Primary culture of avian pancreatic acinar cells for heterologous gene expression

Dear Editor: Cell lines established from pancreatic tumors have provided model in vitro systems to study the cellular and molecular mechanisms controlling pancreatic activity (Frazier and Longnecker, 1993; Oda et al., 1998). One of the best-characterized pancreatic lines is AR4 2J, which synthesizes proteolytic enzymes characteristic of acinar cells and is derived from a primary carcinoma of the rat pancreas (Jessop and Hay, 1980). Although widely utilized in cellular, physiological, and molecular studies the cell lines, including AR4 2J, are all mammalian in origin, and as yet there are no pancreatic cell lines which allow comparable studies in other species. Work in our laboratory has focused on manipulating the pancreas in vivo to increase the repertoire of enzymes synthesized and secreted into the gastrointestinal tract (Hall et al., 1993; All et al., 1996, 1997). The aim is to introduce novel digestive enzymes, including cellulases and hemicellulases, which will allow simplestomached livestock, especially poultry, to benefit directly from diets high in plant structural polysaccharides. To date these studies have been performed in mammalian hosts and similar manipulations in other species, such as the avian, have been impeded by the lack of appropriate pancreatic cell lines. Here we report the establishment of primary cultures of avian pancreatic cells. The avian cells have been characterized as exocrine by their acinar morphology and by the synthesis of amylase. A transient transfection system has been established to introduce DNA encoding a bacterial glycosidase into the cells and to investigate transgene expression. These cells provide an in vitro model with which to study the molecular and cellular mechanisms controlling the expression of avian pancreatic genes, the genetic manipulation of the avian pancreas, and the molecular and cellular effects resulting from such manipulations. Pancreatic tissue was dissected from 25 chick (Ross broiler) embryos, sacrificed by cervical dislocation at 18 d of gestation, and placed in Krebs buffer, containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid and 5 mM glucose, at 37 ~ C. To recover the pancreatic cells the tissue was incubated at 37 ~ C in the above buffer, supplemented with bovine serum albumin (40 rag/ ml), collagenase 0.1% (w/v), and sterilized by filtering through 0.45I. tm filter (Millipore, Watford, PA), for 1 h with gentle shaking. The cells were collected by centrifugation, washed, and suspended in culture medium (Minimal Essential Medium [MEM]; GIBCO-BRL, Paisley, UK) containing penicillin (100 U/ml), streptomycin (100 Ixg/ml), glutamine (4 mM), and 10% chick serum (Sigma, Poole, UK). Cells were seeded into 60-mm culture dishes (3 • 104 per dish), precoated with polylysine (mol. wt. >300,000). After allowing the cells to attach for 1 h, fresh medium was added and the cells incubated at 40 ~ C in an atmosphere of 5% CO2/air. Medium was changed after 24 h to MEM-D-Val (GIBCO-BRL). Cells were harvested/passaged using 0.025% trypsin, 0.02% ethylenediamine-tetraacetic acid treatment for 10 min at a split ratio of 1:3. The avian pancreatic cells attached and grew in a primary culture on polylysine-coated surfaces. On uncoated surfaces the cells did not attach, and on collagen-coated surfaces attachment was reduced by up to 50%. The cells grew in clusters rather than as a monolayer. Cell morphology was very similar to that seen in the AR4 2J cell line (Fig. 1A and B). In preliminary cell isolations, cells with apparent fibroblast characteristics quickly appeared and overgrew the pancreatic cells. In subsequent isolations, these cells were reduced by employing medium containing D-Val, which enables the growth of normal epithelial cells while selectively inhibiting fibroblast proliferation. The growth of the cells was maintained by the use of chick serum and by culturing at 40 ~ C, reflecting the normal physiological temperature of the chicken (41.6 ~ C). The cells were successfully passaged two to three times after which they stopped dividing and started to detach. Using standard immunohistochemical techniques, the avian cells were shown to he of acinar type by their ability to synthesize amylase (Fig. 1C and D). Measurement of amylase activity revealed that the level of enzyme activity was reduced compared to an immortal line AR4 2J (Table 1). The reduced enzyme levels may reflect the embryonic origin of the avian cells and]or the levels of amylase synthesized in the avian compared to the rat. Amylase activity was significantly reduced at passage three, consistent with the degeneration of the primary cells. To investigate whether the avian cells could be engineered to synthesize heterologous proteins, the primary cells were transfected with plasmid DNA containing the ceIE' gene (Fig. 2), which encodes a bacterial endoglucanase, EGE'. The presence of celE' mRNA was detected by reverse transcription-polymerase chain reaction (RTPCR) as evidenced by a DNA band of 0.75 kb (Fig. 3, track 1), following analysis of the RT products by agarose gel electrophoresis. No expression was detected when the cells were subjected to PCR