Morphology and physiologic responsiveness of cultured rabbit lacrimal acini

To assess the morphology and physiologic response of intact rabbit lacrimal acini maintained in culture for as long as 14 days and to compare the characteristics of the cultured acini with untreated fresh lacrimal gland, incubated lacrimal tissue slices, and freshly isolated acini.

Acini were obtained by enzymatic digestion of lacrimal glands of adults male New Zealand white rabbits and cultured for as long as 14 days on Matrigel-coated inserts in DMEM-F12. Untreated fresh lacrimal gland, incubated lacrimal tissue slices, freshly isolated acini, and intact cultured acini were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Maintenance of physiologic responsiveness of the cultured acini was assessed by the comparison of the release of protein in response to muscarinic cholinergic, alpha 1- and beta-adrenergic, and peptidergic stimulation of the cultured acini with the secretory response of incubated tissue slices.

As examined by TEM, the ultrastructure of intact acini cultured for 3 and 7 days was notably similar to fresh tissue and incubated tissue slices. Recovery of the cultured acini from perturbations induced by the digestion procedure resulted in acini that were firmly attached to the filter supports and that exhibited histotypic acinar morphology, including lumenal microvilli, apically situated secretory granules and junctional complexes, lateral desmosomes, and appropriate secretory organelles. Secretory granules and associated organelles also were prominent in 14-day cultures; however, by day 14, as visualized by SEM, the acini had flattened. TEM of acini at this time showed that they had lost much of their acinar organization and cellular polarization. Outgrowths composed of acinar cells could be observed by day 7 by both TEM and SEM, and SEM demonstrated non-acinar cells growing on the filter surface in 14-day cultures. The morphologic and physiologic response of the acinar cells to autonomic agonists was similar in incubated tissue slices and cultured acini. Morphologically, acinar cells in both preparations responded to cholinergic stimulation by releasing secretory granules, resulting in honeycombed regions in the cell. Assay of secreted protein in response to cholinergic, alpha 1- and beta-adrenergic, and peptidergic stimulation indicated that 3-day cultures of acini retain the response to carbachol, phenylephrine, and vasoactive intestinal peptide. However, the response to isoproterenol is diminished when compared with incubated tissue slices.

Under the conditions described, cultures of intact lacrimal acini from New Zealand white rabbits maintain histotypic morphology and physiologic responsiveness similar to fresh lacrimal gland and incubated tissue slices. Culture of the acini affords a useful alternative for the study of chronic, as well as acute, effects of neurotransmitters, neuropeptides, hormones, and cytokines on the structure and function of the gland.