Genetic Engineering & Biotechnology News

NOV1 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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22 | NOVEMBER 1, 2018 | Genetic Engineering & Biotechnology News | Matthew Dallas, Ph.D., Brittany Balhouse, Diana Navarro, Kathy Reid, and Michalina Mackowski Playing a role in a variety of biological pro- cesses, including the promotion of neuronal plasticity, embryonic development, epithelial cell migration, and tumor growth and inva- sion, 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 approximate- ly eight hours at standard mammalian cell culture conditions (37 °C, 5% CO 2 ). With this rate of decay, bFGF activity levels drop substantially after 72 hours at 37 °C (Fig- ure 1A). This instability poses a significant challenge for the use of bFGF in cell culture, often requiring elevated growth factor con- centrations 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 engi- neering 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 stabi- lized 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 hyperac- tivity or off-target signaling, as indicated by qPCR panels (data not shown). The prolonged half-life of Gibco HS bFGF can significantly improve both out- comes 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 com- pensations, however, the bFGF activity levels fluctuate and may negatively impact expan- sion 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 rou- tine medium changes over the course of three weeks, after which culture growth and phe- notype were compared. Both the expansion and phenotype of hu- man 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 pres- ence of unwanted neurite outgrowth in the low-concentration native bFGF condition, whereas HS bFGF cultures retained the ex- pected spread NSC morphology (Figure 2B)—equivalent to that of cultures supple- mented with high levels of native bFGF. Multipotency was confirmed by immuno- cytochemical 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. Me- dia changes in all cultures were performed every 48 hours; some native bFGF cultures were additionally spiked with 10 ng/mL Enhanced 2D and 3D Culture Using Gibco™ Heat Stable Recombinant Human Basic Fibroblast Growth Factor from Thermo Fisher Scientific Improving Cell Culture Outcomes through Stabilized bFGF Bioprocessing Tutorial Figure 2. HS bFGF enhances neural stem cell (NSC) culture. (A) NSCs expanded in HS bFGF (blue) grew significantly faster than those grown with native bFGF (red). (B) Phase-contrast images of NSCs grown in native bFGF (left) and HS bFGF (right). Arrowheads indicate examples of neurite outgrowth. Scale bars: 100 µm. (C) Primary rat NSCs expanded in HS bFGF were allowed to spontaneously differentiate and were stained for markers of neuronal (MAP2), astrocyte (GFAP), and oligodendrocyte (GALC) lineages. Figure 3. HS bFGF improves cancer spheroid proliferation. (A) Phase-contrast images of MCF7 spheroids suggest improved growth at day 8 with HS bFGF (right) relative to native bFGF (left). Scale bars: 250 µm. (B) PrestoBlue™ Cell Viability Reagent confirms the viable cell density in MCF7 spheroids grown in HS bFGF to be higher than those in native bFGF. Figure 1. Gibco TM HS bFGF exhibits superior stability under standard culture conditions. Native bFGF (A) and HS bFGF (B) were incubated at 37 °C (red) or 4 °C (blue) for 72 hours. Stimulation of serum- starved Balb/3T3 fibroblasts was measured via PrestoBlue™ Cell Viability Reagent. Similar testing (C) shows significant loss of activity in competitor bFGF offerings, both native (black) and stabilized (white, gray), compared to HS bFGF (blue). A C A B A B C B

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