TESTING
inflammatory stimuli or immune challenges. ■ Gene expression analysis: Analysing the genetic responses of skin cells to various factors. ■ Cell viability and proliferation tests: Determining the health and growth of skin cells within the culture. ■ Cytotoxicity tests: Assessing the potential toxicity of substances on skin cells. In many of these application fields, assessing
the barrier quality of the skin, transepidermal water loss (TEWL) plays a major role. When the skin is damaged, the close cohesion of skin cells is disrupted. This makes it easier for external irritants to enter, and results in increased evaporation from the skin. Common ‘active’ components that address
these stressors include ceramides, hyaluronic acid, niacinamide and others. Procedures for measuring TEWL on human skin in vivo for assessing the barrier quality have been described in numerous guidelines since 1990.6-8 TEWL measurement on humans has been performed extensively in product tests to proof product claims such as barrier restoring, barrier strengthening, repairing and many more. Since damaging the skin barrier by applying
test substances on humans in vivo again raises ethical concerns and is also rather costly, the demand for replacement methods of these tests is high. Ex vivo and in vitro tests have been developed, many of them still awaiting official approval as a recognized replacement method for in vivo tests on animals or humans.9,10 To observe how TEWL is affected and
improved, ingredients and products are applied to skin models. The results can be used to understand whether a substance is causing harm and compromising the natural line of defence, or to prove that it supports in fact skin barrier recovery. In other words, TEWL measurement offers information about the ‘mildness’/damage potential of ingredients and formulations. For many years already, Franz diffusion
cells have been used for in vitro testing of the diffusion of chemicals into and across excised human or animal skin. OECD guideline 428 describes this test in detail.2 The results of such tests are especially useful for screening the delivery of chemicals from different formulations and the assessment of percutaneous absorption.
Figure 2: Product claims such as repairing or barrier restoring need to be objectively proven
The skin sample separates the two chambers
of the diffusion cell, the upper donor chamber and the lower receptor chamber. The receptor chamber is filled with fluid that is usually heated to a temperature of approximately 32°C, resembling skin temperature. The chemical that is researched, is applied on top of the skin for a specified time at specific conditions, before being removed by a defined cleansing method. The fluid in the receptor chamber is sampled
at different points in time throughout this experiment and analysed for the test product or metabolites. For reliable results, it is essential that the skin that is used for this test is not damaged and has full integrity. TEWL measurement with specially adapted
probes serves for assessing skin integrity of the sample and can also be used for measuring damaging influences on the skin by the chemical. The In vitro Tewameter® probe has been especially developed and is perfectly suited for Franz cell measurement, emulating the donor chamber altogether. This way, it can be placed straight on top of the skin sample, so that the calculated water loss of the skin sample corresponds to TEWL normally measured on the skin surface in vivo in g/h/m2
. The open chamber of the probe offers
the possibility of applying the substance on the skin sample through the measurement cylinder and at the same time can measure the water loss through the skin. For tests requiring higher
amounts of the chemical applied to the skin surface, it is also possible to add a centrepiece that fits exactly on the probe and the receptor chamber of the Franz cell without further fixing adapters.
Cell cultures In the dynamic field of skin testing, the use of cell cultures to reproduce human skin has emerged as a transformative method for advancing cosmetics and promoting skin health. Cell cultures, especially those derived from the outermost layer of the skin’s epidermis, the stratum corneum, provide a controlled environment for studying the effects of cosmetics and skin care products. These in vitro models allow researchers to
observe how different formulations interact with human skin cells, assessing factors such as toxicity, irritation and efficacy. Cultured human cells and skin tissue models are frequently used in microplates, also referred to as multi-well plates.
The rectangular multi-well plates contain
a number of cavities, called ‘wells’, which are isolated from each other in rows and columns. The well plate dimensions are clearly defined with a length of 127.76 mm x 85.48 mm width for fulfilling the needs of automated workstations. Their height is usually also standardized
but may vary to some degrees. Based on these defined dimensions, there is a wide range for
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Figure 3: The In vitro Tewameter emulates the donor chamber of a Franz diffusion cell, fitting perfectly, and can exactly determine the skin integrity before and during the experiment
www.personalcaremagazine.com
Figure 4: With the Tewitro TW 24 the TEWL of cell cultures and skin models in well-plates with 24 wells can be assessed simultaneously
April 2024 PERSONAL CARE
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