April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
A Simpler Method for Lacrimal Gland Acinar Cell Isolation Culture
Author Affiliations & Notes
  • L. T. Malki
    Ophthalmology,
    Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
  • A. Dias
    Ophthalmology,
    Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
  • C. M. Modulo
    Ophthalmology,
    Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
  • W. M. Turato
    Biochemistry and Immunology,
    Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
  • C. Oliver
    Cellular Biology, Molecular and Bioagents Pathogenic,
    Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
  • E. M. Rocha
    Ophthalmology, FMRP-USP, Ribeirao Preto, Brazil
  • Footnotes
    Commercial Relationships  L.T. Malki, None; A. Dias, None; C.M. Modulo, None; W.M. Turato, None; C. Oliver, None; E.M. Rocha, None.
  • Footnotes
    Support  CAPES, FAPESP, CNPq, FAEPA
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4185. doi:
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    • Get Citation

      L. T. Malki, A. Dias, C. M. Modulo, W. M. Turato, C. Oliver, E. M. Rocha; A Simpler Method for Lacrimal Gland Acinar Cell Isolation Culture. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4185.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Lacrimal gland acinar cells primary culture has been used to understand the secretory mechanisms and the physiopathology of dry eye syndrome. Multiple steps and time-consuming protocols for cells isolation have been published. The aim of this study was to describe changes in the isolation method of lacrimal gland acinar cell conditions and to evaluate impact on cell number, morphology and viability.

Methods: : Acinar cells of the lacrimal gland of male Wistar rats were isolated with DMEM supplemented with 1 mM putrescine, 10 µg/ml reduced glutathione, 10 µ/ml dexamethasone, a mix of insulin-transferrin-sodium selenite (5 µg/ml: 5µg/ml: 5ng/ml), 10 ng/ml EGF, and 10% FBS. The tissue was mechanically separated with scissors, enzymatic digested with hyaluronidase, collagenase and DNAase, filtered in 100 and 70 µm cell strainer, and allowed sedimentation in supplemented DMEM and gently cell spinner between those steps. The number, viability and cell morphology were evaluated with Neubauer chamber and microscopy; and positivity for aquaporin-5 on flow cytometry.

Results: : Cell number obtained from exorbital lacrimal gland was 8.4 ± 1.1 x 10 5, viability was 92.2 ± 4.1 %. At the first hours cell shape were round and they tended to group. Cells positive for aquaporin-5 were identified in flow cytometry assay.

Conclusions: : The present work describes a method for lacrimal gland acinar cell isolation whereas several steps as repeated washes, Ficoll gradient and hyperoxygen or warm water incubations were reduced or replaced by simpler steps. The data reveals that it could be reproducible and efficient as previously described methods. It will be useful for future studies on primary acinar culture cells addressing physiopathological mechanisms involved in dry eye syndrome.

Keywords: lacrimal gland • flow cytometry 
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