Protocol for Exosome Isolation from Small Volume of Ovarian Follicular Fluid: Evaluation of Ultracentrifugation and Commercial Kits
The ovarian follicular fluid (FF) is a complex fluid that constitutes the microenvironment of developing follicles and contains factors secreted by the surrounding cells and blood plasma compounds that cross the “blood–follicle barrier.” Upon oocyte retrieval (in human, bovine, and equine) the follicular fluid is normally discarded and represents a repertoire of cellular messages exchanged during follicle development, thus providing a suitable sample for performing oocyte quality diagnostics. Several studies report on the presence of extracellular vesicles (EVs) in FF from human, bovine and equine. Here, we describe the process of FF collection from human and bovine and the enrichment and isolation of EVs that we termed folliculosomes (FFEs), using available commercial kits as well as the traditional ultracentrifugation methods.
Key wordsFollicular fluid Extracellular vesicles Exosomes Folliculosomes Ultracentrifugation
The authors would like to thank Arshia Azizeddin and Bahar Beharouzi for technical assistance, and CReATe IVF laboratory staff for help with sample collection.
Conflict of interest: Shlomit Kenigsberg and Brandon A. Wyse were research associates at Create Fertility Centre while conducting this research and writing the manuscript. Clifford L. Librach is the Medical Director and Owner of Create Fertility Centre. The authors have nothing else to declare.
- 14.Witwer KW et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. doi: 10.3402/jev.v2i0.20360
- 34.Thery C et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3:Unit 3.22Google Scholar
- 40.Van Deun J et al (2014) The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 3. doi: 10.3402/jev.v3.24858