quality assessments and, if it passes, can be used for phototoxicity tests to find out whether a particular chemical compound under investigation becomes toxic when exposed to light. Substances can be tested on a skin model by applying them to the surface of the epidermis and exposing the model to non-toxic doses of UV radiation. The method can be used for aqueous solutions and oils, as well as for some alcoholic extracts.
Whether and to what extent the cells are damaged is assessed using microscopic and spectroscopic methods. Living cells convert phototoxic substrates into coloured products, which enables the researchers to determine the photocytoxic potential of the substance under investigation. A substance is considered phototoxic when the cell viability of the irradiated skin model is 30 percent lower than that of corresponding non-irradiated control cells. The test is so reliable and reproducible that it achieved accreditation and is now available for commercial applications.
“We mainly produce and sell skin models. But we also offer services involving phototoxicity test methods that are ISO certified in accordance with certain guidelines and protocols. Accredited assay procedures are carried out in our own laboratories. The assays can also be carried out in the R&D laboratories of clients who have the necessary equipment. However, in such cases the assays are not accredited procedures,” explains Thude. Accredited test procedures are carried out on behalf of cosmetics producers as well as pharmaceutical companies that produce drugs for administration through the skin. The phototoxicity assay can also be used to assess the photo-cytotoxic effects of herbs and other plant-derived substances. For example, it is known that St. John’s wort and bergamot extracts make the skin more sensitive to UV irradiation.
The skin model may also be suitable for simulating the effects of oral drug administration, as the resulting metabolic products could affect the skin's UV sensitivity. Although such an assay is still a pipe dream, Thude is convinced that it will one day be within reach if the development of organ-on-a-chip technology continues at the current pace. “We are currently working on a number of complex organ-on-a-chip systems. If we manage to model the relevant metabolic processes, I think that phototoxicity assays will one day be suitable for use with orally administered substances.”
At the moment, the team is working on other ideas, including ways to adapt phototoxicity assays to enable the integration of melanocytes, i.e. skin pigment cells. The ability to determine the amount of melanin produced makes the assay suitable for discovering substances such as self-tanners or skin brighteners that stimulate melanin production in the skin. “The pigmented skin model is already used to measure increased melanin production. However, we still have to validate the assay to obtain accreditation for the underlying assay procedure,” says Thude. Although the project is still in the research stage, it could open up other application possibilities. However, the number of quality controls required increases with the complexity of the model, the number of cells used and the number of application possibilities. In short, the more complex the assay procedure, the more extensive the controls. “So we are still looking for accredited partner laboratories that can objectively assess laboratory performance, which is a requirement of external quality assessments,” says Thude who would be delighted to hear from research groups interested in cooperating on this project.