Development of a microfluidic device for mitochondria isolation from cell culture

Mitochondria play a fundamental role in the energy metabolism of most eukaryotes. In the previous studies, the intermediates of the cytosol and the mitochondria are usually sampled together as one pool. Due to the lack of separate experimental data, the dynamics and regulatory mechanism in mitochondria and those at the conjunction of cytosol and mitochondria are thus poorly understood. The difficulties lie partly in the realization of effective methods or devices for fractionation of the cell lysate. Standard methods such as differential centrifugation or electrophoresis cannot produce samples under physiological conditions. For proteomic studies, these methods are not specific enough and require extensive time to be performed. This work introduces a new, affinity based method for isolation of mitochondria on a micro scale after cell disruption. The device comprises of linear micro channels cast in Polydimethylsiloxane (PDMS) and sealed with glass. Using immobilized specific antibodies, mitochondria can be captured in the channel, whereas the remains of the lysate flow out the chip unhindered. Here, successful immobilizations of preconcentrated mitochondria and mitochondria directly from cell lysates are demonstrated. The device was characterized optically using fluorescence microscopy. To better understand the dynamics of the adhesion, a mathematical model was developed. The model was based on the antibody-antigen reaction and included the effect of flow rate on mitochondria immobilization. The model was able to describe the experimental results satisfactorily and is useful for further experimental design. The experimental and the modeling results showed that the yield was influenced by the sampling time, the antibody concentration on the surface, the mitochondria concentration in the sample, as well as the flow rate used during sampling. Further, it is possible to directly observe and analyze the mitochondria on chip without the need to elute them first, e. g. for in-vitro assays on chip. As a proof of principle, in vitro respiration analysis of immobilized mitochondria was performed on chip. The immobilized mitochondria were highly active and able to utilize mitochondrial respiration substrates to produce Adenosine triphosphate (ATP). It was also shown that several segments of the channel can be modified separately from each other. Multiple biomarkers could thus be immobilized in different segments of the channel so that simultaneous observation of different mitochondrial proteins or organelles is possible.