Original Articles Effects of Serum-Free Culture at the Air–Liquid Interface in a Human Tissue-Engineered Skin Substitute Jessica Jean, Ph.D., 1,2 Genevie ` ve Bernard, M.Sc., 1 Alexandra Duque-Fernandez, M.Sc., 1 Franc ¸ ois A. Auger, M.D., 1,3 and Roxane Pouliot, Ph.D. 1,2 Previous studies have reported that well-defined culture conditions can improve keratinocytes terminal dif- ferentiation and reproducibility. The aim of our study was to compare skin substitutes cultured in a complete medium with those cultured in a serum-free medium at the air–liquid interface to optimize the self-assembly method. Skin substitutes, cultured in a serum-free medium over 7, 14, and 21 days, were compared with others cultured in a complete medium (5% serum) over the complete culture period. Masson’s Trichrome staining showed that the substitutes cultured in a serum-free medium generated a well-developed and differentiated epidermis. Immunolabeling analyses between the substitutes cultured without serum and those cultured in complete serum showed similar expression of epidermal differentiation markers, dermo-epidermal junction, and dermal extracellular matrix components. On the basis of our Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) results, the skin substitutes cultured in serum-free condition over 21 days of culture at the air–liquid interface showed lower frequencies of the CH 2 symmetric mode of vibrations, which means a better lipid organization of the stratum corneum. No significant difference in hydrocortisone penetration was observed between serum-free medium substitutes and the controls. Results demonstrate that the absence of serum does not compromise the characteristics of the skin substitutes observed in this study. Introduction S kin substitutes are designed to meet both basic and applied research needs. 1 They can be used in various fields of skin biology, pharmacotoxicology, cellular and molecular biology studies and also as a replacement for human skin in clinical applications. 2,3 They possess various biological and pharmacological properties of human skin that allow and/or promote new tissue growth and optimize the conditions for healing. 4 Among other things, they are designed (1) to provide a mechanical barrier against infection and fluid loss (burn patients), 5 (2) to close wounds (diabetic ulcers), and (3) to replace animal models for dermo- pharmaceutical testings. 1,6 In most tissue-engineering studies, the medium provides the essential nutrients such as amino acids, fatty acids, sug- ars, ions, trace elements, vitamins, cofactors, and molecules necessary to maintain the proper chemical environment for the cells to proliferate accordingly. 7 To produce skin substi- tutes, the addition of animal or human serum is required in the culture medium because serum contains various growth factors, cytokines, hormones, vitamins, and proteins that increase cell growth and cell proliferation. 8,9 Serum is an undefined mixture composed of over 1000 different components, 10 some of whose functions are known, whereas others are not. The primary functions of serum in cell culture are to stimulate cell growth and proliferation by hor- monal factors; to carry hormones, minerals, trace elements, and lipids by transport proteins; and to allow the attachment and spreading of cells by the formation of extracellular matrix components. 8,11 Despite the fact that serum is essential for cell proliferation, it also has many non-negligible disadvantages. First, serum is an ill-defined medium supplement and its composition is inconsistent from one lot to the next, which can increase results variability. 12 If the serum sample is consistent across the experiment, this should improve the reproduc- ibility. 13 Second, serum production is costly and unefficient. Effectively, about 44–144 cows must be slaughtered to obtain 1 L of fetal bovine serum. 14 Third, fetal calf serum can contain different amounts of endotoxins, hemoglobin, and infectious agents (fungi, bacteria, viruses, and prions). 11,15 Finally, high levels of proteins are present in the serum, which can lead to the overgrowth of some unwanted cell types and downstream protein purification. 16 Previous studies have shown that small changes in the concentration of hormones 17 and cholesterol, 18 or large changes 1 Centre LOEX de l’Universite ´ Laval, Ge ´nie tissulaire et re ´ge ´ne ´ration: LOEX—Centre de recherche FRSQ du Centre hospitalier affilie ´ universitaire de Que ´bec, Que ´bec, Canada. 2 Faculte ´ de Pharmacie and 3 Faculte ´ de Me ´decine, Universite ´ Laval, Que ´bec, Canada. TISSUE ENGINEERING: Part A Volume 17, Numbers 7 and 8, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2010.0256 877