Isolation and culture of panning method-enriched Langerhans cells from dispase-dissociated epidermal cells of the mouse

Langerhans cells (LCs) are bone marrow-derived, Ia-positive antigen-presenting cells in the epidermis which constitute 2-4% of the total epidermal cells. We examined the usefulness of a combination of dispase treatment and the panning method for enriching and culturing mouse LCs. Trunk skin was treated with partially purified dispase (Godo Shusei, type II) to separate epidermal sheets and to dissociate epidermal cells. Suspended cells were treated with ascites or culture supernatant containing anti-Ia monoclonal antibody, and LCs were enriched by the Ia-mediated panning method. Per mouse, 3-4 x 10⁵ LCs were recovered with > 95% purity and > 90% viability. Enriched LCs potently stimulated the allogeneic mixed-leukocyte reaction. Ultrastructural observations revealed that enriched LCs contained many vesicles but almost no Birbeck granules. A laminal structure, which was apparently adhesive to the surface of LCs, was observed when ascites were employed as the anti-Ia antibody. These results indicate that a combination of dispase treatment and the Ia-mediated panning method is very useful for isolating high yields of functionally mature murine Langerhans cells with high purity and viability.