Demarcating the membrane damage for the extraction of functional mitochondria

Abstract

Defective mitochondria have been linked to several critical human diseases such as neurodegenerative disorders, cancers and cardiovascular disease. However, the detailed characterization of mitochondria has remained relatively unexplored, largely due to the lack of effective extraction methods that may sufficiently retain the functionality of mitochondria, particularly when limited amount of sample is considered. In this study, we explore the possibility of modulating hydrodynamic stress through a cross-junction geometry at microscale to selectively disrupt the cellular membrane while mitochondrial membrane is secured. The operational conditions are empirically optimized to effectively shred the cell membranes while keeping mitochondria intact for the model mammalian cell lines, namely human embryonic kidney cells, mouse muscle cells and neuroblastoma cells. Unsurprisingly, the disruption of cell membranes with higher elastic moduli (neuroblastoma) requires elevated stress. This study also presents a comparative analysis of total protein yield and concentrations of extracted functional mitochondria with two commercially available mitochondria extraction approaches, the Dounce Homogenizer and the Qproteome ® Mitochondria Isolation Kit, in a range of cell concentrations. Our findings show that the proposed “microscale cell shredder” yields at least 40% more functional mitochondria than the two other approaches and is able to preserve the morphological integrity of extracted mitochondria, particularly at low cell concentrations (5–20 × 10 4 cells/mL). Characterized by its capability of rapidly processing a limited quantity of samples (200 μL), demarcating the membrane damage through the proposed microscale cell shredder represents a novel strategy to extract subcellular organelles from clinical samples.

Extracellular Vesicle- and Mitochondria-Based Targeting of Non-Small Cell Lung Cancer Response to Radiation: Challenges and Perspectives

“…Mitochondria transfer’s impact on cancer is significant [ 10 ]. Various research techniques have been developed and used for different types of cancer, such as breast, kidney, skin, bone, bladder, brain, and lung [ 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 110 , 111 , 112 , 113 , 114 , 115 , 116 ]. The techniques used to extract and study MtO and EVs, as well as their implications for mitochondria transfer-related DNA damage response, can also be explored in NSCLC.…”

“…On the other hand, mechanical methods encompass homogenization, shaking with glass beads, fine grinding, and ultrasonication [ 79 , 80 ]. While mechanical methods generally offer better fragmentation efficiency compared with nonmechanical methods, the high pressure applied during these processes can potentially compromise MtO activity due to significant variations in pressure and temperature [ 81 , 82 ]. A recent development involves a centrifugal device designed for the efficient extraction of functional MtO using a centrifuge commonly found in standard laboratory settings [ 8 ].…”