Mixed cultures comprising syngeneic and allogeneic mouse keratinocytes as a graftable skin substitute

Using inbred mouse strains C3H/He and BALB/c and an anti-class I MHC monoclonal antibody, we examined the graftability of mixed cultures composed of syngeneic and allogeneic mouse keratinocytes. Keratinocytes from neonatal C3H/He (syngeneic donor) and BALB/c (allogeneic donor) mice were cultured for 7 days, both individually and at the inoculum ratio of 1:1, 1:3, or 1:15. The resulting sheets of keratinocytes were implanted subcutaneously into adult C3H/He mice and morphological evaluation was made on the 4th, 7th, and 14th days after grafting. The control cultures of 100% allogeneic keratinocytes were rejected, whereas epidermal reconstruction without typical signs of rejection took place in all grafts derived from the mixed cultures, including the 1:15 cultures. However, immunofluorescence staining showed that, within the grafts, the allogeneic keratinocytes were gradually replaced by syngeneic cells. Thus, these results suggested that graftable skin substitutes may be produced by co-culturing a small amount of autologous keratinocytes with allogeneic keratinocytes, which are readily available.