Purchase this article with an account.
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.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only