Mannosylerythritol lipids inhibit melanogenesis via suppressing ERK‐CREB‐MiTF‐tyrosinase signalling in normal human melanocytes and a three‐dimensional human skin equivalent

Background: Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected. Methods: 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin layers is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data. Results: Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells. Discussion: The MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

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Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model

10.7287/peerj.preprints.2984v1 ◽

2017 ◽

Author(s):

Parvathi Haridas ◽

Jacqui A McGovern ◽

Sean DL McElwain ◽

Matthew J Simpson

Keyword(s):

Basement Membrane ◽

Cell Line ◽

Human Skin ◽

Time Course ◽

Three Dimensional ◽

Melanoma Cells ◽

Skin Equivalent ◽

Radial Spreading ◽

Time Course Data ◽

Human Skin Equivalent

Background: Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected. Methods: 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin layers is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data. Results: Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells. Discussion: The MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

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Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model

PeerJ ◽

10.7717/peerj.3754 ◽

2017 ◽

Vol 5 ◽

pp. e3754 ◽

Cited By ~ 22

Author(s):

Parvathi Haridas ◽

Jacqui A. McGovern ◽

Sean D.L. McElwain ◽

Matthew J. Simpson

Keyword(s):

Basement Membrane ◽

Cell Line ◽

Human Skin ◽

Time Course ◽

Three Dimensional ◽

Melanoma Cells ◽

Skin Equivalent ◽

Radial Spreading ◽

Time Course Data ◽

Human Skin Equivalent

Background Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected. Methods 3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data. Results Both HSE and MSE models are similar to native skin in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells. Discussion The MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details.

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Fundamental in vitro 3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments

4open ◽

10.1051/fopen/2021001 ◽

2021 ◽

Vol 4 ◽

pp. 1

Author(s):

Ayesha Idrees ◽

Inge Schmitz ◽

Alice Zoso ◽

Dierk Gruhn ◽

Sandra Pacharra ◽

...

Keyword(s):

Electron Microscopy ◽

Human Skin ◽

Barrier Function ◽

Culture Conditions ◽

Dermal Fibroblasts ◽

Skin Equivalent ◽

Epidermal Keratinocytes ◽

Normal Human ◽

Human Skin Equivalent

Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro three-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamental in vitro tool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection.

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Desmosomal distribution in the epidermis of a non-contracting human skin equivalent

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124884 ◽

1992 ◽

Vol 50 (1) ◽

pp. 896-897

Author(s):

L.X. Oakford ◽

S.D. Dimitrijevich ◽

R. Gracy

Keyword(s):

Human Skin ◽

Basal Layer ◽

Skin Equivalent ◽

Tissue Component ◽

Intact Skin ◽

Similar Sequence ◽

Sequence Of Events ◽

Suprabasal Keratinocytes ◽

Human Skin Equivalent

In intact skin the epidermal layer is a dynamic tissue component which is maintained by a basal layer of mitotically active cells. The protective upper epidermis, the stratum corneum, is generated by differentiation of the suprabasal keratinocytes which eventually desquamate as anuclear comeocytes. A similar sequence of events is observed in vitro in the non-contracting human skin equivalent (HSE) which was developed in this lab (1). As a part of the definition process for this model of living skin we are examining its ultrastructural features. Since desmosomes are important in maintaining cell-cell interactions in stratified epithelia their distribution in HSE was examined.

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Transformative Encounters and Uncanny Intimacies: Biotechnology and Contemporary Art – A Practitioner's Approach and Perspective

Somatechnics ◽

10.3366/soma.2012.0061 ◽

2012 ◽

Vol 2 (2) ◽

pp. 263-283 ◽

Cited By ~ 1

Author(s):

Svenja J. Kratz

Keyword(s):

Tissue Culture ◽

Human Skin ◽

Contemporary Art ◽

Skin Equivalent ◽

Biomedical Innovation ◽

Equivalent Models ◽

Science Projects ◽

Insight Into ◽

Human Skin Equivalent

Abstract: Presented from an ArtScience practitioner's perspective, this paper provides an overview of Svenja Kratz's experience working as an artist within the area of cell and tissue culture at QUT's Institute of Health and Biomedical Innovation (IHBI). Using The Absence of Alice, a multi-medium exhibition based on the experience of culturing cells, as a case study, the paper gives insight into the artist's approach to working across art and science and how ideas, processes, and languages from each discipline can intermesh and extend the possibilities of each system. The paper also provides an overview of her most recent artwork, The Human Skin Equivalent/Experience Project, which involves the creation of personal jewellery items incorporating human skin equivalent models grown from the artist's skin and participant cells. Referencing this project, and other contemporary bioart works, the value of ArtScience is discussed, focusing in particular on the way in which cross-art-science projects enable an alternative voice to enter into scientific dialogues and have the potential to yield outcomes valuable to both disciplines.

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