Lonza (Germany)

Abstract Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning's Matrigel product. The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology. In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™-2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ vector, and it was shown that the Nucleofection™ process had no deleterious effect on the tube formation potential of HUVEC on Corning's Matrigel product. Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection. VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning's Growth Factor Reduced Matrigel product in comparison to control samples. Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment. Citation Format: Srinivasan Kokatam, Kanchan Tiwari, Jenny Schroeder, Andrea Toell, Lubna Hussain, Preeti Kapoor. Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388A. doi:10.1158/1538-7445.AM2015-1388A

Abstract The fluid flow and suspension characteristics inside small‐scale, stirred, single‐use bioreactors were investigated experimentally and by means of computational fluid dynamics. The required impeller speeds for homogenous suspension were determined for two microcarrier types. The shear stress level and turbulence distribution were predicted using a numerical model, which was verified by particle image velocimetry measurements. In subsequent cultivations of primary mesencymal adipose‐derived stem cells, up to 31.4‐fold expansion in cell number was achieved for serum concentrations as low as 5 %.