While in vivo and traditional cell culture models remain important tools, there is increasing interest in more physiologically relevant culture models, and there is a growth in recent studies employing organotypic skin models . Organotypic Skin Models Researchers have used organotypic models to study skin biology since the 1980s. OSCs are also commonly referred to as human skin equivalents or full-thickness skin models; they typically have dermal and epidermal layers with proper stratification of the epidermis. These models have proven useful for studying skin development, evaluating cytotoxicity, studying wound healing, and more recently as disease and aging models. OSCsare highly customizable and allow for control of organotypic cell populations, genotypes, and culture conditions to enable carefully controlled studies on tissue-level biology. OSCs have the capacity to be used for in depth aging studies without the dangers of human trials or expensive animal models; with long-term culture stability for chronic studies. Most commonly, OSCs contain dermal fibroblasts and keratinocytes and are cultured at an air-liquid interface for epidermal differentiation and stratification. However, with the growth of interest in heterogeneous cell-cell communication, an increasing number of models have been demonstrated with additional cell populations. These include vascular endothelial cells, immune cells, adipose derived stem cells and adipocytes from adipose derived stem cells, embryonic stem cells, melanocytes and melanocytes derived from induced pluripotent stem cells. With this customizability and a growing number of accessible protocols, OSCs represent a useful tool for studying skin aging; exemplar applications are discussed below,drainage planter pot first for disease generally and then with aging specifically.
OSCs have been used in a number of disease studies, both directly and as “hybrid” studies where a humanized OSC is grafted onto immunodeficient mice. Additionally, models have been shown useful for testing potential therapeutic techniques for debilitating skin disorders or injuries. OSC skin disorder models include: psoriasis, recessive dystophic epidermolysis bullosa , xeroderma pigmentosum, vitiglio, Forlin syndrome, lamellar ichthyosis, Netherton syndrome, congenital pachyonychia, Junctional epidermolysis bullosa, and fibrosis. Of these disease models, the fibrosis model by Varkey et al. is especially interesting for its potential to be adapted to use as an aging model. In this study, OSCs were generated using either deep dermal fibroblasts or superficial dermal fibroblasts in combination with normal human keratinocytes. They found that the antifibrotic properties of deep dermal fibroblasts and the fibrotic properties of superficial fibroblasts can influence OSC characteristics. Authors found that when compared to constructs with superficial or mixed fibroblast populations, OSCs with deep fibroblasts had higher levels of interleukin-6, reduced TGF-β1 production, higher PDGF expression, and epidermal formation was less defined and less continuous. This model is potentially interesting as a platform for aging research, as TGF-β is implicated in skin aging through regulation of matrix metalloprotease activity. The work of Varkey et al. highlights the usefulness of OSCs to study signaling between specific cellular subpopulations in a controlled way; an approach that could be readily adapted to aging studies. While there is a great deal of room for OSCs to be used to study the processes of aging in isolation or in combination with disease, several researchers have applied OSCs to aging. Organotypic Skin Models to study aging As OSCs are stable for long culture periods , using the extended culture time to study intrinsic aging is perhaps one of the most straightforward techniques and can be combined with other aging models and/or cell types 20.
With this model, authors demonstrated that extended culture exhibited several age-related aspects similar to those that occur with in vivo aging, including decreases in epidermal thickness, decreases in hyaluronin expression, increases of the aging biomarker p16Ink4a, decreases in keratinocyte proliferation over time, loss of expression of healthy epidermal markers, and basement membrane alterations. Another straightforward application of OSCs in aging is studying the impact of senescent cells. A number of studies have incorporated senescent fibroblasts into OSCs to generate models that recapitulate many of the features of in vivo aged skin. Diekmann and colleagues induced senescence in human dermal fibroblasts and keratinocytes using Mitomycin-C treatment and incorporated the cells into OSCs 84. When compared to mitotic OSCs, the senescent models demonstrated changes similar to aged in vivo skin, including a more compact stratum corneum of the epidermis, reduced dermal fibroblast population, decreased collagen type I and III fiber content, decreased elastin expression and looser elastin structures, increases in MMP1, and disordered epidermal differentiation. Authors also isolated fibroblasts after OSC cultures to re-validate their senescence through aging markers and showed senescent morphology, increased senescence associated β-galactosidase , lower proliferative activity determined by Ki67 expression, upregulated p53 activity, upregulated ROS, and increased concentrations of MMP1 as compared to mitotic fibroblasts. A similar study involving senescent fibroblasts used healthy fibroblasts that were exposed to H2O2 to induce senescence and then cultured the senescent fibroblasts in skin equivalents with healthy keratinocytes. Aging phenotypes were again characterized by changes in proliferation, differentiation of suprabasal epidermal layers, impairments of skin barrier function, and surface property modification. Further, authors found that fibroblasts exhibited senescence-associated secretory phenotype markers including IL-6, GmCSF, and IL-1α, however, this response was blunted in the 3D culture with keratinocytes.
Lower levels of IL-6 were also measured in OSCs generated with fibroblast that underwent doxorubicin induced senescence. Interestingly, Weinmueller et al. observed more Ki67 positive epidermal cells when senescent fibroblasts were present. More research is required to understand senescence in the dermis and how it may effect keratinocyte homeostasis. Serial passaging of fibroblasts has also been employed to simulate aging in OSCs, showing that constructs generated with late passage fibroblasts were similar to in vivo aged skin. OSCs were generated with 15-20% SA-β-gal positive fibroblasts cells in 2D culture prior to 3D seeding. Authors observed few changes in the epidermal compartment while the dermal component of OSCs presented a thinner dermis and increased MMP1, similar to in vivo aged skin. Defects in epidermal-dermal junction in these OSCs were not observed and keratinocytes exhibited a healthy phenotype. Although not shown, authors noted that when greater than 30% Beta-gal positive fibroblast cells in 2D were used to generate OSCs, the fibroblasts did not produce ECM and constructs were not viable likely due to ineffective support for keratinocytes. As Janson et al. found, generating an OSC using senescent cells is technically challenging since the percentage of senescent cells used to generate an OSC can alter skin structure and long-term culture health . Similar studies focused on the aging of the keratinocyte population have also been done. In OSCs generated from primary cells isolated from donors,plant pot with drainage cell donor age is an option for simulating intrinsic aging in vitro . OSCs generated with either keratinocytes isolated from aged individuals or serially passaged keratinocyte cells have been used to examine the effects of replicative senescence. Constructs generated with older keratinocytes exhibited thinner epidermis compared to cells from 1-year old donors. Additionally, there were differences in epidermal organization, where constructs generated with young keratinocytes were well organized with better stratification, while older cells produced more disorganized and less complete stratification. This study also investigated amounts of epidermal stem cell markers. They found that when keratinocytes were pass aged over six times , there was a loss of stemness, indicated by high expression of α6 integrin and low expression of CD71. Likewise, in constructs generated with young keratinocytes, α6 integrin expression was observed in basal cells of epidermis while in constructs generated with adult and elderly cells there was faint andabsent α6 integrin expression . These OSC findings demonstrated in both intrinsic aging and in vitro senescence induced by serial passaging results in depletion of stem cells in the epidermis of skin 86 . Epidermal changes associated with aging have also been shown in models generated through genetically altering expression of key components, for example p16Ink4a 87 . In vivo chronological human aging markers, p16Ink4a and its repressor BM1, are established markers of in vitro aging tissue. p16Ink4a is an inhibitor of cyclin-dependent kinases that blocks the progression from G1 phase to S phase of the cell cycle and promotes senescence onset. In vitro aged skin models can be generated from young donor keratinocytes cells by p16Ink4a overexpression. Conversely, aging phenotypes observed in old donor keratinocytes can be rescued through silencing p16Ink4a.
Aged models resulted in thinner epidermis, loss of stratum corneum , and atrophy. OSCs also allow for studies of matrix and cell-matrix interactions in aging skin. Expression patterns of glycosaminoglycans and proteoglycans are important in skin tissue mechanical integrity, and aging-related changes contribute to frailty in both intrinsically and extrinsically aged skin. Glycation and the presence of advanced glycation end products increase in aging skin, and this has been leveraged in OSCs to create an aged skin model. In this model, collagen was glycated in vitro prior to construction of the OSC. This simulated intrinsic aging of the construct, resulting in modified integrin patterns in the suprabasal epidermal layers, activation of the dermal fibroblasts to increase the production of metalloproteinase, type III procollagen, and type IV collagen 14,32. Authors found that these morphological and molecular changes in the epidermis and dermis could be partially rescued by antiglycation agents such asaminoguandine. More investigation is necessary to understand exactly how GAGs and PGs are affected during skin aging. Open questions include how sex specific hormones may affect concentrations 8 and what downstream effects GAGs and PGs have on the expression of cytokines and growth factors. Building off of the previous OSCs, multi-cellular skin models can help to elucidate aging mechanisms regarding GAGs and PGs and their effect on skin homeostasis. CXyloside is a xyloside derivative that has been investigated as therapeutic to improve dermal-epidermal junction morphology in aging skin. Sok et al. exposed OSCs to CXyloside and investigated the resulting DEJ morphology. C-Xyloside exposure resulted in higher basement membrane protein concentrations, specifically collagen IV, laminin, and collagen VII, and a structure more similar to the microanatomy of healthy human skin. Further, C-Xyloside increased concentrations of dermal proteins such as pro-collagen I and fibrillin, which are key ECM proteins for the maintenance of skin elasticity. Since defects in the basement membrane, DEJ, or elasticity contribute to skin fragility in aging, this model has potential as a test bed for other aging therapeutics. This body of work builds on the framework that previous organotypic skin models have developed and importantly demonstrates further customization through incorporation of different cell types and photoaging studies. We have generated protocols for establishing fully vascularized human skin equivalents alone, and additionally to establish adipose and vascularized human skin equivalents. These OSCs have been analyzed volumetrically and thus, protocols for volumetric imaging of this in vitro skin tissue were developed for optical coherence tomography and confocal microscopy. Although it is an improvement upon the classic histological analysis that OSCs traditionally undergo,volumetric imaging introduces new challenges of analysis, which we have addressed through automated image analysis based on custom algorithms. These end-to-end processes are novel contributions to the tissue engineering field and they importantly enable aging studies because of the skin’s stability over time. Through the generation and analysis of in vitro skin models, multiple image analysis techniques were developed for three-dimensional structures. Specifically, through these algorithms, we were also able to analyze cell monolayers which is demonstrated in chapter 4. PFAS chemicals, or perfluooalkyl substances are very abundant and stable molecules that tend to accumulate in biological systems, or bio-accumulate . These chemicals are especially dangerous in fetal development and have been linked to decreased birthweight but increases in adiposity, and altered lipid profiles in youth and young adults. Through the skills learned in adipose tissue development and automated image analysis, we were able to study the effects that PFAS has on epidermal cell monolayers, adipose derived stem cell monolayers, and the differentiation process of adipose derived stem cells to mature fat cells, adipocytes. This work presents a novel contribution in understanding PFAS mechanistic actions on human cells and how specifically PFAS acts on adipogenesis. Additionally, we have elucidated action of PFAS chemicals on human cells through cytoskeletal disruption.Human skin performs many essential biological functions including acting as an immune/mechanical barrier, regulating body temperature, participating in water retention and sensory roles.