Currently, in vitro models for skin tests range from simple single-layer cultures to more complex three-dimensional co-cultures, up to the use of artificial membranes, all highly standardized models but also far from the complexity of in vivo model. The key challenge of my work was therefore the development of an in vitro system that could be more predictive, going to fill the huge gap that exists between in vitro and in vivo research. The most respective models of reality are excised skin, but it is difficult to use when not preserved in the right conditions, leading to unreliable results. The goal was therefore to develop a system capable of preserving the skin and slowing down its degradation. In this perspective, the use of explanted skin samples (from biopsies or surgical materials) maintained in a fluid dynamic environment would make it possible an in vitro experimentation closer to in vivo physiological conditions. To do this, we used a bioreactor capable of generating a continuous flow of culture medium, and we studied its effects, first, on rat skin excised samples, and later also on human skin from surgeries, through combined microscopic techniques (bright-field microscopy, scanning and transmission electron microscopy) and metabolomic analysis (performed with nuclear magnetic resonance). Once the method was validated, we set-up an inflammatory skin model, using an irritating molecule (i.e., dithranol), having the inflamed skin morphological characteristics altered compared to healthy skin, and therefore a different degree of absorption. On these models, we have thereafter carried out biodistribution studies on fluorescent products differently composed: in one case two poloxamer gels were tested, one of which containing dispersed solid lipid nanoparticles (SLN) and made fluorescent due to the presence of rhodamine; in the second case, we tested poly(lactide-co-glycolide) (PLGA) nanoparticles to which the fluorophore fluorosceinamine was conjugated. Taken together, our results demonstrate the innovation of the system developed as a valid alternative to classic in vitro models to carry out biodistribution and absorption studies, reducing, if not avoiding, the use of laboratory animals. In fact, even using animal skin, from a single animal it is possible to obtain different skin samples on which to test different products or study their kinetics by evaluating different time-points. The use of human skin from biopsies or surgery limits or further avoids the use of animal skin, providing more predictive data, even overcoming interspecies variability, and gaining considerable importance from an ethical point of view.

The main barrier of human body: the skin. An experimental in vitro model to evaluate morphological and functional biodistribution features in healthy and pathological conditions

Cappellozza Enrica
2021

Abstract

Currently, in vitro models for skin tests range from simple single-layer cultures to more complex three-dimensional co-cultures, up to the use of artificial membranes, all highly standardized models but also far from the complexity of in vivo model. The key challenge of my work was therefore the development of an in vitro system that could be more predictive, going to fill the huge gap that exists between in vitro and in vivo research. The most respective models of reality are excised skin, but it is difficult to use when not preserved in the right conditions, leading to unreliable results. The goal was therefore to develop a system capable of preserving the skin and slowing down its degradation. In this perspective, the use of explanted skin samples (from biopsies or surgical materials) maintained in a fluid dynamic environment would make it possible an in vitro experimentation closer to in vivo physiological conditions. To do this, we used a bioreactor capable of generating a continuous flow of culture medium, and we studied its effects, first, on rat skin excised samples, and later also on human skin from surgeries, through combined microscopic techniques (bright-field microscopy, scanning and transmission electron microscopy) and metabolomic analysis (performed with nuclear magnetic resonance). Once the method was validated, we set-up an inflammatory skin model, using an irritating molecule (i.e., dithranol), having the inflamed skin morphological characteristics altered compared to healthy skin, and therefore a different degree of absorption. On these models, we have thereafter carried out biodistribution studies on fluorescent products differently composed: in one case two poloxamer gels were tested, one of which containing dispersed solid lipid nanoparticles (SLN) and made fluorescent due to the presence of rhodamine; in the second case, we tested poly(lactide-co-glycolide) (PLGA) nanoparticles to which the fluorophore fluorosceinamine was conjugated. Taken together, our results demonstrate the innovation of the system developed as a valid alternative to classic in vitro models to carry out biodistribution and absorption studies, reducing, if not avoiding, the use of laboratory animals. In fact, even using animal skin, from a single animal it is possible to obtain different skin samples on which to test different products or study their kinetics by evaluating different time-points. The use of human skin from biopsies or surgery limits or further avoids the use of animal skin, providing more predictive data, even overcoming interspecies variability, and gaining considerable importance from an ethical point of view.
bioreactor, skin, biological barriers, microscopy, inflammation, biodistribution
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1050170
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