74 TESTING
the number of wells that can be used on the well plate. They can come in 6-, 12-, 24-, 48-, 96-, 384- or 1536-well formats. Well plates with fewer wells are preferable
in experiments requiring a larger culture area or more complex conditions. The wells themselves are available in different shapes: F-bottom (flat bottom), C-bottom (flat bottom with minimally rounded corners), V-bottom (tapered bottom) and U-bottom (U-shaped recess). Measurements on cell cultures in multi-
well plates include skin pigmentation which influences various aspects of skin physiology and the response to external agents. Tests on cell cultures that mimic various levels of pigmentation are used, for example, in studies to understand how products interact with melanocytes (pigment-producing cells) and their impact on UV protection and on the regulation of melanin production and distribution. These are relevant to the development
of sun and UV protection as well as to demonstrate the effect of products aimed at pigmentation disorders, such as hyperpigmentation or hyperpigmentation. Moreover, pigmentation is a factor observed in sensitivity and irritation tests and is also important for the efficacy of products for different skin tones. TEWL measurement on cell cultures in wells
is another important parameter to assess the impact of skincare products on skin hydration and integrity, also for product claims such as the improvement or enhancement of the skin barrier. Single channel probes measuring each well separately one after the other will not be able to deliver meaningful results since the cell cultures in the different wells will change quickly after their removal from the incubator. Multi-channel systems such as the Tewitro®
TW 24 offer the advantage to measure all wells simultaneously. It is compatible with standard 24-wells multi-well plates. These well plates offer six rows with four measuring wells each. The probe is set on top so that each of its 24
measurement tips protrudes into one well. The measurement tip is equipped with two pairs of sensors that continuously measure temperature and relative humidity, indirectly capturing the concentration gradient of water vapor from the bottom of the well through the skin model to the surface. To prevent the sensors from touching the
cell tissue, the height of the probe placed above the multi-well plate can be adjusted by fixing different height regulation feet. TEWL measurement is conducted before the
application of the products and then again after the products have been applied and absorbed during an incubation phase and their residue has been removed by rinsing the wells. By following a detailed protocol and using
a laboratory heat plate, product effects on the integrity of the cell cultures can be easily demonstrated in vitro and distinguished from experimental stress occurring in in vivo testing.
Ultrasensitive impedance spectroscopy to replace the Draize test Epithelial barriers form the outside surfaces of
PERSONAL CARE April 2024
Figure 5: The Draize test to classify hazards of chemical substances is performed on rabbits
skin and most organs. To study the chemical safety of substances, the infamous Draize test was developed: The cornea, as the outermost eye layer, acts
as a protective barrier and window for light reception. Therefore, it is only natural that the choice
to predict chemical hazard potential for human skin and eyes (cytotoxicity) and to classify it into reversible and irreversible tissue damage has been done for a long time by irritating albino rabbits’ eyes, the so-called Draize test. Chemicals that are not harmful to the eye
do not require labelling, while those causing reversible or irreversible tissue damage fall under category 2 and category 1, respectively. However, predicting the hazard potential of chemicals is a complex task, given the diverse range of chemicals and their effect on the eye. While animal tests like the Draize-eye test,
even though highly criticised by consumers, are commonly used, non-animal test strategies, such as non-invasive TEER (transepithelial electrical resistance) measurements on cultured tissue well plates, are becoming increasingly important in the industry since they are providing valuable information about integrity and health of epithelial barriers. Electrical impedance spectroscopy is
a known and valuable method to provide quantitative data about tissue barrier integrity and tissue growth after chemical exposure by measuring electrical properties of skin models.11 A low frequency alternating current is applied, and the resistance of the barrier and phase shift of the signal are measured. The easier the current flows between the cells, the lower the TEER value. A reduced TEER value is an indicator of a compromised barrier. However, conventional TEER measurements
are time-consuming and therefore costly, as they are performed by hand well by well.
This makes studies with a high volume of samples laborious and reduces reproducibility of results. Critical timing, as in application of test substances to epithelial models and subsequent TEER measurements, is difficult to retain. Additionally, common stainless-steel or gold electrodes suffer from a high inherent impedance, which often exceeds that of the cell models. To overcome the limitations of common TEER measurement devices mentioned above, with the use of a novel semi-automated impedance spectrometer with Titanium Nitride (TiN) coated electrodes, the TEER can be assessed at 12.5 and 1000 Hz in 24 wells simultaneously as fast as in a total time of 15 seconds.
The method is well suited for real human
epidermis, RHE (reconstructed human epidermis) and thin layer cell cultures. Additionally, a full impedance spectrum
from 1 Hz to 200 KHz is available in less than two minutes, sufficiently sensitive to cell structure integrity on single cell levels. Changes in the spectrum can reflect alterations in the permeability of ions and molecules. Non-invasive TEER analysis can make a
valuable contribution to clear differentiation in the field of chemical risk assessment. With the entire impedance spectrum, even subtle changes in cell cultures, as they are expected in efficacy tests in the cosmetics industry, can be recognised rapidly and reliably.
Conclusion In vitro methods cannot capture all complexities in the body that occur during in vivo tests. Nevertheless, they are an indispensable part of the evaluation process of product effects on the skin. They precede in vivo tests on humans because they are a valuable indicator of the potential behaviour of agents in contact with the tissue.
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