Novel Engineered Basic Fibroblast Growth Factor Improves Stability and Enables Improved Cell Culture Outcomes

Basic fibroblast growth factor (bFGF, also known as FGF2) is widely used in vitro for the maintenance and stimulation of a variety of cells. However, use of native bFGF in cell biology is limited by the fact that bFGF rapidly degrades at physiological temperatures; we demonstrated < 20% activity of native bFGF after 72 hours. We have addressed this problem with an engineered form of bFGF, named Heat Stable bFGF (HS bFGF), which is stable at 37 degrees Celsius. HS bFGF maintains > 90% homology with the native protein and > 80% activity after 72 hours in standard mammalian culture conditions. We have used HS bFGF to enhance expansion of multipotent human and rat neural stem cells (NSCs), using a reduced bFGF concentration and a more user-friendly workflow respectively. Doubling time in both models was also reduced by approximately 30% with the use of HS bFGF versus native bFGF. Critically, differentiation of NSCs into the neuronal, astrocyte-, and oligodendrocyte-lineages was comparable between HS bFGF and native bFGF. The downstream differentiation demonstrates the ability of HS bFGF to be completely removed from the culture when desired. HS bFGF was also tested in the stimulation of MCF7 human breast adenocarcinoma cells in three-dimensional (3D) culture. MCF7 spheroids were cultured in bFGF supplemented serum-free medium for 8 days without media changes so that spheroid morphology was undisturbed. Spheroids cultured in HS bFGF were perceptibly larger than those in native bFGF, starting just three days after seeding and continuing throughout the culture period. HS bFGF enhanced spheroid growth by almost 20% more than the native protein, as measured by PrestoBlue® assay. Because there was no need to change the media, we were also able to observe morphological changes to the spheroids that likely would have been impossible to document with a standard media change regimen. These data reinforce the idea that bFGF is a mitotic agent for MCF7 cells, a point which has previously been debated in the literature, and confirm that HS bFGF can provide stable stimulation in long-term culture. Taken together, these data show HS bFGF has broad applicability in cell biology and will be a valuable technology for a variety of applications moving forward. HS bFGF not only allows for reduced concentrations of bFGF to be used, but also enables a more user-friendly workflow for cell culture maintenance.