Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain.
Departament d'Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain.
J Biotechnol. 2017 Dec 10;263:11-20. doi: 10.1016/j.jbiotec.2017.09.019. Epub 2017 Oct 7.
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 1L 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 (FreeStyleF17+1% of PEM) compatible with cell growth and PEI-mediated transient transfection. In the conditions developed in this work, 0.5×10cells/mL were seeded in 1L bioreactor. Two days later, ∼2×10cells/mL were transfected without medium exchange, using 0.5pg of DNA/cell and 3pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4×10VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved.
人源化 CAP-T 细胞系已被证明是通过 PEI 介导的瞬时转染生产 HIV 病毒样颗粒（VLPs）的强大平台。转染过程的放大是确保临床前和临床测试所需数量的关键。大规模瞬时转染的主要操作挑战之一是转染前通常需要进行培养基替换步骤。在这项工作中，通过在 1L 生物反应器中添加碳酸氢钠和表面通气来培养 CAP-T 细胞，观察到这可以改善转染时的细胞状态。值得注意的是，通过在与细胞生长和 PEI 介导的瞬时转染兼容的培养基（FreeStyleF17+1%的 PEM）中培养细胞，可以避免培养基替换步骤。在本工作中开发的条件下，将 0.5×10cells/mL 的细胞接种到 1L 生物反应器中。两天后，在不进行培养基交换的情况下，使用 0.5pg 的 DNA/细胞和 3pg 的 PEI/细胞进行转染，可获得约 2×10cells/mL 的转染效率。使用 4×10VLPs/mL 的产量，可获得与摇瓶相当的转染效率和 VLPs 生产。该新策略显著简化了大规模瞬时转染，同时实现了合适的细胞生长、转染效率和高质量的 VLPs 生产。
