Publicação
Development of skin equivalents and their characterization for in vitro testing
| Resumo: | Skin is the major organ of the human body and its function is to protect the organism from the environment, constituting a very “impermeable” hydrophobic barrier. This is mainly accomplished by a very specialized avascular external layer – the epidermis followed by the dermis. The epidermis is subdivided in several layers representing different stages of keratinocyte differentiation that is accomplished by their continuous proliferation supported by the dermis, keratinization and death. 2D cell cultures, used worldwide, fail to reproduce the very relevant complexity of in vivo skin microenvironment. Thus, the development of in vitro 3D models that mimic more closely the natural skin architecture is of great relevance. They have become a very important tool as skin research models since the use of animals has been restricted due to ethical issues. The purpose of our work is to develop and validate a noncommercial skin equivalent in our laboratory. In order to accomplish the proposed aim, collagen was extracted from chicken and pig skins using an acid/alkali/enzymatic conjugation method and their performances were compared with commercial calf skin and rat tail collagens. All collagens were characterized by electrophoresis, protein and collagen quantification. After polymerization, the collagens were compared in terms of fibroblast proliferation (BJ-5ta cell line). Optimization experiments were performed. The best pore size of the membrane inserts was defined as 1.0 μm in order to assure the best conditions for fibroblast proliferation. The best fibroblast initial seeding density in the collagen matrices was defined as 6x105 cells/ml. The best collagen concentration for the models development was 3 mg/ml. To validate them, the differentiation of keratinocytes (HaCaT cell line) was evaluated by HE staining and by immunohistochemistry for the expression of keratins 10 and 14 (K10/14 – keratinocyte differentiation markers). For rat and calf collagen matrices, 3 to 5 layers of epidermis were observed and K10 expression was essentially present in the supra-basal layers. K14 had a more uniform expression across the keratinocyte layers, however is less expressed in the outermost layers. Models developed with chicken and pig collagen matrices presented only 1 to 2 discontinuous and disorganized layers. Thus, the rat tail and calf skin collagens proved to be the best choices for the development of a skin equivalent. A better optimization of the collagen extraction protocol is required, and the addition of a biopolymer, such as elastin, to our skin equivalents would also be interesting. Due to the numerous advantages that 3D models present, we believe it is important to continue this work. |
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| Autores principais: | Gonçalves, Anabela Ferreira |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Skin is the major organ of the human body and its function is to protect the organism from the environment, constituting a very “impermeable” hydrophobic barrier. This is mainly accomplished by a very specialized avascular external layer – the epidermis followed by the dermis. The epidermis is subdivided in several layers representing different stages of keratinocyte differentiation that is accomplished by their continuous proliferation supported by the dermis, keratinization and death. 2D cell cultures, used worldwide, fail to reproduce the very relevant complexity of in vivo skin microenvironment. Thus, the development of in vitro 3D models that mimic more closely the natural skin architecture is of great relevance. They have become a very important tool as skin research models since the use of animals has been restricted due to ethical issues. The purpose of our work is to develop and validate a noncommercial skin equivalent in our laboratory. In order to accomplish the proposed aim, collagen was extracted from chicken and pig skins using an acid/alkali/enzymatic conjugation method and their performances were compared with commercial calf skin and rat tail collagens. All collagens were characterized by electrophoresis, protein and collagen quantification. After polymerization, the collagens were compared in terms of fibroblast proliferation (BJ-5ta cell line). Optimization experiments were performed. The best pore size of the membrane inserts was defined as 1.0 μm in order to assure the best conditions for fibroblast proliferation. The best fibroblast initial seeding density in the collagen matrices was defined as 6x105 cells/ml. The best collagen concentration for the models development was 3 mg/ml. To validate them, the differentiation of keratinocytes (HaCaT cell line) was evaluated by HE staining and by immunohistochemistry for the expression of keratins 10 and 14 (K10/14 – keratinocyte differentiation markers). For rat and calf collagen matrices, 3 to 5 layers of epidermis were observed and K10 expression was essentially present in the supra-basal layers. K14 had a more uniform expression across the keratinocyte layers, however is less expressed in the outermost layers. Models developed with chicken and pig collagen matrices presented only 1 to 2 discontinuous and disorganized layers. Thus, the rat tail and calf skin collagens proved to be the best choices for the development of a skin equivalent. A better optimization of the collagen extraction protocol is required, and the addition of a biopolymer, such as elastin, to our skin equivalents would also be interesting. Due to the numerous advantages that 3D models present, we believe it is important to continue this work. |
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