Abstract
For the quantitative analysis of cellular metabolism and its dynamics it is essential to achieve rapid sampling, fast quenching of metabolism and the removal of extracellular metabolites. Common manual sample preparation methods and protocols for cells are time-consuming and often lead to the loss of physiological conditions. In this work, we present a microchip-bioreactor setup which provides an integrated and rapid sample preparation of mammalian cells. The lab-on-a-chip system consists of five connected units that allow sample treatment, mixing and incubation of the cells, followed by cell separation and simultaneous exchange of media within seconds. This microsystem is directly integrated into a bioreactor for mammalian cell cultivation. By applying overpressure (2 bar) onto the bioreactor, this setup allows pulsation free, defined, fast, and continuous sampling. Experiments evince that Chinese Hamster Ovary cells (CHO-K1) can be separated from the culture broth and transferred into a new medium efficiently. Furthermore, this setup permits the treatment of cells for a defined time (9 s or 18 s) which can be utilized for pulse experiments, quenching of cell metabolism, and/or another defined chemical treatment. Proof of concept experiments were performed using glutamine containing medium for pulse experiments. Continuous sampling of cells showed a high reproducibility over a period of 18 h.
Highlights
Fast and dynamic experiments with mammalian cells under real physiological conditions are essential for quantitative and systematical studies of cellular metabolism and its regulation
The separation efficiency of CHO-K1 cells was tested under different flow rates on chip by applying overpressure on the bioreactor setup
Due to the separation of CHO-K1 cells from the exterior medium, a separate analysis of intracellular and extracellular metabolites can be performed off-chip. These results demonstrate that the presented microchip is suitable for dynamic pulse experiments and allows a continuous sample preparation with the integrated bioreactor
Summary
Fast and dynamic experiments with mammalian cells under real physiological conditions are essential for quantitative and systematical studies of cellular metabolism and its regulation. The quantitative determination of intracellular metabolites and metabolic fluxes requires rapid and specific sample preparation under physiological conditions [1,2]. Microfluidic systems can be used to generate controlled environments in which metabolomics and cellomics experiments can be conducted under defined conditions in a reproducible manner [10]. Their key feature is the capability to assemble the necessary components to answer a specific question in one device. Qualitative and quantitative metabolism studies with mammalian cells were conducted by direct integration of electrospray ionization mass spectrometry in a lab-on-a-chip setup [12]
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