Culture of human epidermal Langerhans cells in a skin equivalent

Langerhans cells (LCs) have been cultured in a skin equivalent (SE). Seventy-two SEs were produced by inserting skin biopsies from nine subjects into dermal equivalents consisting of fibroblasts in a collagen matrix. The SEs were cultured in a serum-free medium containing 2-mercaptoethanol with or without 5 ng/mL granulocyte–monocyte colony-stimulating factor (GM-CSF). The SEs were cultured for 12 or 15 days. In the latter case, 0, 1 or 10 μg/mL cyclosporin A (CyA) was added for the last 3 days. The SEs were then snap frozen for immunohistochemistry. The migration of LCs was evaluated by measuring the distances from the inserted skin biopsy in the SEs to the HLA-DR + and CD1a+ dendritic cells localized at the longest distance from the biopsy in the epidermal outgrowth on both sides of the biopsy. The density of these cells was estimated in 15-day-old SEs by counting them on both sides of the inserted skin biopsy and dividing the number of positive cells by the migrated distances. All epidermal outgrowths (range 0.6–3.7 mm) were well differentiated and displayed HLA-DR+, CD1a+ and Lag+ dendritic cells. Only occasionally were CD83+ cells observed. In the 15-day-old SEs cultured with GM-CSF, a few CD86+ cells were seen in the epidermal outgrowths and occasionally CD80+ cells. The median (n  =  4) density of CD1a+ and HLA-DR+ cells in the epidermal outgrowths at day 15 was 5.2 and 9.1 cells/mm, respectively. GM-CSF did not influence migration in 12-day-old SEs, but there was a tendency to increased migration of HLA-DR+ dendritic cells in 15-day-old SEs. CyA did not affect migration or density. We conclude that LCs can be cultured with an in vivo-like density in a SE. They express the phenotype of immature antigen-presenting cells efficient in capturing and processing antigen. This model may be suitable for studies of the initial phase of contact allergic reactions.