November 1, 2018 (Vol. 38, No. 19)
Matthew Dallas Ph.D. Senior Manager Thermo Fisher Scientific
Brittany Balhouse Scientist, Cell Biology Thermo Fisher Scientific
Diana Navarro Scientist, Cell Biology Thermo Fisher Scientific
Kathy Reid Global Project Manager Thermo Fisher Scientific
Michalina Mackowski Global Market Development Manager Thermo Fisher Scientific
Enhanced 2D and 3D Culture Using Gibco™ Heat Stable Recombinant Human Basic Fibroblast Growth Factor from Thermo Fisher Scientific
Playing a role in a variety of biological processes, including the promotion of neuronal plasticity, embryonic development, epithelial cell migration, and tumor growth and invasion, basic fibroblast growth factor (bFGF, also known as FGF2) is one of the most widely studied human growth factors. Like other members of the FGF family, bFGF has been shown to be unstable at physiological conditions, with a half-life of approximately eight hours at standard mammalian cell culture conditions (37 °C, 5% CO2). With this rate of decay, bFGF activity levels drop substantially after 72 hours at 37 °C (Figure 1A). This instability poses a significant challenge for the use of bFGF in cell culture, often requiring elevated growth factor concentrations and, in some cases, daily media changes or bFGF supplementation.
With this limitation in mind, Thermo Fisher Scientific launched Gibco™ Heat Stable Recombinant Human Basic Fibroblast Growth Factor Protein (HS bFGF). Gibco HS bFGF employs patent-pending protein engineering to create a heat-tolerant bFGF, which retains greater than 90% bioactivity after 72 hours at 37 °C (Figures 1B & 1C). This temperature resistance represents a dramatic improvement over not only native bFGF, but also other commercially available stabilized human bFGF offerings, which still lose 50–60% of their activity after similar heat exposure (Figure 1C). Critically, Gibco HS bFGF also maintains over 90% amino acid sequence integrity relative to native human bFGF and exhibits no evidence of hyperactivity or off-target signaling, as indicated by qPCR panels (data not shown).
The prolonged half-life of Gibco HS bFGF can significantly improve both outcomes and workflows in the culture of a wide variety of cell types. We have demonstrated compatibility of HS bFGF with primary cells (human umbilical cord vascular endothelial cells, melanocytes, rat neuronal cells) as well as immortalized mouse and human cell lines. Here, we outline key benefits of Gibco HS bFGF for specific applications in neural stem cell (NSC) and cancer cell culture.
Improved Expansion of Neural Stem Cells
In the NSC expansion workflow, two methods of compensating for the loss of bFGF activity are commonly employed. Scientists supplement NSC culture medium with high bFGF concentrations (e.g., 20 ng/mL) or feed their cultures more often (e.g., every 24–48 hours)1,2; in some cases, both measures may be used. Even with these compensations, however, the bFGF activity levels fluctuate and may negatively impact expansion and/or multipotency of NSCs.
The expansion of human embryonic stem cell–derived NSCs was assessed in Gibco StemPro™ NSC SFM-based culture medium supplemented with reduced concentrations (i.e., 5 ng/mL) of native bFGF or HS bFGF. Cells were passaged twice weekly with routine medium changes over the course of three weeks, after which culture growth and phenotype were compared.
Both the expansion and phenotype of human NSCs were significantly improved by the use of HS bFGF. The doubling time of human NSCs cultured in the presence of 5 ng/mL of HS bFGF was decreased by 35% compared to those of cultures supplemented with 5 ng/mL of native bFGF (Figure 2A). Morphological analysis indicated the presence of unwanted neurite outgrowth in the low-concentration native bFGF condition, whereas HS bFGF cultures retained the expected spread NSC morphology (Figure 2B)—equivalent to that of cultures supplemented with high levels of native bFGF. Multipotency was confirmed by immunocytochemical staining for the NSC marker SOX1 (data not shown).
Similar results were observed in highly bFGF-dependent primary NSCs derived from rat embryos. Following isolation, NSCs were expanded in Gibco DMEM/F-12 supplemented with MEM Non-Essential Amino Acids, 2-mercaptoethanol, N-2, and 10 ng/mL of either native or HS bFGF. Media changes in all cultures were performed every 48 hours; some native bFGF cultures were additionally spiked with 10 ng/mL fresh bFGF every 24 hours, while others were limited only to media changes. Differences in growth rate were apparent as early as passage 1, with HS bFGF cultures exhibiting approximately 15% and 50% more cells than cultures fed with native bFGF daily or every 48 hours, respectively. In addition to significantly reduced expansion levels, cultures fed every 48 hours with native bFGF also showed signs of spontaneous differentiation, as indicated by MAP2 expression (data not shown).
After three-passage expansion, bFGF was removed and rat NSCs were allowed to spontaneously differentiate into the three major neural lineages. Expression of MAP2 (neuronal marker), GFAP (astrocyte marker), and GALC (oligodendrocyte marker) in all HS bFGF cultures (Figure 2C) is indicative of not only the support of multipotency in primary NSCs, but also the absence of residual bFGF activity after media exchange. If HS bFGF were hyperactive or difficult to remove from cultured cells, NSC populations would be expected to continue to expand, show delayed differentiation, and/or demonstrate limited differentiation potential, none of which were observed.
Enhanced Proliferation of Cancer Spheroids
bFGF is known to play an important role in the tumor microenvironment (TME). It is secreted by cancer-associated fibroblasts and is also released from the extracellular matrix by proteolytic enzyme activity.3 bFGF acts on endothelial cells in the TME to enhance angiogenesis,3 but the role of bFGF on the cancer cells directly is less clear. In vitro studies of the effect of bFGF on cancer are conflicting and may be complicated by thermal degradation associated with the native protein.4 The trend toward studying cancer using 3D culture models, and the associated complexities of media changes in such models,5 further complicates the study of the role of bFGF in cancer progression.
In order to investigate the impact of HS bFGF in long-term, 3D culture, MCF7 breast adenocarcinoma cells were seeded into Nunclon™ Sphera™ 96-well U-bottom plates at 500 cells per well in serum-free medium (Gibco DMEM/F-12 with B-27 Plus) supplemented with 10 ng/mL of either native bFGF or HS bFGF. Spheroids formed spontaneously and were imaged regularly to monitor growth. To prevent disruption of spheroids, no media changes or feeds were performed during this period. After eight days of expansion, viable cell density in spheroid cultures was measured via PrestoBlue™ Cell Viability Reagent.
Both image analysis (Figure 3A) and quantitative viability assay (Figure 3B) indicate improved outgrowth in spheroids supplemented with HS bFGF. Direct measurement of metabolically active cells via PrestoBlue shows an increase in viable cell density of nearly 20% from HS bFGF supplementation relative to spheroids grown with native bFGF. This result aligns with imaging, where HS bFGF spheroids were consistently and significantly larger than those in native bFGF conditions. HS bFGF spheroids were also observed to have increased branching and blebbing, both of which of have been associated with an invasive phenotype that may be promoted by bFGF activity.6,7
Gibco HS bFGF Enhances Culture Results
Taken together, the qualities of HS bFGF may deliver not only improved culture outcomes, but also improved workflows, particularly when bFGF activity is critical and routine feeding is problematic. By prolonging the half-life of bioactive bFGF under standard culture conditions, Gibco Heat Stable Recombinant Human Basic Fibroblast Growth Factor provides additional control for bFGF-dependent cultures.
Matthew Dallas, Ph.D., is a senior manager, Brittany Balhouse is a scientist, cell biology, Diana Navarro is a scientist, cell biology, Kathy Reid is a global project manager, and Michalina Mackowski is a global market development manager at Thermo Fisher Scientific.
For additional information, please visit: thermofisher.com/heatstablebFGF.
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