Skin in vitro assays that can replace animal models are in high demand in the skin-care and pharmaceutical industry for toxicology and drug delivery research. Besides being slow and expensive, animal models are not ideal for assessing effects in humans because of species differences, and raise ethical concerns. With the EU 2007 REACH regulation having drastically increased the number of chemicals that need to be evaluated for toxicity, and the EU 2013 ban on the use of animals in cosmetic product testing, there is an urgent need to develop broader and alternative profiling technologies for safety and efficacy skin testing methods.
For in vitro skin safety assays, organotypic skin cultures are tipically used. However, the reconstruction of these skin equivalents is costly, labor intensive, requires substantial cell culture expertise, and suffers from high variability and low barrier function. These 3D models are cultured in tissue culture inserts under static, inefficient, non-physiological conditions, and are not compatible with automated in vitro assays. Microscopy observations are difficult because of the excessive distance from the objective.
Similarly, in vitro efficacy assays require trained personnel, are labour intensive or low-throughput. Moreover, technologies like the Franz diffusion cells tipically used for skin permeation tests are based on the use of excised human skin, whose availability is limited and whose sourcing is regulated.
With the aim to overcome these limitations we integrated skin culture and in vitro testing into one system. We have developed a microfluidic platform that enables us to reconstruct skin equivalents of superior quality under in vivo-like perfusion, when compared to static cultures. The skin equivalent is cultured on a porous support membrane that separates two fluidic compartments. The microfluidic system can easily be converted from a tissue culture reactor to an in vitro analysis system by means of a set of interchangeable lid and insets. Flow of media through the microfluidic compartments provides a dynamic, continuous supply of nutrients and simultaneous removal of metabolic waste products similar to the role of blood vessels in native human skin.
Compared to organotypic skin reconstructed on traditional tissue culture inserts, the skin-on-chip equivalent showed a morphologically superior architecture and improved barrier properties. The epidermal layer is thicker and demonstrates a columnar, polarized basal keratinocytes. The skin-on-chip equivalent also shows more intense expression of involucrin, filaggrin and loricrin, as well as of basement membrane-related proteins laminin-5 and collagen IV, thus demonstrating a tighter anchoring of the epidermis to the dermis.
The use of thin and optically transparent plastic materials and the miniaturized design allow for real-time, non-invasive imaging. This system is scalable and suitable for automation of both the culture and the safety and efficacy experimental protocols. Exploring advanced miniaturization and high-throughput possibilities can minimize the cost of each replicate.
Unlike other skin-on-a-chip approaches, we reconstructed for the first time a full thickness organotypic skin equivalent directly in a microfluidic device made of thermoplastic materials and suitable for mass production.