Jie-Min Wang1,2,3, Yong-Jiang Li1,2,3, Jun-Yong Wu1,2,3, Jia-Xin Cai1,2,3, Jing Wen1,2,3, Da-Xiong Xiang4,5,6, Xiong-Bin Hu1,2,3, Wen-Qun Li1,2,3. 1. Department of Pharmacy, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China. 2. Hunan Provincial Engineering Research Centre of Translational Medicine and Innovative Drug, Changsha, Hunan, China. 3. Institute of Clinical Pharmacy, Central South University, Changsha, Hunan, China. 4. Department of Pharmacy, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China. xiangdaxiong@csu.edu.cn. 5. Hunan Provincial Engineering Research Centre of Translational Medicine and Innovative Drug, Changsha, Hunan, China. xiangdaxiong@csu.edu.cn. 6. Institute of Clinical Pharmacy, Central South University, Changsha, Hunan, China. xiangdaxiong@csu.edu.cn.
Abstract
BACKGROUND: Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells. METHODS: Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. RESULTS: For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods. CONCLUSIONS: For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.
BACKGROUND: Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells. METHODS: Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. RESULTS: For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods. CONCLUSIONS: For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.
Authors: Sonia A Melo; Linda B Luecke; Christoph Kahlert; Agustin F Fernandez; Seth T Gammon; Judith Kaye; Valerie S LeBleu; Elizabeth A Mittendorf; Juergen Weitz; Nuh Rahbari; Christoph Reissfelder; Christian Pilarsky; Mario F Fraga; David Piwnica-Worms; Raghu Kalluri Journal: Nature Date: 2015-06-24 Impact factor: 69.504
Authors: Tamás Baranyai; Kata Herczeg; Zsófia Onódi; István Voszka; Károly Módos; Nikolett Marton; György Nagy; Imre Mäger; Matthew J Wood; Samir El Andaloussi; Zoltán Pálinkás; Vikas Kumar; Péter Nagy; Ágnes Kittel; Edit Irén Buzás; Péter Ferdinandy; Zoltán Giricz Journal: PLoS One Date: 2015-12-21 Impact factor: 3.240