Organ Culture of Human Main and Accessory Lacrimal Glands and their Secretory Behaviour

Abstract

The purpose of this study was to investigate the in vitro viability and secretory behaviour of human main and accessory lacrimal glands using an organ culture technique. We evaluated the influence of the second messengers cAMP and cGMP on secretion. Fragments less than 1mm 3of main and accessory lacrimal glands as well as conjunctiva were cultured for 2–72hr at 37°C in an atmosphere consisting of 50% O 2, 45% N 2and 5% CO 2, using a specially devised culture medium (±cAMP or cGMP). The conjunctival tissue served as negative control. Supernatants were assayed for secretory-component-bound IgA, lactoferrin and lysozyme using ELISA. Cultured tissue pieces were embedded in paraffin, serially sectioned, stained and their volumes calculated using an image-analysis system. This enabled us to differentiate between secretory, connective and fatty tissue. Secreted exudate was correlated to the volume of secretory tissue. Viability of cultured organ pieces was determined by electron microscopic examination. Suitable organ culture conditions for human lacrimal glands were successfully established. Electron microscopic examinations proved that the structural characteristics of the organ and the polarity of the individual cells were well preserved up to 22 days of culture. Culture supernatants were assayed for secretory-component-bound IgA, lactoferrin, and lysozyme and showed that the amount of protein secreted increased with time. Upon addition of cAMP (1×10 -3 m) and cGMP (4×10 -3 m), secretion was elevated in both main and accessory lacrimal glands. An organ culture system for lacrimal glands was developed that maintains their structural and cellular characteristics as well as their secretory function for up to 22 days. We believe that this system mimics the in vitro state of the organ better than monolayer cultures and thus proves to be a valuable tool when examining lacrimal function in vitro. The fact that both cAMP and cGMP enhance secretion may help to shed some light on the cellular pathways human main and accessory lacrimal glands use for signal transduction.