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Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification
Published on: June 2, 2023
MicroPrep: chip-based dielectrophoretic purification of mitochondria
Meike Moschallski1, Monika Hausmann, Anton Posch
1Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.
Abstract:
We have developed a microfluidic system--microPrep--for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.
Insights
We created microPrep, a microfluidic system for cell organelle separation using dielectrophoresis. This system efficiently isolates pure mitochondria from complex samples, outperforming traditional centrifugation methods.
Area of Science:
- Biotechnology
- Cell Biology
- Microfluidics
Background:
- Subcellular fractionation is crucial for studying organelle function.
- Current methods like centrifugation can be time-consuming and may lead to sample contamination.
- There is a need for high-throughput, efficient, and precise methods for organelle isolation.
Purpose of the Study:
- To develop and validate a microfluidic system, microPrep, for high-throughput subcellular fractionation.
- To demonstrate the system's capability for isolating purified mitochondria from human cell homogenates.
- To compare the purity and efficiency of microPrep with conventional centrifugation techniques.
Main Methods:
- Development of a microfluidic chip employing dielectrophoretic sorting for cell organelle separation.
- Utilizing technical measures including BSA coating, mechanical actuators, efficient cooling, wide channels, and serial dielectrophoretic deflectors.
- Monitoring separation via fluorescence microscopy and characterization using western blot analysis.
Main Results:
- The microPrep system demonstrated robust, high-throughput, and continuous operation for up to 60 hours.
- Tens of micrograms of enriched and purified mitochondria were obtained within hours.
- Western blot analysis revealed a six-fold reduction in endoplasmic reticulum contamination compared to centrifugation methods.
Conclusions:
- MicroPrep offers a novel and efficient microfluidic approach for subcellular fractionation.
- The system provides high purity and yield of isolated mitochondria, surpassing current state-of-the-art centrifugation.
- This technology has significant potential for advancing cell biology research and diagnostics.
