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Incorporation of Vasculature and Fat in Human Akin Equivalents, Validated Through Volumetric Analysis to Improve Aging Models


Aging Associated Diseases (AADs) represent a continuing and increasing burden on elderly patients and their families. AADs have many commonalities, such as inflammation and reduced healing, and are difficult to study with current animal and in vitro models. The overarching aim of this work was to develop in vitro tissue models that act as platforms for AAD research. Focus of these studies is on the development of tissue engineered human skin equivalents that more closely re-capitulate human systems. To this end, we have developed and validated culture methods for human skin equivalents that incorporate dermal vasculature and subdermal adipose. The developed protocols are highly customizable, and we have demonstrated success using multiple human cell lines. Further, we improve upon the gold standard of analysis, histological sectioning and staining, by utilizing volumetric analysis which aids in understanding these complex three-dimensional tissues. Imaging through confocal microscopy and optical coherence tomography allow for quantification of several parameters including many that cannot be obtained through two-dimensional studies: vascular network diameter and diffusion length, volumetric fraction of specific cells/structures, thickness measurements of skin epidermis and adipose, and protein expression/intensity throughout a sample rather than on a single cross section. Further, automated algorithms have been developed to quantify and characterize the tissue engineered samples outlined. Similar techniques have also been applied to separate toxicity studies. Skin and fat cells were used as two-dimensional models to understand the toxic mechanisms of perfluoroalkyl substances (PFAS), commonly referred to as “forever chemicals” due to their environmental persistence. The underlying mechanisms of PFAS at the human cell level are not well understood. In these studies we show that PFAS dysregulate the cytoskeleton and enhance adipogenesis .

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