Genetic Engineering & Biotechnology News

NOV1 2018

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GENengnews.com | Genetic Engineering & Biotechnology News | NOVEMBER 1, 2018 | 23 fresh bFGF every 24 hours, while others were limited only to media changes. Differ- ences in growth rate were apparent as early as passage 1, with HS bFGF cultures exhibit- ing 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, cul- tures fed every 48 hours with native bFGF also showed signs of spontaneous differen- tiation, 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 mark- er), and GALC (oligodendrocyte marker) in all HS bFGF cultures (Figure 2C) is indica- tive of not only the support of multipotency in primary NSCs, but also the absence of re- sidual bFGF activity after media exchange. If HS bFGF were hyperactive or difficult to re- move from cultured cells, NSC populations would be expected to continue to expand, show delayed differentiation, and/or demon- strate 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 en- hance 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 ther- mal 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 mod- els, 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 me- dium (Gibco DMEM/F-12 with B-27 Plus) supplemented with 10 ng/mL of either na- tive 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 Pre- stoBlue™ Cell Viability Reagent. Both image analysis (Figure 3A) and quantitative viability assay (Figure 3B) indi- cate improved outgrowth in spheroids sup- plemented with HS bFGF. Direct measure- ment of metabolically active cells via Presto- Blue shows an increase in viable cell density of nearly 20% from HS bFGF supplemen- tation relative to spheroids grown with na- tive 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 cul- ture outcomes, but also improved work- flows, 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. Terumo BCT combines deep expertise and innovative solutions to help you automate your processes early—and take scientific ingenuity even further. ©2018 Terumo BCT, Inc. / PN 306650541A For decades, Terumo BCT has provided critical tools for visionary researchers, developers and manufacturers. Today, we're bringing that partnership to new levels with our automated cell processing technologies. Our team of automation experts can help you integrate the equipment you need to pursue new standards of quality, to improve consistency and to scale your next-generation cell and gene therapies—potentially lowering your cost and risk in the process. And even more innovation is on the way. Let Terumo BCT help you achieve therapeutic firsts for the patients who need them most. Learn more at terumobct.com/machineefficiency HUMAN INGENUITY. MACHINE EFFICIENCY. Bioprocessing 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. References available online.

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