In situ freeze-fracture of monolayer cell cultures grown on a permeable support

The growth of cultured epithelial cells on permeable supports allows increased cell differentiation and the assessment of a variety of transcellular and paracellular transport processes. The need to assess the corresponding ultrastructural characteristics of these cells under identical conditions prompted this laboratory to develop a reliable method for producing freeze-fracture replicas of these cultures. Sections of filter inserts with the cell-side facing up are placed between layers of polyvinyl alcohol with a strip of mylar positioned on the layer of polyvinyl alcohol. Following freezing, the monolayer is fractured by lifting the mylar strip from the assembly. The result is a consistent fracture of the apical membrane sufficient for analysis of tight junction sealing strands, microvilli distribution, and intramembranous particle (IMP) distribution between apical and lateral membrane domains. This method utilizes standard equipment and readily available materials and, most importantly, allows the freeze-fracture and replication of an undisturbed cell monolayer.