Monolayer culture of rat parotid acinar cells without basement membrane substrates

Abstract

Acinar cells have been difficult to maintain in primary or secondary cultures over extended periods of time. The most successful monolayer culture system reported to date requires basement membrane substrates. We report here a technique for culture of rat parotid acinar cells which does not rely upon basement membrane supports for maintenance and growth. The procedure involves gland excision, treatment to chelate metal ions, enzymatic digestion with collagenases and hyaluronidase, removal of fat and red blood cells by gravimetric separation, and nylon mesh filtration to yield a homogeneous suspension of small aggregates and single cells. The cells were examined for: a) morphology, identity, and growth; b) macromolecular synthesis; and c) secretory output. They were healthy, peroxidase positive, and growing for up to 10 d. Protein synthesis increased from the point of cell layer formation at 3 to 4 d, through 10 d, while DNA synthesis decreased. As in other studies, amylase secretion fell sharply between 2 and 4 d in culture and remained low. Although previous studies indicated that the initial isolation protocol left these acinar cells unable to thrive in monolayer culture except in the presence of basement membrane substrates, the modified technique reported herein allows these cells to attach, spread, and grow on a wide variety of commercially available plasticware. This method lends itself readily to long-term analysis of rat parotid acinar cell metabolism without the complications of dedifferentiation, cell loss through culture manipulation common in suspension cultures, or complex interactions between bioactive supports and cell surfaces.