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 microg/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.