Rat pancreases were minced and treated with collagenase or collagenase supplemented with chymotrypsin to yield a mixture of ducts, islets, acinar cell clusters, blood vessels, and nerves . Histologically and ultrastructurally, the isolated tissues resembled their in situ counterparts in most respects, the major difference being the destruction of the basement membranes (basal laminae) . Ducts ranging in size from the common bile/main pancreatic duct to the intercalated ducts were identified in the digest, although interlobular ducts were most frequently observed .Acinar tissue fragments were separated from nonacinar structures either by flotation through discontinuous gradients of Ficoll or by sieving, the latter technique being the more efficient . Common bile/main ducts, interlobular ducts, and blood vessels were selected manually from the nonacinar fractions . Biochemical analyses showed that the entire nonacinar fraction, as well as isolated ducts and blood vessels, contained larger alkaline phosphatase, carbonic anhydrase, and Mg-ATPase specific activities than acinar tissue, whereas acinar tissue contained larger y-glutamyltranspeptidase and amylase activities . However, >63% of the total recovered activity of each enzyme was associated with the acinar tissue . Both the association of the majority of each of these enzyme activities with the acinar tissue and the similarity in specific activities associated with ducts and blood vessels indicate that none of the enzymes tested is a unique marker for interlobular and larger ducts of the pancreas of the rat.Pancreatic ducts comprise a simple cuboidal to columnar epithelium (12,16,21) and a layer of connective tissue . The ducts serve as a conduit for the movement of exocrine enzymes from the acinar cells to the duodenum and they also produce at least part of the secretin-stimulated bicarbonaterich pancreatic fluid secretion (9, 44) . The role of the ducts in fluid/electrolyte secretion has been supported by ultrastructural (24) and micropuncture (29, 43) studies and by studies of the relatively unimpaired fluid/electrolyte secretion in rats whose acinar cells have been destroyed (14) .Carbonic anhydrase in the dog (4), (Na+K)-ATPase in the rabbit (40, 48), and HC03-ATPase in the dog and cat (45) have been implicated in pancreatic fluid secretion by experiments in which specific inhibitors (acetazolamide, ouabain, and thiocyanate, respectively) reduced fluid secretion by the intact pancreas . Histochemical studies have demonstrated the presence in the duct epithelium J. CELL BIOLOGY C The Rockefeller University Press

Interlobular and intralobular ducts isolated from the pancreas of the rat by digestion with collagenase and chymotrypsin were cultured in an agarose matrix containing CMRL-1066 supplemented with insulin, dexamethasone, L-glutamine, soybean trypsin inhibitor, antibiotics, and fetal bovine serum. The cut ends of most interlobular ducts sealed to create enclosed lumina. Some ducts retained their original cylindrical organization; others enlarged to varying degrees, resulting in structures that ranged from cylindrical to spherical in shape. The duct walls consisted of viable epithelium and connective tissue, although the amount of connective tissue declined with age. Both epithelial and connective tissue cells became flattened in the enlarged ducts. Intralobular and small interlobular ducts often remained associated with the larger interlobular ducts. These duct fragments have been cultured for as long as 6 weeks.

Rat and hamster pancreatic ducts were isolated by digestion with collagenase plus chymotrypsin and were cultured for eight weeks in an agarose matrix. Freshly isolated and cultured ducts were characterized morphologically and biochemically. The in vivo morphology of the ducts was maintained in vitro, although certain differences were noted. Both interlobular and intralobular ducts could be identified. gamma-Glutamyltranspeptidase and Mg-ATPase were stable enzymatic activities of the ducts of both species; alkaline phosphatase persisted only in the hamster ducts. Carbonic anhydrase and (Na + K)ATPase were minor activities of the rat ducts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the rat ducts suggested that actin was the major duct peptide and that the major zymogens were greatly diminished. These results demonstrate that pancreatic ducts can be maintained in vitro and can be used for biochemical studies of this minor pancreatic tissue type.