May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Bioengineering an Artificial Lacrimal Gland: Transepithelial Bioelectrical Properties of Rabbit Acinar Cell Monolayers on Polyester Membrane Scaffolds
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
Author Affiliations & Notes
  • S. Selvam
    Ocular Surface Center, Doheny Eye Institute, Los Angeles, California
    Mork Family Department of Chemical Engineering and Materials Science, USC, Los Angeles, California
  • P. B. Thomas
    Ocular Surface Center, Doheny Eye Institute, Los Angeles, California
  • H. J. Gukasyan
    La Jolla Laboratories, Pfizer Inc., San Diego, California
  • D. Stevenson
    Ocular Surface Center, Doheny Eye Institute, Los Angeles, California
  • A. S. Yu
    Medicine,
    Keck School of Medicine, USC, Los Angeles, California
  • M. D. Trousdale
    Ocular Surface Center, Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine, USC, Los Angeles, California
  • J. E. Schechter
    Ophthalmology,
    Cell and Neurobiology,
    Keck School of Medicine, USC, Los Angeles, California
  • A. K. Mircheff
    Ophthalmology,
    Physiology and Biophysics,
    Keck School of Medicine, USC, Los Angeles, California
  • R. E. Smith
    Ocular Surface Center, Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine, USC, Los Angeles, California
  • S. C. Yiu
    Ocular Surface Center, Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine, USC, Los Angeles, California
  • Footnotes
    Commercial Relationships S. Selvam, None; P.B. Thomas, None; H.J. Gukasyan, None; D. Stevenson, None; A.S. Yu, None; M.D. Trousdale, Doheny Eye Institute, P; J.E. Schechter, None; A.K. Mircheff, Doheny Eye Institute, P; R.E. Smith, Doheny Eye Institute, P; S.C. Yiu, Doheny Eye Institute, P.
  • Footnotes
    Support Supported by EY15457, EY03040, EY10550, EY12689, DK062283 and grant from RPB. CR: None
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1883. doi:
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      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.

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Abstract

Purpose:: To demonstrate active ion fluxes across rabbit lacrimal acinar cell (RLAC) monolayers on polyester membrane scaffolds.

Methods:: 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.

Results:: 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.

Conclusions:: 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.

Keywords: lacrimal gland • cornea: tears/tear film/dry eye • electrophysiology: non-clinical 
© 2007, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2025 Association for Research in Vision and Ophthalmology.
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