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Human plasma-derived bilayered skin substitutes were successfully used by our group to produce human-based in vitro skin models for toxicity, cosmetic, and pharmaceutical testing. However, mechanical weakness, which causes the plasma-derived fibrin matrices to contract significantly, led us to attempt to improve their stability. In this work, we studied whether an increase in fibrin concentration from 1.2 to 2.4 mg/mL (which is the useful fibrinogen concentration range that can be obtained from plasma) improves the matrix and, hence, the performance of the in vitro skin cultures. The results show that this increase in fibrin concentration indeed affected the mechanical properties by doubling the elastic moduli and the maximum load. A structural analysis indicated a decreased porosity for the 2.4 mg/mL hydrogels, which can help explain this mechanical behavior. The contraction was clearly reduced for the 2.4 mg/mL matrices, which also allowed for the growth and proliferation of primary fibroblasts and keratinocytes, although at a somewhat reduced rate compared to the 1.2 mg/mL gels. Finally, both concentrations of fibrin gave rise to organotypic skin cultures with a fully differentiated epidermis, although their lifespans were longer (25-35%) in cultures with more concentrated matrices, which improves their usefulness. These systems will allow the generation of much better in vitro skin models for the testing of drugs, cosmetics and chemicals, or even to "personalized" skin for the diagnosis or determination of the most effective treatment possible.

Effect of Fibrin Concentration on the In Vitro Production of Dermo-Epidermal Equivalents