Generation of Self-assembled Vascularized Human Skin Equivalents

Abstract

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4-and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

Advancements in Extracellular Matrix-Based Biomaterials and Biofabrication of 3D Organotypic Skin Models

“…To date, collagen remains the most popular ECM-based biomaterial for dermal matrix biofabrication. These collagens are typically derived from animal sources such as bovine collagen ,, or rat tail collagen. − Alternatively, avian collagen, ovine collagen, porcine collagen, , and duck feet collagen are used, albeit to a lesser extent for dermal matrix development. Although human collagen can also be derived from skin biopsies and cadaveric tendons, research into its use in organotypic skin models remain elusive.…”

Organotypic cultures as aging associated disease models

“…OSCs are 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 (typical culture lengths of 8-12 weeks) [91][92][93]. Most commonly, OSCs contain dermal fibroblasts and keratinocytes and are cultured at an air-liquid interface for epidermal differentiation and stratification.…”

“…Lower elasticity, increased fragility, and wrinkle formation [47,50,53,54] Increased collagen disorganization, accumulation of advanced glycation end products, and changes in (GAG) and (PG) concentrations/organization [49,53,[55][56][57][58][59][60][61] Flattening of the dermal epidermal junction [50,52] Decreased dermal vasculature [62] Reduced subcutaneous fat volume [50] Increased cellular senescence [49,63] Decreased cell population and turnover, including melanocytes, epidermal cells, dermal fibroblasts, and immune cells [50,63,64] Reduced barrier function coupled with changes in the stratum corneum, lipid composition, and filaggrin expression [65][66][67][68][69] [ 71,94,95]. These include vascular endothelial cells [92,93,[96][97][98][99][100][101], immune cells [102][103][104][105], adipose derived stem cells and adipocytes from adipose derived stem cells [106][107]…”

“…Similarly, changes in vasculature are prevalent in aged skin, but OSCs often lack vascular cells. While progress has been made in vascularizing OSCs and related models [92,93,96,97,99,100,141,[144][145][146], there is still a great deal of work to be done in applying this to aging questions.…”

“…However, with the growth of interest in heterogeneous cell-cell communication, an increasing number of models have been demonstrated with additional cell populations [ 71 , 94 , 95 ]. These include vascular endothelial cells [ 92 , 93 , 96 – 101 ], immune cells [ 102 – 105 ], adipose derived stem cells and adipocytes from adipose derived stem cells [ 106 – 108 ], embryonic stem cells [ 71 ], melanocytes [ 109 – 111 ] and melanocytes derived from induced pluripotent stem cells [ 112 ]. With this customizability and a growing number of accessible protocols, OSCs represent a useful tool for studying skin aging; exemplar applications are discussed below, first for disease generally and then with aging specifically.…”