Characterization of Keratinocytes, Fibroblasts and Melanocytes Isolated from Human Skin using Gene Markers

Sains Malaysiana 51(5)(2022): 1425-1436 http://doi.org/10.17576/jsm-2022-5105-13 

ABSTRACT Cells isolated from skin have wide applications in studies of the pathogenesis of skin-related diseases and the construction of 3D skin equivalents. This study aimed to isolate keratinocytes, fibroblasts, and melanocytes from human foreskin and characterize the purity of the cell types. Keratinocytes, fibroblasts, and melanocytes from human foreskin were isolated by differential trypsinization and media selection. The purity of the cell types was characterized based on the expression of gene markers. The assessment of gene marker expression involved RNA extraction, primer design, quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Our results showed that in cocultures of keratinocytes and fibroblasts isolated from the dermis, fibroblasts could be separated from keratinocytes by quick trypsinization and culture in Dulbecco’s modified Eagle’s medium. The remaining keratinocytes are cultured in Epilife medium. Melanocytes in cocultures of melanocytes and keratinocytes isolated from the epidermis could be selected by changing Epilife medium to M254 medium. Gene marker results suggested that cytokeratin 14 (CK14) is a suitable marker for keratinocytes, elastin (ELN) is a suitable marker for fibroblasts, and tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are suitable markers for melanocytes. In conclusion, keratinocytes, fibroblasts, and melanocytes can be isolated from the same human foreskin sample by differential trypsinization and media selection techniques and characterized by suitable gene markers. This finding will aid in the isolation of pure skin cell types for various applications in regenerative medicine and toxicity studies.

INTRODUCTION Human skin consists of three layers, namely, the epidermis, dermis, and hypodermis (Aasen & Belmonte 2010). Keratinocytes and melanocytes can be isolated from the epidermis, whereas fibroblasts can be isolated from the dermis (Henrot et al. 2020). Cells isolated from the skin can be used to study the pathogenesis of skinrelated diseases and as models for cosmetic toxicity tests. In addition, keratinocytes, fibroblasts, and melanocytes isolated from skin can be used to generate 3D skin equivalents (Gledhill et al. 2015). The isolation of cells from skin tissues involves the separation of epidermis from dermis using mechanical force or enzymatic digestion with dispase, thermolysin, and trypsin followed by dissociation of the tissue into a single cell suspension by proteolytic enzymes, such as collagenase and trypsinEDTA (Henrot et al. 2020; Hybbinette et al. 1999). In addition, various culturing protocols for isolated cells have been reported; for example, isolated keratinocytes can be cultured on feeder cells (Rheinwatd & Green 1975) or in serum-free defined media (Tenchini et al. 1992). Defined media have the advantage of avoiding feeder cell contamination, but cell proliferation is slower in defined media compared with serum- or feeder-based media.

A common problem associated with cell isolation is the contamination of other cell types from skin tissues (Szymański et al. 2020). For example, keratinocytes, and fibroblasts are typically isolated together from the dermis, whereas keratinocytes and melanocytes are isolated together from the epidermis. A combination of differential trypsinization and media selection techniques could be used to isolate keratinocytes, fibroblasts, and melanocytes from the skin based on their differential adhesive strength in culture flasks and nutrient requirements (Caneparo et al. 2020). In the coculture of keratinocytes and fibroblasts isolated from the dermis, a quick trypsin-EDTA incubation could dissociate the fibroblasts from the coculture while leaving the keratinocytes attached to the culture flask because keratinocytes attached more strongly than fibroblasts to the flask (Zuliani et al. 2013). The separated cell types could be cultured in specific media that promote their growth, namely, Epilife medium with growth supplements for keratinocytes and DMEM/FBS for fibroblasts. In the coculture of keratinocytes and melanocytes isolated from the epidermis, the use of melanocyte medium, such as M254 with appropriate growth supplements, could promote the growth of melanocytes while suppressing keratinocytes proliferation.

Several genes have been reported to be useful for cell type identification based on their function in that particular cell type. Cytokeratin 14 (CK14) is expressed in dividing basal keratinocytes and is involved in maintaining the shape of epidermal cells and resisting mechanical stress (Byrne et al. 1994). Cytokeratin 19 (CK19) is expressed in basal keratinocytes and has been suggested as a marker for epidermal homeostasis and as an indicator of young growing skin (Pontiggia et al. 2009). E-cadherin (CDH1) is a calcium-dependent transmembrane glycoprotein that is involved in keratinocyte intercellular adhesion and stratification (Charest et al. 2009; Hines et al. 1999). Elastin (ELN) is a major fibrillary protein component of elastic fibres produced by fibroblasts (Giro et al. 1985). Fibronectin (FN1) and collagen (COL1A1) are components of the wound healing extracellular matrix produced by fibroblasts (Werner & Grose 2003). Melanocytes are characterized by melanocyte-specific markers, such as tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase-related protein 2 (TYRP2) activities (Cichorek et al. 2013). Hence, these markers could be used to characterize the purity of isolated cells from the skin. Accurate markers are important for the isolation of pure cell types for various applications, i.e. for the study of skin-related diseases and models used for cosmetic toxicity tests, in addition to other applications in regenerative medicine. This study aimed to isolate and culture keratinocytes, fibroblasts and melanocytes from the same human foreskin sample using differential trypsinization and media selection techniques and to characterize the purity of the cell types based on their respective gene markers.

Sains Malaysiana 51(5)(2022): 1425-1436 http://doi.org/10.17576/jsm-2022-5105-13