title: Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles.
abstract: Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.
authors: Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.|Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.|Department of Orthopaedics, University Medical Center Utrecht, Utrecht, Netherlands.
authors: Janneke
authors: J
authors: Boere
authors: Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.|Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.
authors: Chris H A
authors: CH
authors: van de Lest
authors: Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.
authors: Sten F W M
authors: SF
authors: Libregts
authors: Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.|Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.
authors: Ger J A
authors: GJ
authors: Arkesteijn
authors: Department of Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht, Netherlands.
authors: Willie J C
authors: WJ
authors: Geerts
authors: Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.
authors: Esther N M
authors: EN
authors: Nolte-'t Hoen
authors: Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.|Department of Orthopaedics, University Medical Center Utrecht, Utrecht, Netherlands.
authors: Jos
authors: J
authors: Malda
authors: Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.
authors: P René
authors: PR
authors: van Weeren
authors: Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands; m.h.m.wauben@uu.nl.
authors: Marca H M
authors: MH
authors: Wauben
keywords: CD44; cryo-TEM; equine; extracellular vesicles; high-resolution flow cytometry; hyaluronidase; isolation; joint; standardization; synovial fluid