Review Organotypic cell cultures and two-photon imaging: Tools for in vitro and in vivo assessment of percutaneous drug delivery and skin toxicity Sari Pappinen a , Evgeny Pryazhnikov b, c , Leonard Khiroug b, c , Marica B. Ericson d , Marjo Yliperttula e , Arto Urtti f, ⁎ a Department of Pharmaceutics, University of Eastern Finland, Kuopio, Finland b Neuroscience Center, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland c Neurotar Ltd., Viikinkaari 4, 00790, Helsinki, Finland d University of Gothenburg, Sweden, Box 100, S-405 30 Gothenburg, Sweden e Division of Biopharmacy and Pharmacokinetics, Faculty of Pharmacy, University of Helsinki, Finland f Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Finland abstract article info Article history: Received 2 January 2012 Accepted 6 March 2012 Available online 13 March 2012 Keywords: Transdermal drug delivery Cell culture Permeability Imaging Two-photon microscopy Skin irritation The outermost protective layer of the skin, the stratum corneum, is responsible for skin impermeability to- ward external medications and potentially harmful chemicals. Stratum corneum is the target for physical and chemical approaches to enhance drug permeation. These approaches are commonly investigated in the field of drug delivery, but the drug absorption enhancement is often linked with local toxicity. In this review we are discussing two emerging technologies for drug and chemical studies in the skin: organotypic cell cultures and non-invasive two-photon microscopic imaging. Even though several cell culture based ‘skin equivalents’ have been introduced and validated for skin irritation testing, they are usually leaky and inade- quately characterized in terms of permeation. Rat epidermal culture model (ROC) has been thoroughly char- acterized and it shows comparable barrier properties with the human skin thereby being useful in drug permeation and toxicity studies. In vitro and in vivo visualizations of permeants and skin structures are now feasible due to the rapid development of two-photon microscopy that allows improved depth scanning and direct in vivo visualization of the permeating compounds and adverse reactions in the skin structures. In summary, the new tools in percutaneous drug delivery studies will provide new insights to the permeation process and local toxicity. These tools may facilitate development of effective and safe transdermal drug delivery methods. © 2012 Elsevier B.V. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657 2. Structure of the skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657 3. Drug permeation in the skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658 4. Organotypic keratinocyte cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658 4.1. The barrier function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659 4.2. SC lipid organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659 4.3. Diseased skin models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 659 4.4. Drug permeation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 4.5. Permeation enhancer effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 5. Chemical irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 5.1. Use of reconstructed skin models in chemical irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660 5.2. Overview of two-photon microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662 5.3. Applicability of TPM to transdermal drug delivery research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662 5.4. Use of TPM in transdermal drug delivery research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662 5.5. TPM as a tool for time- and space-resolved visualization of drug–tissue interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 663 Journal of Controlled Release 161 (2012) 656–667 ⁎ Corresponding author at: Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, 00014 University of Helsinki, Finland. Tel.: + 358 40 5402279. E-mail address: arto.urtti@helsinki.fi (A. Urtti). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2012.03.005 Contents lists available at SciVerse ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel