| 【Objectives】In traditional Chinese medicine, chicken embryo eggs were used as medicated diet supplements to improve human immune. The effects of different extraction methods on the isolation of chicken embryoderived exosomes were studied to further analyze the physiological functions of chicken embryo eggs.【Method】12 Huiyang chicken embryo eggs with embryonic age of 13 days were selected (3 groups, 4 eggs/group) and pretreatment was carried out by enzyme digestion, differential centrifugation and filtration, then the obtained samples divided into four parts. Subsequently, the four parts were processed by four different exosomes isolation methods: ultracentrifugation (UC) , sucrose density ultracentrifugation (SDUC) , membrane affinity (exoEasy) and polyethylene glycol precipitation (ExoQuick) and labeled from A to D, respectively. The protein concentration, morphology, size and protein expression of the isolated exosomes were compared by using four methods: BCA protein assay, transmission electron microscopy (TEM) , nano-flow cytometry (nanoFCM) and fluorescence activated cell sorting（FACS）.【Result】The results showed that all the 4 isolation methods could produce extracellular vesicles within the expected size range (40–200 nm) and the vesicles had typical microcapsule structure of exosomes.The exosomal protein concentration and particle concentration varied under different isolation methods (D ＞ C ＞ A ＞ B) , in which D contained other aggregate-contaminating proteins. Flow cytometry confirmed the expression of two marker proteins, CD63 and CD81, in samples isolated by all methods (B ＞ D ＞ C ＞ A) .【Conclusion】Considering the exosomes purity, morphology, size and expression of the marker protein comprehensively, the sucrose density ultracentrifugation or membrane affinity methods may be preferred according to the source of tested samples, experimental conditions and purposes. The sucrose density ultracentrifugation is preferred when high-purity exosomes are required, while the membrane affinity method is recommended for limited samples and the unavailability of ultracentrifugation equipment. |
