Core Biogenesis FGF-2-STAB^® is produced by recombinant expression of the human sequence of FGF-2 in plant seeds of Camelina sativa. Modifications to the sequence for improved expression, bioactivity and stability are applied but are proprietary. None of the components or raw materials employed for the production of FGF-2-STAB^® are derived or extract from animal or human origin.

Uniquely Engineered for Stem Cell Production:

FGF-2 STAB^® engineered with a novel nine amino acid substitution to improve stability is thermostabilized growth factor that enables more efficient FGF-2-dependent cell cultures with fewer media changes.

Wild Type FGF-2	FGF-2-STAB®

Product Information:

Specifications: 

Product bioactivity measured by proliferation assay of NIH/3T3 cells with FGF-2 STAB^® :

Human embryonic stem cell cultures need FGF2 in the media to remain in an undifferentiated and pluripotent state. Since FGF2 is highly unstable (its functional half-life is only 9 h at 37°C), daily media changes with fresh FGF2 are required, which can be both time- and money-consuming. This product is a stable variant of 155-amino-acid human FGF2 (FGF2- STAB^®), exhibiting a half-life increase of 25-fold compared to the wild-type. This unprecedented stabilization was achieved using a computer-assisted protein engineering strategy for designing new proteins with new or improved properties.

In this study, a proliferation assay using NIH/3T3 cells was performed in order to demonstrate that FGF2-STAB^® can maintain stable cell growth and pluripotency. NIH/3T3 cells were seeded in a density of 40,000 cells/cm² in 190 μl of medium per well (DMEM 31966, Gibco^® + P/S + 10 % newborn calf serum). After 24 hours, the media was changed for starvation (DMEM 31966, Gibco^® + P/S + 0.5 % newborn calf serum). After 16 hours, FGF2-STAB^® or FGF2 wild-type were diluted in sterile water to final concentrations of 0.01 – 20 ng/ml and added to the cells, which were cultured for an additional 48 hours at 37°C. Cell proliferation was measured using CyQuant^® fluorescence assay.

Comparison of FGF2-STAB^® with human FGF-2 wild-type revealed that after 48-hour incubation at 37°C the wild-type has lower capacity to promote 3T3 cell proliferation than engineered FGF2-STAB^®. FGF2-STAB^® exhibited an ED₅₀ as much as 5-fold lower than the wild-type protein, demonstrating that FGF2-STAB^® has increased thermal stability. The ED₅₀ for FGF2-STAB^®, i.e., the concentration of FGF2-STAB^® that produces one-half the maximal response, as determined in a proliferation assay of NIH/3T3 cells, is 0.6-1.1 ng/ml.

FGF-2-STAB^® is a stabilized growth factor that offers a novel way to grow FGF-2-dependent cell cultures more efficiently, with fewer media changes. FGF2-STAB^® retains full biological activity even after twenty days at 37°C. The stable level of FGF-2 in culture allows for a more homogenous, undifferentiated stem cell culture, while saving researchers valuable time and money because repeated supplementation with FGF-2 and a daily medium change is not required. 

The ability of FGF-2 variants (WT and STAB^®) were tested to induce the proliferation of NIH/3T3 cells under serumfree basal media at 10ng/mL. Cell were pre-incubated at 37°C for 6 h, 12 h, 24 h, 48 h and 72 h in triplicates for each condition. At each time point, cells were collected and lysates were Western blotted for phosphorylated ERK1/2. Blots per each protein variant were analyzed and band densities were plotted as a function of pre-incubation time.

Image of SDS-PAGE gel showing purity of isolated FGF2-STAB^®. Protein marker: 116, 66, 45, 35, 25, 18, 14 kDa. Medium was supplemented with 10 ng/ml FGF2 and incubated at 37°C for 6, 12 and 24 hours, and 2, 6, 10, 16, 18, and 20 days. Then, FGF2-starved hPSC were treated with CM containing pre-incubated FGF2 for two hours and Western blotted for phosphorylated ERK1/2. While the biological activity of the wild-type declined with time of heatpreincubation, the stabilized FGF2-STAB^® retained full biological activity even after twenty days at 37°C.

Realize tangible benefits of FGF-2 STAB^® protein stability: significant reductions in the amount of media required to feed cells, as well as reductions in the number of feedings.

Quality Standards:

Core Biogenesis is currently in the process of establishing a GMP compliant production facility to produce biorisk-free cGMP growth factors and cytokines in our facility in Strasbourg, France. Our current biorisk-free products nonetheless represent a significant improvement in safety over products produced using traditional production platforms based on bioreactors and other expression hosts.

Prior to filling of the sterile container, FGF-2-STAB^® is manufactured in liquid form. It is then filtered at 0.2 μm following aseptic technique and following standard operation procedures.

Disclaimer: This product is not intended for any therapeutic or diagnostic use in humans or animals. Do not use internally or externally in humans or animals. For Research Use Only.

• FGF2-G3 was developed by Dvorak et al., 2018. Biotechnology and Bioengineering

Dvorak, P., Bednar, D., Vanacek, P., Balek, L., Eiselleova, L., Stepankova, V., Sebestova, E., Kunova Bosakova, M., Konecna, Z., Mazurenko, S. and Kunka, A., 2018. Computer‐assisted engineering of hyperstable fibroblast growth factor 2. Biotechnology and bioengineering, 115(4), pp.850-862.

• B8 media protocol established by Kuo et al., 2020. Stem Cell Reports 

Kuo, H.H., Gao, X., DeKeyser, J.M., Fetterman, K.A., Pinheiro, E.A., Weddle, C.J., Fonoudi, H., Orman, M.V., Romero-Tejeda, M., Jouni, M. and Blancard, M., 2020. Negligible-cost and weekend-free chemically defined human iPSC culture. Stem Cell Reports, 14(2), pp.256-270.

• updated in Lyra-Leite et al., 2021. STAR Protocols 

Lyra-Leite, D.M., Fonoudi, H., Gharib, M. and Burridge, P.W., 2021. An updated protocol for the cost-effective and weekend-free culture of human induced pluripotent stem cells. STAR protocols, 2(1), p.100213.

• Review of FGF 2 stabilization by Benington et al., 2020. Pharmaceutics

Benington, L., Rajan, G., Locher, C. and Lim, L.Y., 2020. Fibroblast growth factor 2—A review of stabilisation approaches for clinical applications. Pharmaceutics, 12(6), p.508.

• Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat, Kaplan et al., 2021.

Stout, A.J., Mirliani, A.B., Rittenberg, M.L., Shub, M., White, E.C., Yuen Jr, J.S. and Kaplan, D.L., 2022. Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat. Communications Biology, 5(1), p.466.

 Thermostable Recombinant FGF-2 (FGF-2 STAB) (Product Information)

 Thermostable Recombinant FGF-2 (FGF-2 STAB) (Brochure)

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 FGF-2 Human (Product Information)

 FGF-2 Bovine (Product Information)