Enhanced predictive capacity using dual-parameter chip model that simulates physiological skin irritation

Highlights

• In vitro skin irritation assessment method using a dual-parameter chip model has been developed and evaluated.

• Dual-parameter (cell viability and tight junction) can distinguish between irritants and nonirritants.

• Predictive values of 80% sensitivity, 80% specificity, and 80% accuracy are achieved.

• Skin on a chip based model can mimic physiological skin irritation, including endothelial components.

• Statistical logistic regression models can help improve in vitro alternatives to animal testing.

Abstract

Current alternatives to animal testing methods for skin irritation evaluation such as reconstructed human epidermis models are not fully representing physiological response caused by skin irritants. Skin irritation is physiologically induced by the dilation and increased permeability of endothelial cells. Thus, our objectives were to mimic physiological skin irritation using a skin-on-a-chip model and compare predictive capacities with a reconstructed human epidermis model to evaluate its effectiveness. To achieve our goals, the skin-on-a-chip model, consisting of three layers representing the epidermal, dermal and endothelial components, was adapted. Cell viability was measured using the OECD TG 439 protocol for test substance evaluation. The tight junctions of endothelial cells were also observed and measured to assess physiological responses to test substances. These parameters were used to physiologically evaluate cell-to-cell interactions induced by test substances and quantify model accuracy, sensitivity, and specificity. Based on in vivo data, the classification accuracy of twenty test substances using a dual-parameter chip model was 80%, which is higher than other methods. Besides, the chip model was more suitable for simulating human skin irritation. Therefore, it is important to note that the dual-parameter chip model possesses an enhanced predictive capacity and could serve as an alternative to animal testing for 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.