Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a “gas pedal”, based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a “brake pedal”. A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the “gas and brake pedals”, enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.

中文翻译：

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用于生物制造的工程哺乳动物细胞生长动力学

精确控制哺乳动物细胞生长动力学是生物制药制造中的重大挑战。在这里，我们提出了一种多层次的细胞工程策略，用于调节哺乳动物细胞的生长阶段。最初，我们通过使用 CRISPR/Cas9 敲除促凋亡蛋白 Bax 和 Bak 来设计哺乳动物死亡阶段，通过提高细胞活力和延长培养物寿命来显着减弱细胞凋亡。第二阶段引入了类似于“油门踏板”的生长加速系统，该系统基于调节 cMYC 基因表达的脱落酸诱导系统，能够快速增加细胞密度和控制细胞周期。第三阶段的重点是静止阶段诱导系统，类似于“制动踏板”。基于 BLIMP1 基因的四环素诱导遗传回路在激活后导致细胞生长停止并停滞细胞周期。最后，我们开发了一种双可控系统，结合了“油门和制动踏板”，能够动态、精确地协调哺乳动物细胞的生长动力学。这项工作例证了合成生物学工具和组合细胞工程的应用，为操纵哺乳动物细胞生长提供了复杂的框架，并为重新编程细胞行为以增强生物制药制造和进一步的生物医学应用提供了独特的范例。