Human mammary gland morphogenesis in vitro: The growth and differentiation of normal breast epithelium in collagen gel cultures defined by electron microscopy, monoclonal antibodies, and autoradiography

Abstract

Fragments of ducts and terminal lobuloalveolar units have been prepared by the digestion of normal human breasts with collagenase. Immediately following preparation, the fragments comprise luminal epithelial cells and myoepithelial cells. The cells retain their normal, in vivo topological organisation but are devoid of basement membranes and stromal contaminants. The epithelial structures (organoids) have been embedded within the three-dimensional matrix of rattail collagen and cultured in vitro. The events reported herein are limited to the first 9 days of such cultures because the most dramatic morphological changes were observed to occur during this period. Initially, the organoids undergo a distinct phase of cellular reorganisation which is completed during the first 36 hr following embedding. In the presence of hydrocortisone, insulin and epidermal growth factor, proliferative activity (identified by [ 3H]thymidine pulse labelling) is stimulated. The numbers of cells incorporating [ 3H]thymidine within each organoid sharply increased during the second 24 hr in culture. On Day 5 of the cultures, the proportion of cells within the original organoids labelling with [ 3H]thymidine is 8.3 ± 0.8%, whereas the proportion of cells labelling within the proliferating outgrowths is 11.5 ± 2.3%. Subsequent morphogenesis of the cultures depends exclusively upon the physical state of the collagen gel matrix. Epithelial organoids cultured within floating (released) collagen gels give rise to branched cylindrical structures. In contrast, similar organoids obtained from identical preparations and cultured in the presence of the same humoral additives but within nonreleased collagen gels do not demonstrate this type of morphogenesis. These organoids do not proliferate as large branched tubular structures. Instead they penetrate the collagen as irregular, multilayered sheets of cells. The directions in which the proliferating branched tubular structures extend into the surrounding gel, and the sites of subsequent branching appear to be dictated by the mechanical forces set up within the gel. Thus, following attachment of the epithelial cells to collagen fibrils, proliferative activity within an organoid gives rise to traction forces which result in a local mechanical distortion of the gel matrix. Wherever two or more organoids are plated in sufficient proximity to allow transmission of the distorting forces from one organoid to another, then subsequent outgrowths from the two organoids tend to converge along these lines of distortion. Subsequent to proliferation, the outgrowths undergo partial internal differentiation. The cells in the core of the cylinders separate from one another and simultaneously begin to express some of the differentiation features of luminal breast epithelium which are identified using ultrastructural criteria and by monoclonal antibodies raised to the human milk fat globule membrane. Our findings indicate that the culture of normal human breast epithelial cells within the three-dimensional matrix of a collagen gel gives rise to structures which are morphologically homologous with the ducts of the intact tissue. This is a useful model with which to study in vitro human breast epithelial cytodifferentiation and duct morphogenesis.