IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal; ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
IBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal.
J Biotechnol. 2020 Jan 10;307:139-147. doi: 10.1016/j.jbiotec.2019.10.004. Epub 2019 Nov 4.
Adaptive laboratory evolution (ALE) has been extensively used to modulate the phenotype of industrial model organisms (e.g. Escherichia. coli and Saccharomyces cerevisae) towards a specific trait. Nevertheless, its application to animal cells, and in particular to insect cell lines, has been very limited. In this study, we describe employing an ALE method to improve the production of HIV-Gag virus-like particles (VLPs) in stable Sf-9 and High Five cell lines. Serial batch transfer was used for evolution experiments. During the ALE process, cells were cultured under controlled hypothermic conditions (22 °C instead of standard 27 °C) for a prolonged period of time (over 3 months), which allowed the selection of a population of cells with improved phenotype. Adapted cells expressed up to 26-fold (Sf-9 cells) and 10-fold (High Five cells) more Gag-VLPs than non-adapted cells cultured at standard conditions. The production of HIV Gag-VLPs in adapted, stable insect Sf-9 cell lines was successfully demonstrated at bioreactor scale. The Gag-VLPs produced at 22 °C and 27 °C were comparable, both in size and morphology, thus confirming the null impact of adaptation process and hypothermic culture conditions on VLP's quality. This work demonstrates the suitability of ALE as a powerful method for improving yields in stable insect cell lines producing VLPs.
适应性实验室进化（ALE）已被广泛用于调节工业模式生物（例如大肠杆菌和酿酒酵母）的表型，使其朝向特定性状发展。然而，它在动物细胞中的应用，特别是在昆虫细胞系中的应用非常有限。在这项研究中，我们描述了一种使用 ALE 方法来提高稳定 Sf-9 和 High Five 细胞系中 HIV-Gag 病毒样颗粒（VLPs）产量的方法。连续分批传代用于进化实验。在 ALE 过程中，细胞在受控的低温条件下（22°C 而不是标准的 27°C）长时间培养（超过 3 个月），从而选择了具有改善表型的细胞群体。适应细胞的表达量比在标准条件下培养的未适应细胞高 26 倍（Sf-9 细胞）和 10 倍（High Five 细胞）。在生物反应器规模上，成功地在适应的稳定昆虫 Sf-9 细胞系中展示了 HIV Gag-VLPs 的生产。在 22°C 和 27°C 下生产的 Gag-VLPs 在大小和形态上是可比的，这证实了适应过程和低温培养条件对 VLP 质量没有影响。这项工作证明了 ALE 作为一种提高产生 VLPs 的稳定昆虫细胞系产量的有效方法的适用性。
