engleski

Human Limbal Epithelial Cells Cultured on Different Scaffolds

The aim of the study was to investigate the impact of electrospun polyurethane and polycaprolactone nanoscaffolds, before and after hydrolytic surface modification, on viability and differentiation of cultured human eye epithelial cells, in comparison with natural scaffolds. The scaffolds used were: amniotic membrane, fibrin, polyurethane (PU) and polycaprolactone (PCL) nanoscaffolds, with or without prior NaOH treatment. Human placenta was taken at elective caesarean delivery. Fibrin scaffolds were prepared from commercial fibrin glue kits. Nanoscaffolds were fabricated by electrospinning. Limbal cells were isolated from surpluses of human cadaveric cornea and seeded on feeder 3T3 cells. Scaffolds morphological characterization was performed using scanning electron microscopy. Human limbal cells were identified by immunofluorescence confocal laser scanning microscope, using markers for limbal stem cells p63 and differentiated cells of the cornea CK3. There was statistically significant difference in viability of cells cultured on plastic and cells cultured on all other scaffolds. Cells grown on fibrin and amnion showed higher viability compared to the electrospun nanoscaffolds. On the other hand, electrospun PU, PCL and electrospun PCL treated with NaOH had more than 80% of limbal cells positive for stem cell marker p63. SEM photographs of all tested scaffolds showed good colony spreading of seeded limbal cells. Although compared to natural carriers, the higher porosity and larger surface of nanoscaffolds and their NaOH treated versions, promised superior cell attachment and spreading, this was not the case. They showed lower cell viability compared to fibrin and plastic. On the contrary, high percentages of p63 positive cells on these scaffolds still makes them good candidates as efficient delivery systems for therapeutic purposes. Modifications in nanoscaffold surface properties should be more thoroughly tested to achieve higher level of viability and proliferation.

limbal stem cells therapy; limbal stem cell deficiency; scaffolds; electrospun scaffolds; viability

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