کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
4753485 | 1416980 | 2017 | 10 صفحه PDF | دانلود رایگان |
- CAP-T cells were successfully cultured and transfected in 1âL bioreactor.
- VLPs were obtained in bioreactor with the expected quantity and quality.
- Medium exchange prior transfection was avoided using a specific media combination.
Human-derived CAP-T cell line has been demonstrated to be a powerful platform for high-titer production of HIV virus-like particles (VLPs) by PEI-mediated transient transfection. Scale-up of transfection processes is key to ensure the necessary quantities for pre-clinical and clinical testing. One of the major operational challenges of large-scale transient transfection is the medium replacement step that is often required before transfection. In this work, CAP-T cells were cultured in 1Â L bioreactor with addition of sodium bicarbonate and surface aeration, which were observed to improve cell state for transfection. Remarkably, the medium replacement step was avoided by culturing the cells in a combination of media (FreeStyleF17Â +Â 1% of PEM) compatible with cell growth and PEI-mediated transient transfection. In the conditions developed in this work, 0.5Â ÃÂ 106Â cells/mL were seeded in 1Â L bioreactor. Two days later, â¼2Â ÃÂ 106Â cells/mL were transfected without medium exchange, using 0.5Â pg of DNA/cell and 3Â pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4Â ÃÂ 1010Â VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved.
Journal: Journal of Biotechnology - Volume 263, 10 December 2017, Pages 11-20