Considerations for extracellular vesicle and lipoprotein interactions in cell culture assays

Abstract

With an exponential increase in extracellular vesicle (EV) studies in the past decade, focus has been placed on standardization of experimental design to ensure inter-study comparisons and validity of conclusions. In the case of in vitro assays, the composition of cell culture media is important to consider for EV studies. In particular, levels of lipoproteins, which are critical components of the interstitial fluid, should be taken into consideration. Results from this study reveal that lipoprotein levels in cell culture medium impact the effects that EVs have on recipient cells. Additionally, evidence of EV binding and fusion to lipoprotein-like structures in plasma is provided. However, it is unclear whether the impact of lipoproteins in cell culture is due to direct interactions with EVs, indirect effects, or a combination of both mechanisms. Taken together, cell culture studies performed in the absence of physiological levels of lipoproteins are unlikely to reflect interactions that occur between EVs and recipient cells in an in vivo environment.

FIGURE 1

Levels of lipoproteins in foetal bovine serum (FBS) and impact of low‐density lipoprotein on extracellular vesicle (EV) effects. (a) Very‐low‐density lipoprotein (VLDL)/LDL cholesterol and high‐density lipoprotein (HDL) cholesterol in regular FBS, EV‐depleted FBS (Exo‐FBS), Exo‐FBS supplemented with LDL (500‐μg/mL apoB‐100), and MDA‐MB‐231‐derived EVs (10¹⁰/mL). b) Association of fluorescently labelled MDA‐MB‐231, BrM2‐831, A375, and M12‐derived EVs (10⁹/mL) with human brain microvascular endothelial cells (HBMECs) and THP‐1 human monocytes. Adapted with permission from Busatto et al. (2020). (c) mRNA levels of tumour necrosis factor α (TNF‐α; normalized to β‐actin) in THP‐1 monocytes exposed to MDA‐MB‐231, BrM2‐831, A375, and M12‐derived EVs (10⁹/mL) and/or LDL (50‐μg/mL apolipoprotein/apoB‐100). (d) Cryogenic transmission electron microscopy images of crude human plasma. EVs (black arrows) are seen fusing/binding to lipoprotein‐like structures (white arrows) that display crystallization in polygonal‐like faceted shapes (example shown with double arrows). Scale bars, 100 nm. Cell studies were performed in Exo‐FBS. Data represent mean ± standard deviation (SD) of three replicates. Statistics by analysis of variance (ANOVA) with the Tukey's multiple comparison post hoc analysis. *p < 0.05; **p < 0.01; ****p < 0.0001. #p < 0.05 compared to untreated cells (without added EVs and LDL). ns, not significant