Ultrastructural changes in keratinizing epithelium following trypsinization, epidermal detachment and apposition to mesenchymes

Abstract

The cellular effects of trypsinization, a commonly used method for separating epithelial from mesenchymal tissues, were examined with the electron microscope. Specimens were fixed after each step of the trypsinization process in 1 % OsO₄ in double strength Tyrode solution and after 1–7 days of culture following reapposition of the epithelium to its own mesenchyme or collagen-rich 12-day embryonic quadriceps tendon. Controls consisted of specimens identically prepared from limb bud skin derived from the same site from s-day normal embryos to hatching. A second series of controls were 5-day skin explants cultured for 1-7 days in vitro following identical preparation except for the omission of trypsin. Trypsin caused transient cytoplasmic protrusions (‘blebs’) on the deep aspect of the basal cells, cellular shrinkage and increase in lysosomes. The basal lamina was disrupted overlying the blebs and remained attached to the basal cells only between the blebs. Continuity of the basal lamina was restored within 24 h after the onset of cultunng and developed precociously thereafter, regardless of whether it was reapposed to its own collagen-poor mesenchyme or the collagen-rich quadriceps tendon. In the latter, focal excessive basal lamina formation was observed. Epithelial cell differentiation proceeded equally precociously in trypsinized and non-trypsinized cultures. Disruption of the basal lamina by trypsinization did not produce detectable alteration in the synthesis of tonofilaments, desmosomes, granular reticulum or corpuscula cnbriformia. Keratohyaline bodies were not found in any of the cultures and keratinized cells did not develop.