Background: Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction\r\nof cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that\r\nrapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty\r\nacid free, low calcium medium that is replaced every 24 hrs.\r\nMethods: Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of\r\ntissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely\r\nconfluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested\r\nevery 24 hrs. over a 7ââ?¬â??10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask\r\nto form another confluent monolayer over 2ââ?¬â??4 days. This new culture, in turn produced additional cell suspensions\r\nthat when serially passed expand the cell strain over 2ââ?¬â??3 months, without the use of enzymes to split the cultures.\r\nThe cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size\r\nand glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte\r\ndifferentiation, and characterized by immunohistochemistry.\r\nResults: The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used\r\napproximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells\r\n(8ââ?¬â??10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and\r\n15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The\r\nePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing).\r\nConclusions: Using this primary culture technique, large quantities of epithelial cells can be generated without the\r\nuse of the enzyme trypsin to split the cultures. The cells are small in diameter and have basal cell progenitor/ââ?¬Âstemââ?¬Â\r\n(P/SC) cell characteristics induced by daily feeding with larger than normal medium volumes. The ePUK epithelial\r\ncells have the potential to be used in regenerative medicine and for basic studies of epithelia P/SC phenotype.
Loading....