RT info:eu-repo/semantics/article
T1 Successful consecutive expansion of limbal explants using a biosafe culture medium under feeder layer-free conditions
A1 López Paniagua, Marina
A1 Nieto Miguel, Teresa
A1 De La Mata Sampedro, Ana
A1 Galindo de la Rosa, Sara
A1 Herreras Cantalapiedra, José María
A1 Corrales, Rosa María
A1 Calonge, Margarita
K1 Cell culture; Culture media; Limbal cells; Ocular surface; Stem cells
AB Purpose: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers.Materials and methods: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence.Results: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished.Conclusion: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.
PB Taylor and Francis Ltd.
SN 0271-3683
YR 2017
FD 2017
LK https://uvadoc.uva.es/handle/10324/64741
UL https://uvadoc.uva.es/handle/10324/64741
LA eng
NO Current Eye Research. 2017;42(5):685-695
NO Producción Científica
DS UVaDOC
RD 24-nov-2024