Establishment of primary human skin cell culture and soft agar transformation assay

Abstract

The skin, the largest organ of the human body, is a complete closed system with various functions, including physical protection from the exterior hostile environment, sensing outside signals via its complex sensory system and initiating immunologic reactions when the initial physical protection fails. These functions are possible because of the highly elaborate structure of this multilayered organ, made up of various kinds of cells which maintain the homeostasis of skin. In this study, we optimized the isolation procedures and culture conditions of primary human melanocytes and keratinocytes from the epidermis and primary human fibroblasts from the dermis. These three cell types are especially important in cancer research. Skin cancers arise mainly from melanocytes and keratinocytes. However, fibroblasts are used widely in transformation assays, which are indispensible elements of cancer research. Using the isolated primary human fibroblasts, we established the soft agar colony formation assay, an important tool for determining the transformation potential of genetically altered cells. To transform primary fibroblasts we knocked-down p53 expression using a lentiviral transduction system and in combination we expressed oncogenic forms of hRas or cMyc. The transduction efficiency of the lentiviral system was over 90 per cent, and the tumor suppressor genes p53 and Bax were knocked down successfully using shRNA based lentiviral constructs. Colonies of potentially transformed primary fibroblasts grew in attachment independent manner in soft agar upon p53 knock-down in combination with either cMyc or hRas oncogene expression, implying a successful transformation. Next, the transformation ability of the newly generated anti-ASC shRNA vector will be tested in combination with the oncogenes cMyc or hRas using primary human fibroblasts and melanocytes.