Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction
Authors
Zala Luzˇnik . Claudia Breda . Vanessa Barbaro . Stefano Ferrari . Angelo Migliorati . Enzo Di Iorio . Barbara Ferrari . Carlo Griffoni . Andrea Grassetto . Hossein Mostafa Elbadawy . Marina Bertolin
Institution
Eye Hospital, University Medical Centre, Department of Molecular Medecine, University of Padua & Department of Pharmacology and Toxicology , College of Pharmacy, Taibah University
Country
Slovenia
Year
2017
Journal
Cell and Tissue Banking
Abstract
Isolated limbal epithelial stem cells (LESCs)
were cultured with or without a 3T3 murine fibroblast
feeder-layer (FL) in 4 different culture media on culture
plates or on denuded human amniotic membrane (AM)
support and fibrin gel support: (1) control medium
supplemented with fetal bovine serum; (2) control
medium supplemented with the synthetic serum ‘‘XerumFree
TM XF205’’ (XF); (3) CnT-20 medium supplemented
with ‘‘XerumFreeTM XF205’’ (CnT-XF) and (4)
CnT-20 medium supplemented with human AB serum
(CnT-AB). The three xenogeneic media were compared
to standard condition (control ? FL) and parameters
assessed included cell morphology, proliferative potential,
number of passages, assessment of clonogenic and
abortive colonies, life span, DNp63a expression and
epithelial morphology on AM. During serial cultivation
of LESCs, most of the tested xeno-free media supported
similar numbers of cell passages, total colony number,
cumulative cell doublings (CCD) rates and expression of
DNp63a compared to control. The conditions cultivated
with a FL showed a non-statistically significant higher
number of cell passages and CCD rates before senescence
when compared to the same conditions cultured
without FL. Except for the control medium, only XF
medium enabled the growth of cells on AM. The
expression of DNp63a was comparable in all the cultures
grown ontoAM, when compared to the controls on fibrin
gel. In conclusion, the xeno-free media enabled LESC
culture both on plastic and on denuded human AM.
Despite the analyses were carried out in a statistically
low number of samples and need re-assessment in a
larger cohort, our results suggest that the production of a
completely xeno-free LESC graft could be beneficial for
future clinical applications.