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Denise Loya, Eduardo Camacho-Martinez, Judith Zavala, Julio C Hernandez, Jorge E Valdez; Enhancing pterygium fibroblasts culture: a serial explant technique using extracellular matrix coating. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3939.
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© ARVO (1962-2015); The Authors (2016-present)
The lack of commercial cell lines and the need of primary tissue explants to culture pterygium fibroblasts are obstacles in the study of pterygia. The use of extracellular matrix for culture optimization with encouraging results has been described for epithelial cells but not for fibroblasts. The aim of this study is to analyze the feasibility of a serial explant culture technique using extracellular matrix coating as a viable option for enhancing human pterygia fibroblasts culture for its subsequent use in research
Pterygium tissue was obtained from the pathology department of Hospital San José Tecnológico de Monterrey (Monterrey, México) and fractionated into small pieces of approximately 3mm2 each. Tissue was cultured in a 24 well microplate previously coated with 20µl of extracellular matrix based hydrogel (Matrigel, Corning), and just enough culture medium D-MEM F-12 (Gibco, Ref: 12400-016) to cover the explant and the extracellular matrix. Explants were incubated under standard conditions (37 C, 5% CO2) with medium being refreshed twice per week. Once cells were confluent subculture and explant transfer was done. Cells from each well were subcultured in 3 different wells in a new coated 24 well microplate. Explants were also transferred to new 24 well microplates coated with extracellular matrix based hydrogel
After the primary culture, a total of four explant transfers were done. Confluence was achieved 27 days later for the primary culture, 4 weeks later for the first explant, 3 weeks later for the second and third explant transfers, and 4 weeks later for the fourth explant transfer. Subcultures were made for each plate once 80% confluence was achieved on a 1:3 basis, with subsequent passages as were needed. These passages showed decreased time period till confluence as they were expanded
The use of serial explants cultured with extracellular matrix coating proved to be a feasible way of increasing the number of cells that can be obtained from a single pterygium tissue sample. Fibroblastic cells obtained using this culture technique allowed preservation of cell morphology, replication rate and growth pattern in the explant transfers and subcultures. Further molecular characterization of pterygium fibroblasts will validate the stability of the obtained cells for their subsequent use in basic and translational research.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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