Abstract
The quantification of metabolite leakage from damaged mammalian cells to the surrounding medium is of high interest for the processing of samples for metabolomic analysis. It is also of relevance to know the typical time span which is required for a promoted metabolite release through a selectively permeabilized cell membrane. The real-time observation of such a process is difficult since small metabolites cannot be observed directly by optical methods and other more indirect assays can disturb the metabolite concentration itself. However, the diffusion based loss of metabolites from the cytoplasm can be predicted on the basis of reference measurements taken from an easy-to-detect molecule with known diffusion coefficient. In this work, we use green fluorescent protein (GFP) as a marker and model its release from damaged cells using the finite-element method. A correlation between the disrupted membrane area fraction, A d , the distribution of membrane ruptures and the rate of GFP efflux, k e , has been established. k e has been determined experimentally for Chinese hamster ovary cells, which have been damaged mechanically by passage through a micronozzle geometry in a microfluidic system. The immediate GFP release downstream of the micronozzles has been observed in real-time and the corresponding membrane damage has been predicted. On this basis, we calculated the expected times required for the drainage of freely diffusable cytosolic glucose and found a loss of ≈90% within 1 s for a disrupted membrane area fraction of ≈5%. Hence, even minimal membrane damage would lead to a rapid loss of cytosolic metabolites by diffusion unless membrane resealing processes take place.
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Acknowledgments
We are grateful to Prof. Dr.-Ing. J. Mueller and his coworkers of the Institute of Microsystems Technology at the University of Technology Hamburg for cooperation with respect to the manufacturing of the microfluidic systems. This project has been funded partially by the German Ministry of Education and Research (BMBF, FZ. 0315555A) and the Deutsche Forschungsgemeinschaft (DFG, ZE 542/4-1).
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Wurm, M., Zeng, AP. Estimation of protein and metabolite release rates from damaged mammalian cells by using GFP as a marker molecule. Metabolomics 8, 1081–1089 (2012). https://doi.org/10.1007/s11306-012-0413-9
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DOI: https://doi.org/10.1007/s11306-012-0413-9