Keratinocytes

Keratinocytes are the epithelial cells lining the epidermis; the uppermost layer of the skin and the mucosa. Approximately, 95% of the cells found in the epidermis constitute this outer most layer of the skin. It is first line of defense. Under stimulation from pathogens, keratinocytes under stimulation express toll like receptors (TLR) able to stimulate the secretion of cytokines, chemokines and keratinocyte growth factor leading to an inflammatory response. Therefore, these skill cells are not only a physical barrier but actually do take part in the process of pathogen recognition. 

Today human keratinocytes are being used in skin patients. The keratinocytes are extracted from small biopsies of skin. The skin could ideally be from any part of the body. The keratinocytes are extracted carefully by mechanically dislodging the epidermis from the dermis and culturing the cells in cell culture flaks. The cells are then transplanted in patients with skill burns. This is a process that has been successfully used in several labs and burn centre around the world. As grafts often leave scars or patchy skill, the use of keratinocytes can be routinely used for skin regeneration.

The undifferentiated or stem cell keratinocyte reside in the basal layer (deepest layer of the epidermis). They don’t divide as much and forms transient amplifying cells. Transient amplifying cells are defined as those cells that undergo rapid division before they terminally differentiate into mature keratinocytes and move in the epidermis. During the differentiation process, they synthesize keratohyalin granules that play a role in keratinization. Other major protein present in the keratinocytes includes the filamentous keratins, the cytoskeleton of keratinocytes (constituent structural proteins). Keratins are usually of 2 types; alpha-Keratins present in the hair and horns nails and claws of animals. The beta-keratins are present in the nails, scales or claws of reptiles, feathers, beaks, claws of birds. 

Cultured Keratinocytes

In labs skill biopsies can be placed in a 0.2% trypsin solution that mechanically separates the epidermis and dermis in about 20 hours. The keratinocytes are then washed with buffered saline and plated out in cell culture flasks at a density of 2 million cells. Media is regularly changed to flush other contaminating cells. After 10 days the cells can then be used in experiments or transplanted into burns or wounds. 

Alternatively, skin biopsies are treated overnight with dispase. The dermis from the epidermis is dissociated using of a solution of EDTA/trypsin for 10 minutes at 37 degrees. The action of trypsin is stopped by using DMEM and 10% fetal calf serum. The cells are then centrifuged and re-suspended in media.