Title
Establishing Optimal Seeding Density and Ideal Starvation Conditions of Sf9 and NIH/3T3 Cell Lines
Document Type
Grant
Publication Date
9-1-2023
Department
Biology
Abstract
Fibroblast Growth Factors (FGFs) are a family of structurally related polypeptide cytokines. 22 FGF genes (FGF1-14, FGF16-23) are found in the human genome. FGFs have various known intracrine, paracrine, and endocrine functions and are essential for the development and wound repair in organisms through these mechanisms. The role of FGFs in viruses has become an area of piqued interest in the field of pathology as several virus families have genomes that encode one or more growth factor homologues. It has been suggested that a virally encoded ortholog of FGF (vFGF) identified in the viral families of Baculoviridae and Iridoviridae is involved in the movement of these viruses across the basal lamina in the midgut of insect hosts to shift from primary infection to systemic infection (Means and Passarelli 2010). It was found that the Baculoviridae encodes a viral fibroblast homolog (vfgf) expressed as an early gene in the beginning stages of viral infection (Katsuma et al. 2006). Despite the evidence of the involvement of vFGF in cell migration, there is no published research on its role in cell proliferation, even though many FGFs are known to be mitogens. The purpose of our research is to produce recombinant FGF from two baculoviruses, AcMNPV and CfMNPV, and test their effect on cell proliferation of multiple cell lines. A part of this process includes finding the optimal seeding densities and optimal starvation periods of each cell line used. The optimal seeding density was found by seeding 96-well plates of SF9 and NIH/3T3 cells at various ranges of seeding densities correlated to their growth curves. The data was then analyzed using CCK-8 and crystal violet proliferation assays to observe where cells appeared to begin to plateau, indicating that they were overgrown. We found the optimal minimal seeding densities of SF9 cells to be about 50,000 cells/well, and about 4,000-4,500 cells/well for NIH/3T3 cells. We then took 96-well plates seeded at 3,000, 5,000, and 7,000 cells/well and treated them with media containing a range of 0-10% and 0-2% newborn calf serum to observe which concentration of serum allowed for cells to remain viable while being starved. It was found that serum containing 1% newborn calf serum allowed cells to remain the most viable during starvation at a seeding density of around 5,000 cells/well. This data will be used to set up plates of SF9 and NIH/3T3 cells to be starved and then treated with various concentrations of vFGF to observe the effects on cell proliferation.
First Advisor
Finnerty, Casey M
