Enzymatic dissociation of hamster tracheal epithelial cells

Abstract

Enzymatic dispersal of tissue is a popular means of providing monoceUular preparations that are used for histological and metabolic studies. Collagenase, pronase, trypsin and hyaluronidase all have been successfully used for cell dissociation of various tissues, such as the heart, liver, pancreas, trachea, intestine and the adrenal glands (1-5). A recent study by Frazier et al. (6) compared the effects of various enzymes on lung tissue dispersion. The perfusion technique employed by Frazier, however, was tedious and yielded a variety of contaminated cells, including leukocytes, vascular endothelial cells, tracheal epithelial cells, eosinophils and lymphocytes. Our study illustrates a simple technique for dissociating tracheobronchial epithelia, primarily ciliated columnar epithelia, without apparent damage to the cells or the cilia. With increasing emphasis on toxicological research, improvement of existing enzymatic dispersal techniques is necessary in order to provide specialized cell cultures for biochemical and cytological studies. The precise obj ective of our dissociation technique is to isolate ciliated epithelia for use in studying toxicological effects on respiratory epithelia, utilizing either in vivo or in vitro exposure systems. Two PRECAUTIONS are important when conducting these experiments: (a) Sterile conditions must be maintained during surgery and cell culture. (b) Cell viability determinations must be conducted in a serum-free medium to avoid false positive results. If even 5% serum is added to the culture medium, errors in viability determination may occur due to the high affinity of the serum for trypan blue dye.