Enrichment of unlabelled human Langerhans cells by modified discontinuous Ficoll-metrizoate density medium and following Langerhans cell culture.

Abstract

Highly enriched and specifically unlabelled human Langerhans cells (LC) from epidermal cell suspensions (ECS) are indispensable for the intensive study of LC function. Discontinuous Ficoll-metrizoate density gradient centrifugation was found to be a satisfactory method of enriching LC from ECS. In contrast to a previous study, however, the majority of LC floated on Ficoll-metrizoate with a density of 1.057 g/cm3 instead of 1.068 g/cm3. The concentration of unlabelled LC can be enriched to as high as 92% from the original 1.8-3.1% LC in ECS. Approximately 40-50% of original LC can be harvested. The procedure was also simplified by using a Schick razor instead of a dermatome to obtain thin epidermal sheets (about 0.25 mm in thickness), omitting the density gradients of 1.089 and 1.10 g/cm3 and using the avidin-biotin complex method to identify LC. LC are non-proliferative cells in in vitro culture systems. The viability of LC dropped to 20-30% after 3 days in the culture medium. It was also observed that LC tended to attach to aggregated keratinocytes in the culture system.