Purchase this article with an account.
S. Selvam, P. B. Thomas, H. J. Gukasyan, D. Stevenson, A. S. Yu, M. D. Trousdale, J. E. Schechter, A. K. Mircheff, R. E. Smith, S. C. Yiu; Bioengineering an Artificial Lacrimal Gland: Transepithelial Bioelectrical Properties of Rabbit Acinar Cell Monolayers on Polyester Membrane Scaffolds. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1883.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To demonstrate active ion fluxes across rabbit lacrimal acinar cell (RLAC) monolayers on polyester membrane scaffolds.
Purified RLACs at a density of 5 x 105/ml were seeded onto polyester membrane inserts. Confluent cell monolayers were stained with anti-occludin antibody. Ultra-thin sections were also prepared for TEM studies to evaluate the morphological properties of the cells. To evaluate the bioelectrical properties, cell monolayers with transepithelial resistances (TER) in the range of 500-1500 ohms.cm2 were studied in Ussing chambers under short-circuit conditions. Cells were stimulated with basal-lateral (BL) addition of carbachol (CCh, 100 µM). Active ion fluxes were evaluated by inhibiting the short circuit current (Isc) with a Na,K-ATPase inhibitor, ouabain (100 µM, BL) and under Cl--free buffer conditions following CCh stimulation. Regulated protein secretion was also evaluated by measuring the ß-hexosaminidase catalytic activity in the apical (AP) culture medium in response to 100 µM CCh.
TEM of sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of AP secretory granules, microvilli and junctional complexes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Cell monolayers spontaneously generated a small baseline Isc in the BL→AP direction. However, stimulation with CCh induced a large Isc (20-60 µA/cm2) in the AP→BL direction. Inhibition of BL Na,K-ATPase with ouabain completely abolished Isc. Furthermore, replacing both the AP and BL fluids with Cl--free buffer solution returned Isc back to baseline values. CCh stimulation increased AP protein secretion 3.85-fold (P<0.05) over the resting values of the cultured cells.
The generation of a Cl- dependent-, ouabain-sensitive AP→BL Isc in response to CCh demonstrates that RLACs are capable of establishing continuous epithelial monolayers that generate active ionic fluxes consistent with current models for Na+-dependent Cl- secretion. We believe the results indicate great promise for the fabrication of a fluid-secreting lacrimal gland device.
This PDF is available to Subscribers Only