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Decreased Oxygen Consumption after Cellular Detachment not Trypsin Specific
Investigators at the Catholic University of Louvain in Belgium have published an interesting study suggesting that the previously known inhibition in Oxygen Consumption Rate (OCR) following trypsinizing detachment is not enzyme specific. Instead, in may be that other methods of enzymatic cell detachment may also skew OCR and may therefor affect metabolic studies.
The authors compared OCR in murine melanoma (B16F10-luc) cells, and human umbilical vein endothelial cells (HUVEC) cells both prior to enzyme detachment, and then again following detachment protocols using trypsin and alternatively, collagenase. Baseline OCR rates were measured by electron paramagnetic resonance (EPR) of cells attached to microbeads and cultured in suspension.
Potential mitochondrial loss from trypsin or collagenase harvesting was determined with the detection of mouse mitochondrial complex IV subunit 1 (COXI) by western blotting. Cellular ATP production, glucose metabolism and cell death was compared in both groups.
Trypsinization of B16F10-luc murine melanoma cells induced an approximately 60% decline in OCR, while similar detachment of HUVECs resulted in approximately a 40% reduction of OCR. This inhibition of respiration was slightly less pronounced when collagenase was used to detach the cells, but still measured a decrease of 35% on OCR with B16F10-luc cells and 30% with HUVECs. COXI protein measurements showed approximately equal declines in both groups of detached cells as compared to adherent controls. Trypsinized cells demonstrated significant decreases in glucose consumption but little change in lactate production when compared to adherent cells. In contrast, collagenase-treated cells showed similar levels of glucose consumption compared to adherent cells, but an increase in lactate. Both detachment groups experienced high levels of cell death beginning within three hours of detachment.
The study suggests that cell adhesion promotes cell respiration and traditional cell detachment protocols increase mitochondrial uncoupling. Post-detachment, a net reduction in intracellular ATP was observed, as well as, after significant detachment time, a higher glycolytic index. This latter effect suggests a shift away from glucose as a significant source of lactate production.
This experimental demonstration of the previously known trypsin effect on OCR to collagenase detachment leaves open the significant question as to whether other, and perhaps even all, cell detachment methods affect cellular respiration. While causes such as alteration of membrane organization and changes in cell morphology cannot be excluded as contributing to this decreased OCR, it remains that enzymatic methods of cell detachment could be responsible for influencing possible results in important metabolic studies. The limitations of many of our classical in vitro techniques are again highlighted.