Chapter 21 Culture of Human Pancreatic Ducts

METHODS IN CELL BIOLOGY,VOLUME ZIB

Chapter 21 Culture of Human Pancreatic Ducts RAYMOND T. JONES

AND BENJAMIN

F. TRUMP

Department of PatboZogy and Matykand Institute for Emergency Medical Services, University of Matykand Scbool of Medicine, Baltimore, Maryland

GARY D. STONER Human Tissue Studies Section, Laboratmy of Experimental Patbology, N a t i w l Cancer Institute, Betbesda, Maryland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . .

A. Ductal Explants . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ductal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Xenotransplantation Studies . . . . . . . . . . . . . . . . . . . . . 111. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. E x p l a n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1V.Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Current Applications and Findings . . . . . . . . . . . . . . . . . . B. Future Studies . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction There is a paucity of studies involving cultured human pancreatic ductal and exocrine tissues in the literature. A few attempts have been made to establish pancreatic cell lines from fetal tissue (Hay, 1975; Wellman et al., 1975) and 429 Copyright 0 1980 by Academic Ress, Inc. All rights of reproduction in any fom reserved. ISBN 0-12-561140-0

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Carrel and Lindberg (1938) described in vitro culture of cat pancreas for 4 days. The development of several clonal lines of epithelial cells from adult bovine pancreatic duct was described by Stoner et al. (1978). The small number of studies involving cultured human pancreatic tissues as well as pancreatic tissues from experimental animals is perhaps due to the misconception that pancreatic tissue is extremely labile, undergoing autolysis at a very rapid rate which therefore makes it difficult to obtain and culture pancreatic tissues. However, a study by Jones and Trump (1975) demonstrated that rat pancreatic slices maintained ultrastructural evidence of viability for up to 3 hours under complete ischemic conditions at 37°C. This study led to the development of a bovine pancreatic ductal system which was the model for the successful long-term culture of human pancreatic ductal explants (Jones et al., 1977). The present chapter will discuss the culturing of ductal elements of the human pancreas (culture of human islets is discussed in Chapter 6).

Materials and Methods

11.

A. Ductal Explants 1. SOURCE Human pancreas was obtained either at immediate autopsy (Trump et al., 1975), at routine autopsy (within 3 hours of death), or at surgery and returned to the laboratory. Under sterile conditions and using sterile techniques, the main duct was opened the entire length of the pancreas, dissected out and placed in cold L-15 [Grand Island Biological Co. (GIBCO), Grand Island, NY] medium. Some interlobular ducts remained attached to the main duct and were removed with it. 2.

CULTURE CONDITIONS

Aftter the glandular part of the pancreas was dissected away from the duct, the duct was cut into pieces approximately 1 cm long. These pieces were placed in 60-mm plastic petri dishes (Falcon Plastics, Oxnard, CA). Three pieces of duct were placed with the epithelial surface toward the gas-medium interface in each dish and 5 ml of supplemented CMRL 1066 (GIBCO, Grand Island, NY) was added. The CMRL 1066 was supplemented with 1 p g insulidml, 0.1 p g hydrocortisone hemisuccinate/ml, 2 mM L-glutamine, 100 U penicillin G/ml, lOOpg streptomycidml, and 5% heat-inactivated fetal bovine serum. The petri dishes were maintained in a controlled atmosphere chamber (Bellco Glass Co.,

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Vinland, NJ) which was gassed with a mixture of 45% 02,50% Nz, 5% CO,. The chambers were placed on rocker platforms (Bellco Glass Co., Vinland, NJ) in a 37°C incubator and rocked 10 timedminute. All explants and isolated cells (see Section II,B) were treated for morphologic study by techniques previously described (Jones et al., 1977).

B.

Ductal Cells 1.

SOURCE A N D ISOLATION

Three organs removed at immediate autopsy were used for the isolation of human pancreatic ductal cells. Two isolation techniques were used, one a modification of a procedure for isolation of bovine pancreatic ductal cells described by Jones er al. (1979). This technique is as follows: The main pancreatic duct was cannulated at both the head and tail with an 18-gauge needle at the head and a 22-guage needle at the tail. Both needles had the pointed ends removed. Pieces of silicon tubing were connected to the needles through which 100 ml of Eagle's minimal essential medium (MEM-F- 14 GIBCO, Grand Island, NY) was perfused through the duct (inlet at the tail, outlet at the head) to wash cell debris and mucus from the duct as well as to locate branches of the duct which needed to be clamped with hemostats. Following the wash, 50 ml of enzyme solution 0.1% dispase in Hank's balanced [0.1% Worthington Type 111 collagenase salt solution (GIBCO)] was continually perfused through the duct for 40 minutes. The enzyme solution was then washed out of the duct with 150 ml of MEM-F-12 cell culture medium (GIBCO, Grand Island, NY) and collected. The cells were then centrifuged at 600 g for 5 minutes and washed twice with MEM-F- 12. The second technique to isolate human pancreatic ductal epithelial cells was by permitting epithelial cells to grow out from explants onto culture dishes and removing the explants from the dishes before outgrowth of fibroblasts. In this manner, epithelial cultures could be obtained which were relatively free of fibroblasts.

+

2.

CULTURE CONDITIONS

The cells were grown in 250-ml Falcon plastic flasks containing 12 ml of MEM-F- 12 supplemented as follows: 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G, 100 pg/ml streptomycin, 0.25 p/ml fungizone, 1 pg/ml insulin, 0.292 mg/ml L-glutamine, 2.2 gdliter NaHC03, and 10 mM HEPES buffer. The cells were maintained at 37"C, 95% relative humidity with 5% C 0 2 in air.

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C. Xenotransplantation Studies Human pancreatic ductal explants were cultured for 1 week under conditions described in Section II,A,2 prior to xenotransplantation into athymic “nude” mice. Since this is the subject of a manuscript in preparation by Valerio (1980), no further methods will be described in the present chapter.

111. Results

A . Explants Human pancreatic duct obtained for explant culture consists of the main duct which is composed of columnar epithelial cells (Fig. 1) as well as a few secondary ducts which are attached to the main duct. By transmission microscopy, ducts obtained at autopsy or surgery show evidence of cell injury (Fig. 2). The dilated endoplasmic reticulum, swollen mitochondria, clumped chromatin, and blebbing of the plasma membrane (all examples of reversible cell injury) were all repaired by the culture conditions (Fig. 3). However, numerous autophagic vacuoles were seen in epithelial cells of the explants following 3 weeks in culture (Fig. 4). Alcian-blue/periodicacid-Schiff (PAS)-positivematerial could still be seen in epithelial cells of the explants and in lumens of the secondary ducts at 5 weeks in culture (Fig. 5 ) . Any acinar or islet cells which were attached to the main duct prior to the culturing of the explants were lost during culture. Ductal epithelium grew over the necrotic tissue as well as onto the petri dish. This could be seen quite dramatically by scanning electron microscopy (Fig. 6 ) . Viable human pancreatic ductal cells were maintained in vitro by means of this explant procedure for up to 2 months (Fig. 7). Human pancreatic ductal explants have been successfully xenotransplanted into athymic “nude” mice. The success of this technique can be seen in Fig. 8, a plastic embedded section of a human pancreatic duct after 123 days in a nude mouse. There is excellent preservation of the architecture of the duct. Normal appearing epithelium with goblet cells have been maintained in this system.

B . Cells To date, only preliminary data have been obtained from ongoing studies of human pancreatic duct cells in culture. Cells isolated by the digestion technique described and grown in tissue culture for 18 days are shown in Fig. 9. These epithelial-like cells have material suggestive of mucus present in the culture. We have also cultured human pancreatic ductal epithelial-like cells that grew

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FIG I . Scanning electron micrograph (SEM) of a piece of human pancreatic duct (HPD) fixed at immediate autopsy. Note the blebbing of some of the columinar epithelial cells and areas where cells have sloughed. Courtesy of Dr.H. Sanefugi. FIG.2. Transmission electron micrograph (TEM) of a HPD fixed prior to culture. Cells show evidence of reversible injury: dilated endoplasmic reticulum, swollen mitochondria, blebbing of the plasma membrane, and chromatin clumping.

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Fic. 3. HPD maintained in explant culture for 8 days. Cell injuries similar to that seen in Fig. 2 have been reversed by the culture conditions. FIG.4. HPD cultured for 3 weeks. Numerous autophagic vacuoles are present (mows).

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FIG. 5 . Light micrograph of a paraffin-embedded alcian bluePAS-stained section of a HPD which was cultured for 5 weeks. Alcian blue-positive material is on the apex of the ductal epithelial cells as well as within a lumen of a secondary duct. FIG.6. SEM of a HPD explant also cultured for 5 weeks. The epithelial cells have migrated over the substrate. Numerous microvilli and cilia can be seen in the epithelial cells. Courtesy of Dr. H. Sanefugi.

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FIG. 7. HPD cultured for 2 months. Note that the organelles are within normal limits. FIG. 8. Plastic-embedded toluidine blue-stained section of a HPD which was xenotransplanted into a nude mouse for 123 days. The ductal epithelial cells show excellent preservation of their normal characteristics. Note the presence of goblet cells. Courtesy of Dr. M. G . Valerio.

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FIG. 9. Phase contrast micrograph of HPD epithelial-like cells that have been isolated by techniques described in the text and maintained in primary culture for 40 hours. FIG. 10. Phase contrast micrograph of HPD cells that migrated onto a pehi dish from an explant.

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out from explants in primary culture. These cells are similar in appearance (Fig. 10) to those which were obtained by the digestion technique.

IV.

Discussion

The observations presented here demonstrate that it is possible to culture human pancreatic ductal tissues obtained at autopsy within 4 hours of somatic death for extended periods since many injuries to the tissues are reversed by the culture conditions. Pancreatic ductal explants maintain excellent ultrastructural preservation for up to 60 days and we feel that it is possible to culture human pancreatic duct for at least twice this long since we have been able to culture bovine pancreatic ductal explants for longer than 4 months. The present studies have also demonstrated the feasibility of obtaining epithelial-lke cell cultures from human pancreatic duct by either an enzyme digestion technique or from outgrowths from explant cultures. Although these cell isolation and culture studies are preliminary, we feel these techniques will enable one to obtain cell cultures of pancreatic ductal epithelial cells for a variety of investigations.

V. A.

Perspectives

Current Applications and Findings

The utilization of human pancreatic ductal tissue in long-term organ and cell cultures has made it possible to study the effects of chemical carcinogens on this important target tissue by our laboratories (Jones et at, 1977; Harris et al., 1977), and hopefully these techniques will make it possible to study the pathogenesis of human pancreatic cancer under in vitro conditions.

B.

Future Studies

It is our opinion that both explant and cell culture methods for human pancreatic ducts described in this chapter make it possible for functional studies to be carried out in order to determine the role of these tissues in disease processes and in the normal physiology of digestion. For example, the preparations that are discussed in this chapter could be used to study diseases such as cystic fibrosis. The ease with which human pancreatic ductal explants can be xenotransplanted into nude mice and the excellent maintenance of normal cellular

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structure also makes it possible to utilize this technique to study the pathophysiology of human pancreatic ducts. It is also our opinion that the methodology now exists for culturing human pancreatic exocrine tissue (as single cells or as acini) for several weeks. Rat acinar cells have been isolated and cultured by Oliver (1978) for up to 4 weeks. Hopefully, the long-term culture of human exocrine pancreatic tissue with preservation of secretory function will soon be a reality.

ACKNOWLEDGMENTS These studies were supported in part by Public Health Service Contracts NO 1-CP-43237 and NO I-CP-75947 from the Division of Cancer Cause and Prevention, National Cancer Institute. This is publication #774 from the Department of Pathology of the University of Maryland School of Medicine.

REFERENCES Carrel, A,, and Lindberg, C. A. (1938). “The Culture of Organs,” p. 151. Hoeber, New York. Harris, C. C., Autrup, H., Stoner, G., Wang, S. K., Leutz, J. L., Gelboin, H. V., Selkirk, J. K., Connor, R. J., Barrett, L. A., Jones, R. T . , McDowell, E. M., andTrump, 9 . F. (1977). Cancer Res. 37,3349. Hay, R. 1. (1975). Adv. Exp. Med. Biol. 53, 23. Jones, R. T., Barrett, L. A , , van Haaften, C., Harris, C. C., and Trump, 9. F. (1977). J. Null. Cancer Inst. 58, 557. Jones, R. T., and Trump, 9. F. (1975). Virchows Arch. B. Cell Pathol. 19, 325. Jones, R. T., Lakshmanan, M., and Stoner, G. D. (1979). In Vitro 15, 215. Oliver, C. (1978). J. Cell Biol. 79, 68a. Stoner, G. D., Hams, C. C., Bostwick, D. G., Jones, R. T., Trump, 9. F., Kingsbury, E. W., Fineman, E., and Newkirk, C. (1978). In Virro 14, 581. Trump, B. F., Valigorsky, J. M., Jones, R. T., Mergner, W. J., Garcia, J. H., and Cowley, R. A. (1975). Hum. Purhol. 6, 499. Valerio, M. G. (1980). Unpublished studies. Wellman, K., Amsterdam, D., and Valk, B. (1975). Arch. Pathol. 99, 424.