Tumor cell-derived exosomes have been proposed as non-cellular nanomeric vaccine which could induce potent anti- tumor immune response in mice. In order to develop the protocols to prepare tumor cell-derived exosomes for basic research and clinical trail, we isolated exosomes from ovalbumin (OVA)-expressing thymoma cells EG.7-OVA by various preparation methods. We demonstrate the non-sedimentation method is simple, rapid, efficient with higher yield and purity of exosomes. EG.7-OVA-derived exosomes are 40-100 nm in diameter sequestered by lipid bi-layer, and contain rich heat shock protein (HSP) and OVA. The result of the size distribution determination is consistent with the calculation by the visual microscopic inspection, with 90.4% particles at the range of 50-90 nm. Moreover, as a model antigen of the EG.7 cells, OVA concentra- tion in EG.7-derived exosomes can be regarded as a good quality control parameter. Therefore, we have established a platform to efficiently prepare exosomes for tumor immunotherapy.
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