Three-dimensional culture of human keratinocytes on a dermal equivalent as a model system to study environmental modulation of epidermal physiology in vitro: effect of air-exposure

Abstract Mini-abstract: Application of a three-dimensional culture system with air exposure facilitates the formation of large cell spheres possessing cribriform glands and producing mucin in the collagen gel. Transmission electron microscopy revealed the formation of microvilli and junctional complexes at the apical side of the cell. This study aimed to reproduce the characteristics of original adenocarcinoma tumors in vitro. The pancreatic cell line, SUIT-58, derived from a moderately differentiated adenocarcinoma of metastatic pancreatic cancer was used. The cells have a sheet structure in conventional cell culture without forming glands or exhibiting mucin production in the lumen. First, the necessity of scaffolds to create an adenocarcinoma-like microenvironment for SUIT-58 pancreatic cancer cells was assessed. Compared with conventional culture plates, the use of type I collagen as a scaffold played an important role in the formation of densely congested microvilli, as observed through scanning electron microscopy. As gland formation is one of the features of adenocarcinoma, we also assessed gland formation. Use of a recently developed three-dimensional culture system with air exposure resulted in the formation of large cell spheres possessing cribriform glands, which released mucin into the lumen. Transmission electron microscopy also revealed the formation of microvilli in the lumen of the glands and junctional complex at the intercellular part, which were similar to those observed in xenografts. These findings indicate that an in vitro three-dimensional culture system with air exposure reflects the intrinsic features of the original tumor, suggesting that this culture system could be useful for preliminary research of certain cancers.

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A Novel In Vitro Culture Model System to Study Merkel Cell Polyomavirus–Associated MCC Using Three-Dimensional Organotypic Raft Equivalents of Human Skin

Viruses ◽

10.3390/v13010138 ◽

2021 ◽

Vol 13 (1) ◽

pp. 138

Author(s):

Amanda S. W. Loke ◽

B. Jack Longley ◽

Paul F. Lambert ◽

Megan E. Spurgeon

Keyword(s):

Culture System ◽

Three Dimensional ◽

Human Keratinocytes ◽

Merkel Cell Polyomavirus ◽

Three Dimensions ◽

Human Polyomavirus ◽

Merkel Cell ◽

Raft Culture ◽

Dermal Equivalent

Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies.

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