Enhanced predictive capacity using dual-parameter chip model that simulates physiological skin irritation
Introduction
The 3R's (replacement, reduction, and refinement) are the principles for the ethical use of animals in scientific research. The 3R's minimize animal use and support high-quality science, which is delivering new research models, tools, and approaches to improve animal welfare and scientific predictive value (Graham and Prescott, 2015). Several alternatives to animal testing such as the Draize skin irritation test have been developed since the 1940s (DRAIZE et al., 1944; Purchase, 1997). In particular, alternative in vitro skin irritation tests using reconstructed human epidermis (RhE) models that mimic the three-dimensional structure of the human epidermis, such as EpiSkin™, EpiDerm™, and SkinEthic™, have been accepted as part of OECD TG 439 based on a European Center for the Validation of Alternative Methods validation study (Alepee et al., 2010; Fentem et al., 2001; Kandarova et al., 2004).
Epidermal models are verified for skin irritation evaluation. However, RhE models do not include endothelial cells and evaluate irritation based on cell viability (CV) (OECD, 2015; Spielmann et al., 2007). Since chemically induced skin irritation is primarily the result of a series of events caused by edema when the test substance passes through the skin, and because this edema is induced by the dilation and increased permeability of endothelial cells, it is crucial to include endothelial cells when evaluating test substances for skin irritation (OECD, 2015; Wachtel et al., 1999; Welss et al., 2004).
Cell-to-cell interactions between endothelial cells can be used to assess edema, as they can simulate the dilation and increased permeability. One of the cell-to-cell junctional complexes arranged in the lateral plasma membrane—a tight junction (TJ)—is generally considered as an inert barrier that regulates paracellular solute and water flux (Anderson and Van Itallie, 2009; Schneeberger and Lynch, 2004). When TJs are dissociated by test substances, cellular permeability increases, resulting in edema. These microenvironments and physiological responses cannot be fully imitated by current RhE models. Thus, the development of new in vitro human skin models as an alternative to an animal testing method is needed.
Recently, the emerging concept termed “organ on a chip” that mimics the functions of human cells inside a microfluidic chip has been extensively investigated. These microfluidic cell culture devices are a better model of physiological cell-cell interactions and cellular microenvironments. Huh and Ingber's research team have developed a microfluidic chip called “lung on a chip,” which mimics various mechanical and physical stimuli of the human lung (Huh et al., 2010). Additionally, there are many other organs on a chip, such as a kidney and gut, in development (Bhatia and Ingber, 2014; Jang et al., 2013; Kang et al., 2016; Kim and Ingber, 2013; Yoon et al., 2016). Furthermore, endothelial cell functions have been previously mimicked in microfluidic chips (Hattori et al., 2014; Osaki et al., 2018; Smith and Gerecht, 2014; Um Min Allah et al., 2017). These various chip models can reproduce important aspects of cellular interactions to contribute to a better understanding of human physiology (Zhang et al., 2018).
Since human skin is a complex organ in which different cell types within different skin layers interact, a skin-on-a-chip microenvironment that includes endothelial cells was needed to overcome the limitations of current RhE models. Moreover, it was necessary to evaluate CV as well as cellular responses to physiological edema, such as quantified tight junction ratio (TJR), to help better understand the response of various skin cells after exposure to skin irritants.
Thus, we adapted a skin-on-a-chip model to physiologically replicate skin irritation and assessed its effectiveness by comparing the predictive capacities of the RhE model, single-parameter model 1 (CV only), single-parameter model 2 (TJR only), and dual-parameter model (CV and TJR) to evaluate skin irritation induced by test substances.
Section snippets
Molding procedure
A computer-aided design program was used to design the skin-on-a-chip model. After the SU-8100 photoresist was spin-coated at 1100 rpm onto a 3.5-in. silicon wafer, it was baked first for 1 h at 65 °C and then for 5 h at 95 °C. After it was spin-coated at 1100 rpm again, it was baked using the same parameters to construct a 500-μm photoresist. The photoresist was covered with a previously designed film mask, and UV light was emitted onto it for 50 s at 15 mJ/cm2. After exposing the photoresist
Assessment of the chips
The inspection of the chips was performed according to the methods described above. The measured diameter and height of the main chamber were 17 mm and 500 μm, respectively. Because lithography was used, the sizes of the inner chamber were consistent, with tolerances after assembly of less than 0.5 mm. Some fabricated chips failed inspection, primarily because of the deformation of the membrane after heating and bonding. Ones that passed inspection were used for skin cell culture.
Single-parameter model 1 analysis
The mean CV
Discussion
In the present study, we have developed and evaluated a skin-on-a-chip based in vitro alternative to animal testing for skin irritation. As described in our previous study, the skin-on-a-chip model was fabricated as an alternative to animal testing (Wufuer et al., 2016). The chip was further developed and specialized for skin irritation testing due to its ability to mimic physiological responses of skin irritation. As mentioned earlier, the current RhE model cannot imitate physiological skin
Conclusion
The dual-parameter chip model that physiologically simulates skin irritation was developed and evaluated using various test substances. This model was able to classify irritant and nonirritant substances with 80% accuracy based on in vivo data. Moreover, when human data were considered as reference values, the model seemed more suitable for simulating human skin irritation. Thus, the developed dual-parameter chip model provides a new approach to the development of an alternative to animal
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This research was supported by grants (16182MFDS526 and 18182MFDS462) from the Ministry of Food and Drug Safety in 2016 and 2018. This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2017R1D1A1B03030163).
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