Keratinocytes are the main components of skin epidermis constituting more than 90% of it which are responsible to regulate skin regeneration during external or internal injury. These cells can be found in a heterogenic form containing proliferative, terminally differentiated and transit amplifying (TA) cells located in basal layer, outer keratinized layer and intermediate layers, respectively when isolated from an adult skin. Efforts are going on to characterize keratinocytes precisely as in comparison with widely used stem cells, mesenchymal stem cells (MSCs) and their application in cutaneous wound healing as a translational approach in regenerative medicine. In this work, we have applied a comprehensive approach to identify and characterize keratinocytes which are a valuable tool in wound healing. Keratinocytes have successfully been isolated from adult skin and the ratio of proliferative, terminally differentiated and TA cells based on the expression of α6-integrin and CD71 was studied using flow cytometry after 7, 14 and 21 days of their culture in vitro. RT and qRT-PCR was applied to study the change in genetic expression and relativity of cytokeratin markers with the passage of time. PopUp culturing and population doubling time was performed to study the proliferative potential of heterogenic population of cells. Their colonogenicity and wound healing potential were also studied to explain their healing behaviour during the time of injuries. 47% keratinocytes after 7 days, 62% after 14days and 93% keratinocytes after 21 days of culturing. Expression of CD71 was also observed in cells as, 5% after 7 days, 22% after 14 days and 66% of keratinocytes after 21 days were positive for CD71 expression. Gradual increased expression of genetic markers, K10, K14, INV and P63 was observed using qRT-PCR. Studying comparative data, it can be concluded that keratinocytes after 14 days of culturing are better to use for clinical purposes. This comprehensive protocol could be a valuable addition in cutaneous biology helping researchers to identify keratinocytes and to use them for their pre-clinical studies

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