Abstract
Purpose :
Primary pterygium fibroblasts are required for translational studies. Therefore, it is critical to establish optimal culture conditions for their expansion. Using an experimental model in vitro, we tested the effect of different Fetal Bovine Serum (FBS) concentrations on the cell proliferation of primary subculture of human pterygium fibroblasts.
Methods :
A primary subculture of human pterygium fibroblasts was expanded in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 5% FBS and 1% Penicillin-Streptomycin. At confluence, the culture was trypsinized and cells were seeded at a concentration of 600k cells per T25 flask. After 24 hours, medium was changed to DMEM/F-12 supplemented with different FBS concentrations (0%, 5%, 10% and 15%). Cell counts were registered in triplicates at day 2,3 and 4 post supplementation. In order to determine the real proliferative effect of FBS, our statistical analysis was performed using unpaired T student test after subtracting the proliferation due to other medium factors (0% FBS cultures). Morphological changes were documented at 10x magnification using an inverted microscope.
Results :
Cultures supplemented with 15% FBS showed less proliferation when compared to 5% and 10% FBS cultures at day 2 (p-value=0.014). At day 3 post supplementation, the positive proliferative effect seen in 5% FBS cultures was statistically significant compared to 10% and 15% FBS cultures (p-value=0.003). However, there was no significant difference between the three FBS concentrations at day 4 post treatment. Moreover, our morphological evidence showed that the higher FBS concentration, the higher confluence was reached.
Conclusions :
We found that an optimal human pterygium fibroblasts proliferation can be achieved using 5% FBS at day 3. Although no significant difference was found at day 4, the increasing confluence could be explained by the combination of contact inhibition and a hypertrophic effect. Image analysis will corroborate these possible morphological changes. Further assays are being conducted with human pterygium fibroblasts and non pathological fibroblastic cell lines to confirm our findings. These preliminary results will allow us to optimize the culture of pterygium fibroblasts for future research.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.