Lamellar Granule Extrusion and Stratum Corneum Intercellular Lamellae in Murine Keratinocyte Cultures

Lamellar granules are specialized epidermal organelles containing stacks of membranous disks that are extruded into the intercellular spaces in the upper portion of the granular layer. The extruded disks are believed to undergo biochemical and biophysical changes to form the stratum corneum intercellular lipid sheets that constitute the epidermal permeability barrier. Little is known about this important component of epidermal differentiation, in part due to lack of a suitable in vitro model. We have demonstrated microscopically the presence of characteristic lipid membrane structures in a primary keratinocyte culture system which shown morphologic differentiation comparable to that seen in vivo. A basal cell-enriched fraction of isolated neonatal mouse keratinocytes was plated into Vitrogen-coated 30mm Millicell (Millipore, Bedford, Massachusetts) wells, fed daily with Medium 199 containing 10% fetal bovine serum, 10 μg/ml each of insulin and hydrocortisone, and kept at 32°C in a 5% CO2/95% air atmosphere in a humidified incubator. Three days after plating, cultures were placed on living, epidermis-free mouse dermis at the air/liquid interface. At 2 wk, histologic examination showed multiple well-organized cell layers, including a distinct granular layer and a well-developed stratum corneum. Transmission electron microscopy demonstrated numerous lamellar granules and extrusion of their contents into the intercellular space. After fixation with ruthenium tetroxide, stacked intercellular lamellae in the stratum corneum were seen. Both the presence of dermis and growth at the air/liquid interface were necessary to achieve complete differentiation. This system conclusively demonstrates the formation of complex epidermal lipid structures in vitro and should allow the mechanisms and regulation of their synthesis to be elucidated