Abstract
Skin plays an important role in defense against infection and other harmful biological agents. Due to its fragile structure, skin can be easily damaged by heat, chemicals, traumatic injuries and diseases. An autologous bilayered human skin equivalent, MyDerm™, was engineered to provide a living skin substitute to treat critical skin loss. However, one of the disadvantages of living skin substitute is its short shelf-life, hence limiting its distribution worldwide. The aim of this study was to evaluate the shelf-life of MyDerm™ through assessment of cell morphology, cell viability, population doubling time and functional gene expression levels before transplantation. Skin samples were digested with 0.6% Collagenase Type I followed by epithelial cells dissociation with TrypLE Select. Dermal fibroblasts and keratinocytes were culture-expanded to obtain sufficient cells for MyDerm™ construction. MyDerm™ was constructed with plasma-fibrin as temporary biomaterial and evaluated at 0, 24, 48 and 72 hours after storage at 4°C for its shelf-life determination. The morphology of skin cells derived from MyDerm™ remained unchanged across storage times. Cells harvested from MyDerm™ after storage appeared in good viability (90.5%±2.7% to 94.9%±1.6%) and had short population doubling time (58.4±8.7 to 76.9±19 hours). The modest drop in cell viability and increased in population doubling time at longer storage duration did not demonstrate a significant difference. Gene expression for CK10, CK14 and COL III were also comparable between different storage times. In conclusion, MyDerm™ can be stored in basal medium at 4°C for at least 72 hours before transplantation without compromising its functionality.
Highlights
Skin is the largest and highly complex organ that covers the outer part of human body [1]
When the cells attached to the culture dish, their morphology changed to polygonal shape for keratinocytes and spindle shape for fibroblasts
The efficiency of cell adherence was judged by comparing the cell density from MyDermTM of different storage times
Summary
Skin is the largest and highly complex organ that covers the outer part of human body [1]. And functionally, it consists of two layers: the superficial epidermal layer which is mainly made up of keratinocyte and the deeper dermal layer which is made up of fibroblast [2,3,4]. The epidermal layer acts as the front line defense against infection and other harmful biological agents [1,2] It plays important roles in regulating body heat, preventing the loss of moisture and nutrients from the body. Due to its soft and fragile structure, skin can be damaged under certain circumstances such as heat, chemical, traumatic injuries and certain diseases such as diabetes when the healing ability is significantly diminished. [1]
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