Response to letter to the editor

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Response to “Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells”

Dear Editor,

We thank Dr Fiandaca and colleagues for commenting on our article in which we proposed optimized protocols for the removal of extracellular RNA from foetal bovine serum (FBS) for studies on cell culture EV [1]. We fully agree that continuing research efforts and discussions on the preparation and use of EV-depleted media are essential to the field of EV research. Dr Fiandaca and colleagues commented on two aspects of our work. First, they argue that in their hands after prolonged ultracentrifugation a large number of particles (10E9/ml) stay behind in the supernatant and can be detected by Nanoparticle Tracking Analysis (NTA) [2]. This is an expected result because particles other than EV are detected using this methodology as well. As we and others have shown, many of the (RNA-containing) structures that remain in the supernatant after ultracentrifugation of FBS represent lipoprotein particles (LPP) [1,3,4]. These LPP are estimated to largely outnumber EV in plasma and serum and cannot be distinguished from EV using NTA because of their overlap in size [5,6]. Hence, it is recommended to remove LPP from EV populations prior to quantification by NTA [7]. This can for example be done using ultracentrifugation followed by density gradient centrifugation, as we show in Figure 2 of our article. We performed fluorescent labelling and high-resolution flow cytometric quantification of these purified EV to prove that our optimized depletion method removed the far majority of EV from FBS [1]. Moreover, we show that LPP-associated miRNAs (such as miR-122) were not detected in purified populations of cell culture EV. So, despite the fact that LPP-associated miRNAs were not depleted by ultracentrifugation, further purification steps allowed analysis of EV without contaminating LPP.

A second point raised by Dr Fiandaca and colleagues is that depletion of EV from FBS used to supplement culture media can affect the growth of certain cell types and that the field should increase its focus towards the use of serum-free media. We fully agree that any change in cell culture medium preparation should first be tested for negative effects on the phenotype and functional behaviour of cultured cells; we included several examples of how FBS-EV depletion changes cell growth and function [8–10] in the introduction of our article [1]. Yet, the use of serum-free media comes with its own challenges. First, some cell types, e.g. certain immune cell subsets, change their functional behaviour when cultured in serum-free media (e.g. [11]). Second, also serum-free media may contain particles or contaminating proteins/RNAs derived from supplemented growth factors or other additives. The use of this type of media therefore does not eliminate the need to include medium controls in each individual cell culture EV study. In our article, we show how such controls can be used to estimate the background levels of contaminant RNAs from FBS. General guidelines on the use of medium controls to assess the influence of medium-derived components on EV quantification and characterization are provided in the Minimal information for studies of extracellular vesicles 2018 (MISEV2018) statement [12], which is endorsed by a large group of international EV scientists.