Submitted on May 16, 2007
Accepted on August 12, 2007
Transepithelial Bioelectrical Properties of Rabbit Acinar Cell Monolayers on Polyester Membrane Scaffolds
Shivaram Selvam1, Padmaja B Thomas2, Hovhannes J Gukasyan3, Alan S. L. Yu4, Douglas Stevenson2, Melvin D Trousdale5, Austin K Mircheff6, Joel E Schechter7, Ronald E Smith5, and Samuel C Yiu8*
1 Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, California, United States; Ocular Surface Center, Doheny Eye Institute, Los Angeles, California, United States
2 Ocular Surface Center, Doheny Eye Institute, Los Angeles, California, United States
3 La Jolla Laboratories, Pfizer Inc., San Diego, California, United States
4 Division of Nephrology, Department of Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, United States
5 Ocular Surface Center, Doheny Eye Institute, Los Angeles, California, United States; Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
6 Physiology & Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California, United States; Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
7 Cell & Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States; Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
8 Ophthalmology, USC / Doheny Eye Institute, 1450 San Pablo Street, Room 5704, Los Angeles, California, 90033, United States; Physiology & Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
* To whom correspondence should be addressed. E-mail: syiu{at}doheny.org.
In our quest to develop a tissue engineered tear secretory system we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy (TEM) and immunofluorescence staining for Na,K-ATPase and the tight junction associated protein, occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli and junctional complexes. Na,K-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short circuit current (Isc) with a Na,K-ATPase inhibitor, ouabain (100 µM, basal-lateral, BL), and under Cl—-free buffer conditions after carbachol stimulation (CCh, 100 µM). The directional apical secretion of Cl— was demonstrated through pharmacological analysis, using amiloride (1mM, BL) and bumetanide (0.1mM, BL), respectively. Regulated protein secretion was evaluated by measuring the 
-hexosaminidase catalytic activity in the AP culture medium in response to 100 µM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl— -dependent, ouabain-sensitive AP→BL Isc in response to CCh, consistent with current models for Na+-dependent Cl— secretion.
