Rat pancreatic interlobular duct epithelium: isolation and culture in collagen gel

Interlobular duct fragments from the pancreas of the rat were isolated by collagenase digestion and filtration, embedded in a matrix of rat-tail collagen, and cultured in a 1:1 mixture of Dulbecco's minimal essential and Ham's F12 media supplemented with cholera toxin (CT, 100 ng/ml) and epidermal growth factor (EGF, 10 ng/ml) in addition to supplements used previously, thereby improving the yield of ducts by a factor of two compared with previous results. The ducts were harvested by digestion of the collagen matrix with collagenase and were then dissociated by treatment with EDTA in divalent cation-free salt suspended in collagen and cultured as were the ducts. Numerous cysts appeared as a function of time and some of these enlarged dramatically. Some of the larger cysts exhibited secondary tubular processes extending into the surrounding collagen. The addition of bovine pituitary extract (BPE, 50 micrograms/ml) doubled the number of cysts, whereas omission of serum or CT + EGF reduced the number. BPE or forskolin could substitute effectively for CT. Agents that stimulate (secretin) or inhibit (e.g., ouabain or acetazolamide) fluid-electrolyte secretion in vivo had no effect on the number or average diameter of the cysts. The cysts were 83 to 88% epithelial with the balance of the cells being fibroblastic in appearance. Some cysts consisted only of epithelium. The proliferative capacity of the cystic epithelium was shown by the presence of mitotic figures and by an autoradiographic labeling index of 22 to 30% after a 24-h exposure to [3H]thymidine. The labeling index was reduced by the omission of CT + EGF. Transmission electron microscopy showed that the cysts exhibited morphologic features of duct epithelium in vivo, including apical microvilli, lateral interdigitations of the plasma membrane, and typical cytoplasmic organelles.