April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Optimization of primary cultures of rabbit limbo-corneal cells for their use in preclinical experimentation in limbal deficiency
Author Affiliations & Notes
  • Noelia Andollo
    Cell Biology and Histology, School of Medicine and Dentistry, University of The Basque Country, BioCruces Health Research Institute, Leioa, Spain
  • Raquel Hernáez-Moya
    Cell Biology and Histology, School of Medicine and Dentistry, University of The Basque Country, BioCruces Health Research Institute, Leioa, Spain
  • Vanesa Freire
    Cell Biology and Histology, School of Medicine and Dentistry, University of The Basque Country, BioCruces Health Research Institute, Leioa, Spain
    R & D Dept., Instituto Clinico Quirurgico de Oftalmologia, Bilbao, Spain
  • Juan A Duran
    Ophthalmology, School of Medicine and Dentistry, University of The Basque Country, BioCruces Health Research Institute, Leioa, Spain
    R & D Dept., Instituto Clinico Quirurgico de Oftalmologia, Bilbao, Spain
  • Jaime Etxebarria
    Cell Biology and Histology, School of Medicine and Dentistry, University of The Basque Country, BioCruces Health Research Institute, Leioa, Spain
    Ophthalmology, University Hospital of Cruces, BioCruces Health Research Institute, Barakaldo, Spain
  • Footnotes
    Commercial Relationships Noelia Andollo, None; Raquel Hernáez-Moya, None; Vanesa Freire, None; Juan Duran, None; Jaime Etxebarria, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 517. doi:
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      Noelia Andollo, Raquel Hernáez-Moya, Vanesa Freire, Juan A Duran, Jaime Etxebarria; Optimization of primary cultures of rabbit limbo-corneal cells for their use in preclinical experimentation in limbal deficiency. Invest. Ophthalmol. Vis. Sci. 2014;55(13):517.

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

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Abstract

Purpose: In order to establish an animal model for preclinical transplantation of limbo-corneal epithelial cells for the treatment of limbal deficiency, we have tried to optimize the in vitro primary culture of rabbit cells to maintain their undifferentiated phenotype.

Methods: Stemness was measured in terms of number of cell colonies with holoclone morphology as well as immunocytochemical staining. Duplication number and viability of cells was also analyzed in each cell passage. We studied the culture of limbal explants versus enzyme-digested cells from limbal rings. Several substrates were used for culture, such as a mitomycin-inactivated feeder-layer of 3T3 murine fibroblast, amniotic membrane (AM), AM upon a 3T3 feeder-layer or cell-treated plastic. In addition, two different culture medium were assayed, a hormone-based medium with 10% FBS and the serum-free and low-calcium medium KSFM.

Results: Our results show that culture methods modify cell phenotype and influence the maintaining of cell stemness. Undifferentiated characteristics of cells are better preserved by culture of cells onto a mitomycin-inactivated feeder-layer of 3T3 murine fibroblast with the serum-free and low-calcium medium KSFM. AM favors holoclon formation but doesn’t allow cell passaging.

Conclusions: Similarly to the culture of human limbo-corneal epithelial cells, the election of culture methods is critical to maintain undifferentiated features of cells during ex vivo expansion prior to transplantation. For cell culture, we prefer the use of other substrates instead of AM, although it could be a very useful substrate for cell transplantation.

Keywords: 480 cornea: basic science • 482 cornea: epithelium • 721 stem cells  
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