Various Optimization Strategies for the Isolation of Mitochondria from Sprague-Dawley Rat Liver Tissue

Mitochondria are key organelles that establish a very large free energy drop for the ATP hydrolysis reaction in the cytoplasm of cells, which provides energy required for the cell maintenance of homeostasis. They are commonly isolated from living tissue as it makes them easier to interrogate at the biochemical level. Thus, isolated mitochondria of sufficient quality are desired to improve translational impact. To gain a greater insight into the isolation process and optimize our current isolation protocol for quality, we implemented various fine-tuning modifications to our standard homogenization, centrifugation, and purification steps with isolated rat hepatocyte mitochondria. The following modifications we tested were: i) different homogenization speeds (10,000, 14,000, and 18,000 rpm) with varying time intervals (10, 20, and 30 sec), ii) addition of an additional purification spin, and iii) use of density gradients to further purify the isolated mitochondria from non-mitochondrial contaminants. Mitochondrial quality was approximated using the well-established respiratory control ratio (RCR). The data reveal that our original protocol yields isolated mitochondria with acceptable quality and the optimization attempts produced similar or worse mitochondrial isolates. In addition, an extra purification spin decreased RCR values and therefore is not recommended. We note that the use of density gradients did not improve the RCR, but it did remove presumed peroxisomal contamination. While this protocol can be further enhanced using additional metrics, the data indicate that our current isolation protocol is sufficiently effective since additional modifications did not yield major improvements.