1. Research Techniques Made Simple: Organotypic Skin Culture Ji Won Oh 1,2, Tsai-Ching Hsi 1, Christian Fernando Guerrero-Juarez 1, Raul Ramos 1, Maksim V. Plikus 1 1. Department of Developmental and Cell Biology, Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, CA, USA. 2. Department of Immunology, School of Medicine, Kyungpook National University, Daegu, Korea.

2. Overview of Organotypic Culture Systems (OCSs) OCSs allow for in vitro growth of complex biological tissues in a way that replicates part of their normal physiology and function. OCSs are capable of partially replicating the normal function and physiology of complex biological tissues.

3. OCSs enable human skin to be studied with approaches, such as genetic manipulations, otherwise unsafe and unethical in human subjects. Skin OCSs are powerful as an experimental platform in pre-clinical dermatological research, helping to validate mechanisms of diseases and test the therapeutic potential of candidate drugs. Overview of Organotypic Culture Systems (OCSs)

4. OCSs in Epidermal Research Epidermal OCSs allow for stratified epidermis to form They are created by: 1. Populating the extracellular matrix scaffold with fibroblasts, then 2. Seeding epidermal cells on top, and finally 3. Elevating the scaffold to induce stratified epidermal differentiation [Figure 2 goes here]

5. From Barker et al. (J Invest Dermatol 123: 892-901, 2004) Epidermal OCSs reconstituted with keratinocytes and fibroblasts derived from a human donor with psoriasis (far right) displayed high expression of TNFα and IFNγ cytokines in epidermis (pink color, arrows), similar to psoriasis skin (arrows). IL-6 remains high both in normal and psoriasis OCSs.

6. Human hair follicles can continue to grow new hair in vitro for up to two weeks Microdissected hair follicles are cultured in a free-floating state Hair elongation rate is recorded by means of time- lapse photography and morphometry [Figure 3 goes here] OCSs in Hair Follicle Research

7. From Kwack et al. (J Invest Dermatol 132: 43-49, 2012) IL-6 is elevated in human dermal papilla cells derived from balding scalp hair follicles. Addition of recombinant human IL-6 into human hair follicles cultures significantly inhibits hair shaft elongation rate comparing to control (a). Number of proliferating cells in the matrix of cultured humans hair follicles, as measured by immunostaining for Ki-67 marker, significantly decreased in the presence of IL-6 (b). Also compare Ki-67 expression in d (IL-6 treated) vs. c (control).

8. Advantages of Skin OCSs Limitations Easy to create in standard laboratory settings; Detailed protocols are readily available; Allows for the study of complex in vivo-like behavior of humans skin cells in vitro; Research techniques otherwise unsafe and unethical in human subjects can be used; Enables screening therapeutic potential of new drug compounds Recapitulates only part of normal skin organization and function; Do not support complex, systemic responses, such as wound healing; Size of individual cultures is constrained by inefficient diffusion of nutrients; Often require tissues from disease-affected donors; Data from OCSs needs support from in vivo studies;