A novel lab-on-a-chip is presented for the high-throughput preparation of mammalian cell samples formetabolic studies associated with cell compartmentation. The chip is directly connected to an overpres-surized bioreactor, for a continuous harvest and manipulation of cells during the cultivation process. Theproposed on-chip preparation technique allows the rapid cell permeabilization and separation of sub-cellular components as cytoplasm and mitochondria, both primarily responsible for cell metabolism. Themicrofluidic chip comprises two temperature zones (37◦C to ensure physiological conditions and 4◦C forcell quenching), which are thermally decoupled. Cells are manipulated in a continuous flow process in fiveconnected functional modules. The key modules of the integrated chip are a rapid split-and-recombinemicromixer for biological system perturbation and chemical lysis, a chaotic advection “stirred” incuba-tion channel for reduced residence time distribution, and a spiral channel for efficient particle separationand media exchange. The bioreactor-chip system was characterized with Chinese hamster ovary cells. Itexhibits a high efficiency in the lysis process and separation of sub-cellular components.