Application of a Three Dimensional Culture Method for the Growth and Differentiation of Normal Human Bronchial Epithelial Cells

Abstract

Lung cancer accounts for more deaths in the world than any other malignancy. The majority of these cancers arise in the epithelial lining of the airways of the lung (MMWR, 1989; Averbach et aL, 1961). Sophisticated methods have been developed to culture normal airway epithelial cells in order to better study the mechanisms regulating their conversion to their neoplastic counterparts. However, normal human bronchial epithelial cells (NHBE) in vitro from normal explant tissue rapidly lose their differentiated phenotype and have a flattened, prolate spheroid morphology. NHBE cells respond to the presence of serum and transforming growth factor-b (TGF-b) and undergo terminal squamous differentiation. These cells have none of the mucociliary characteristics of the cells in vivo. Experiments have been conducted to determine conditions that would allow NHBE to express the mucociliary, fully differentiated phenotype. Such conditions would also allow investigation of the pathways involved during proliferation, regulation, and differentiation. This system would also allow investigations on interactions between stromal cells and matrix molecules and their influence on the residing epithelium. This approach was adapted for these studies since it has been demonstrated in several laboratories that epithelial cells cultured on a collagen matrix (three-dimensionally) often do not express the same characteristics as when grown on tissue culture plastic (two-dimensionally). In order to more closely recapitulate a tissue-like structure, collagen gels were cast containing fibroblasts which gather the collagen fibers and contract the gel into a tissue-like matrix. Investigations into the role of various media components and additional structural and regulatory proteins on the contraction ability of the gels by fibroblasts were initiated. The concentration of calcium in the media plays a role in contraction. In addition, casein, fetuin, and collagen type IV and V enhance contraction by fibroblasts. The effects of these components on the resident epithelial cells was not investigated. Our results indicate that components in serum promote contraction of the gel by fibroblasts, and that chemically denatured serum (CDS) and transforming growth factor-b (TGF-B) can replace the serum component. Further investigations demonstrated the ability of transformed bronchial epithelial cell lines to grow, form colonies, and express a differentiated phenotype when maintained in this system. Histological sections demonstrated an epithelial-like morphology and mucin production, as well as cellular organization. This demonstrates that the matrix provides conditions that allow the cells to differentiate, and that the cells, maintained in replicative culture, had not lost their ability to undergo mucous differentiation. Somewhat surprisingly, NHBE cells plated on the surface of gels in the presence of TGF-b maintain proliferative potential and differentiate into mucin producing cells instead of the squamous, terminally differentiated forms found in two dimensional culture. However, complete differentiation was not observed in vitro in that cilia were never observed.