Generation of full‐thickness skin equivalents using hair follicle‐derived primary human keratinocytes and fibroblasts

Löwa, Anna; Vogt, Annika; Kaessmeyer, Sabine; Hedtrich, Sarah

doi:10.1002/term.2646

Cited by 17 publications

(13 citation statements)

References 45 publications

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“…Described HSEs are generated using commercially available cell lines that are not bound by ethical or logistic issues and can be used at least up to 14–18th passage. The results of histology and immunofluorescence analysis of our constructs were also similar to those obtained by other researchers [ 24 , 25 ]. Many of the HSE described by others were constructed from the cells isolated from patients.…”

Section: Discussionsupporting

confidence: 90%

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture

Szymański

Jęderka

Cios

et al. 2020

IJMS

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An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 μM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80–200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 μM and below was not cytotoxic to the keratinocytes, and 150 μM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

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Section: Discussionsupporting

confidence: 90%

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture

Szymański

Jęderka

Cios

et al. 2020

IJMS

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“…While developing the first healthy tissue engineered model of skin in 1978, Green H. reported the importance of adding cAMP stimulator, such as cholera toxin (CT) or isoproterenol (ISO), to induce the proliferation of cells in culture [15]. Therefore, skin models are produced using culture media supplemented with a stimulator of the AC system, which is crucial for stimulating the colony growth of human keratinocytes and thus essential to obtain a fully differentiated epidermis [15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

A Tissue-Engineered Human Psoriatic Skin Model to Investigate the Implication of cAMP in Psoriasis: Differential Impacts of Cholera Toxin and Isoproterenol on cAMP Levels of the Epidermis

Simard

Morin

Rioux

et al. 2020

IJMS

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Pathological and healthy skin models were reconstructed using similar culture conditions according to well-known tissue engineering protocols. For both models, cyclic nucleotide enhancers were used as additives to promote keratinocytes’ proliferation. Cholera toxin (CT) and isoproterenol (ISO), a beta-adrenergic agonist, are the most common cAMP stimulators recommended for cell culture. The aim of this study was to evaluate the impact of either CT or ISO on the pathological characteristics of the dermatosis while producing a psoriatic skin model. Healthy and psoriatic skin substitutes were produced according to the self-assembly method of tissue engineering, using culture media supplemented with either CT (10−10 M) or ISO (10−6 M). Psoriatic substitutes produced with CT exhibited a more pronounced psoriatic phenotype than those produced with ISO. Indeed, the psoriatic substitutes produced with CT had the thickest epidermis, as well as contained the most proliferating cells and the most altered expression of involucrin, filaggrin, and keratin 10. Of the four conditions under study, psoriatic substitutes produced with CT had the highest levels of cAMP and enhanced expression of adenylate cyclase 9. Taken together, these results suggest that high levels of cAMP are linked to a stronger psoriatic phenotype.

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“…In particular, the basement membrane appeared thicker in glycated RHS, although RHS in general lacks the sub-basal laminae of the basement membrane [ 25 ]. This alteration is well in accordance with the increased collagen-IV expression in glycated RHS [ 23 ], but the ultrastructural analysis identified longitudinal clusters of finest collagen fibrils to mimic a thick lamina fibroreticularis (fibroblast derived) of the basement membrane ( Figure 3 ).…”

Section: Discussionmentioning

confidence: 99%

Ultrastructural and Molecular Analysis of Ribose-Induced Glycated Reconstructed Human Skin

Rigon

Kaessmeyer

Wolff

et al. 2018

IJMS

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Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix–cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.

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Generation of full‐thickness skin equivalents using hair follicle‐derived primary human keratinocytes and fibroblasts

Cited by 17 publications

References 45 publications

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture

A Tissue-Engineered Human Psoriatic Skin Model to Investigate the Implication of cAMP in Psoriasis: Differential Impacts of Cholera Toxin and Isoproterenol on cAMP Levels of the Epidermis

Ultrastructural and Molecular Analysis of Ribose-Induced Glycated Reconstructed Human Skin

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