Search

Myocardial infarction is the leading cause of mortality worldwide. Reperfusion is the primary therapy for myocardial infarction. Paradoxically, reperfusion causes further cell death beyond the injury caused by ischaemia alone. Exosomes are a type of extracellular vesicles released from cells. Exosomes from stem cells have been reported to confer cardioprotection from ischaemia and reperfusion injury. However, since it is currently challenging to obtain exosomes of high purity and quantity, it is difficult to know whether exosomes or residual contaminants are responsible for cardioprotection. In this thesis, I report my studies comparing different techniques to isolate exosomes and developing a novel affinity method to isolate more pure exosomes. I show that exosomes separated by size-exclusion chromatography are not as pure as affinity-purified exosomes. I found that exosomes obtained from a commercial collaborator using a combination of by size exclusion chromatography and tangential flow filtration were highly variable. Therefore, a novel isolation method was established in which exosomes were affinity purified by immune-pulldown of an epitope tag transiently expressed in cells in culture. This method allows the collection of more pure exosomes. Exosomes purified by this method could be used to understand the role of exosomes in cardioprotection. Finally, I investigated several previously published cell models of cardiac ischaemia and reperfusion injury focusing on mitochondrial injury, but these had low replicability. more...