Adipocyte-derived extracellular vesicles: the quest for a proper isolation protocol

Abstract

Background: In obesity, excessive adipose tissue (AT) accumulation leads to ectopic lipid deposition, insulin resistance (IR) and dysfunctional interorgan crosstalk, in which extracellular vesicles (EVs) became known as important mediators. In the obese state, adipocyte-derived EVs (adEVs) might play a distinct role in the development of obesity-related IR. However, a broad research interest yet large methodological heterogeneity and lack of prior optimization studies emphasizes the need for uniform, sourcespecific adEV isolation protocols. Our study aims to standardize the approach for adEV isolation in future studies. Methods: Human mature adipocytes were isolated from paired visceral and abdominal subcutaneous AT biopsies. Adipocytes were incubated using different modalities (falcon vs membrane cultures), media compositions and timings. EV isolations were performed using either ultracentrifugation (UC) or 10kD ultrafiltration combined with size exclusion chromatography (UF-SEC). Obtained adEVs were characterized following MISEV18 guidelines, including TEM, NTA and western blot. Results: After 3h incubation of visceral mature adipocytes, UC-based isolation of adEVs resulted in a notable lower yield as opposed to UF-SEC-based isolation (mean yield: 2,0E8 ± 3,8E6 vs 1,1E10 ± 2,8E7; mean size: 166nm vs 152nm, respectively), most likely due to the inability of UC to pellet high-lipid containing floating EVs. Isolation of adEVs from conditioned media of mature adipocytes, incubated without EV-depleted FBS supplementation, resulted in concentrations below the accuracy limit of the Zetaview. Subcutaneous mature adipocytes produced less adEVs as compared to visceral mature adipocytes (n=1; yield: 2,6E9; mean size: 162nm). However, in contrast to mature adipocytes, similar whole tissue EV concentrations were observed for visceral and subcutaneous AT (mean yield: 1E10 vs 1,4E10; mean size: 218nm vs 216nm, respectively). Conclusion: Even though adEVs are advocated for their role in regulating metabolism, further optimization with regard to incubation modalities for EV release from mature adipocytes is required to improve quality of obtained adEV samples.