Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media

www.nature.com/scientificreports OPEN Received: 7 October 2018 Accepted: 18 March 2019 Published: xx xx xxxx Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media Bettina Mannerström 1, Riku O. Paananen2, Ahmed G. Abu-Shahba Riitta Seppänen-Kaijansinkko 1 & Sippy Kaur 1 1,3 , Jukka Moilanen2, In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EVdepleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EVdepleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA, Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media. Fetal bovine serum (FBS) contains essential factors required for cell growth, metabolism, attachment, and stimulation of proliferation1. Thus, it is the most widely used supplement for culturing human and animal cells. One of the major concerns that has become evident recently is the presence of large amounts of extracellular vesicles (EVs) in FBS2–4. Secreted by most cell types, EVs are mediators of cell-to-cell communication and immune regulation5,6. They carry the genetic material (DNA and RNA) as well as proteins, lipids and other molecules, the transfer of which can alter the functions of the recipient cells. EVs present in FBS are co-isolated with cell-derived EVs and therefore act as contaminants that, affect the reliability of the read-out from cell culture experiments, as FBS-derived EVs are structurally and functionally similar to cell-derived EVs7. In addition, FBS-EVs taken up by cultured cells cause substantial physiological effects2. To overcome this problem, different methods are used to deplete EVs from FBS. Ultracentrifugation (UC) is commonly used, but this method removes EVs only partially3,4,8. Commercially available depleted FBS are produced using proprietary protocols to reach higher purity levels. Also, commercially available defined, serum-free and animal protein free (xeno-free) media developed for clinical cell therapy purposes are an alternative to the largely undefined FBS in culture media. However, we and others have shown that EV-depleted FBS produced by UC or even commercial EV-depleted FBS are not completely free of FBS-derived EVs3,4. Recently we have developed a simple, standardized, cost- and time-effective protocol to produce EV-depleted FBS by means of ultrafiltration (UF) for cell culture purposes that support cell viability and proliferation9. 1 Department of Oral and Maxillofacial Diseases, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. 2Helsinki Eye Lab, Ophthalmology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. 3 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tanta University, Tanta, Egypt. Correspondence and requests for materials should be addressed to S.K. (email: ) Scientific Reports | (2019) 9:5538 | https://doi.org/10.1038/s41598-019-41772-3 1 www.nature.com/scientificreports www.nature.com/scientificreports/ Acronym Culture media Basal media Serum manufacturer Serum content Supplementation Price/50 ml (excl. VAT) FBS-1 FBS-1 media DMEM/F12 + Glutamax cat# 31331093 Sigma, ref. 10270106, lot. 42F8554K 10% FBS 1% pen-strep 4 eur FBS-2 FBS-2 media DMEM/F12 + Glutamax cat# 31331093 Gibco, ThermoFisher cat# 10270106 lot. 42G8468K 10% FBS 1% pen-strep 4 eur UC-dFBS-1 Ultracentrifugation EVdepleted FBS media DMEM/F12 + Glutamax cat# 31331093 Sigma, ref. 10270106, lot. 42F8554K 10% UC-dFBS 1% pen-strep 36 eur UC-dFBS-2 Ultracentrifugation EVdepleted FBS media DMEM/F12 + Glutamax cat# 31331093 Gibco, ThermoFisher cat# 10270106 lot. 42G8468K 10% UC-dFBS 1% pen-strep 35 eur UF-dFBS-1 Ultrafiltration EVdepleted FBS media DMEM/F12 + Glutamax cat# 31331093 Sigma, ref. 10270106, lot. 42F8554K 10% UF-dFBS 1% pen-strep 53 eur UF-dFBS-2 Ultrafiltration EVdepleted FBS media DMEM/F12 + Glutamax cat# 31331093 Gibco, ThermoFisher cat# 10270106 lot. 42G8468K 10% UF-dFBS 1% pen-strep 52 eur SBI-dFBS-1 Commercial Exosome depleted FBS media DMEM/F12 + Glutamax cat# 31331093 System Biosciences, ref. EXO10% SBI-dFBS FBS-50A-1 lot.170501-001 1% pen-strep 39 eur SBI-dFBS-2 Commercial Exosome depleted FBS media DMEM/F12 + Glutamax cat# 31331093 System Biosciences, ref. EXO10% SBI-dFBS FBS-50A-1 lot.170718-001 1% pen-strep 39 eur StemPRO Commercial StemPro MSC Serum free medium StemPro MSC SFM Basal Medium Gibco, Thermo Fisher, A1067501 0.3% pen-strep, 1% StemPro MSC CFM Xenofree supplement, GlutaMAX Supplement 37eura ® — ™ Table 1. Suppliers and pricing information of culture media used for small ncRNA sequencing. Pricing information include consumables and supplementation needed for preparation of 50 ml of complete media. aculture vessels need to be coated with CELLstart Substrate (Thermo Fisher cat# A1014201) 1:50 dilution 3.6 eur/ml. ™ In the EV research field, identifying the ‘RNA contaminants’ derived from media itself is of high importance when developing RNA biomarkers. To address this question, we undertook this study to characterize the extracellular small non-coding (nc)RNA contaminants present in FBS, EV-depleted FBS, commercially available EV-depleted FBS as well as serum- and xeno-free defined media to assess their small ncRNA content and diversity. Results EV characterization. EVs were isolated from FBS, ultracentrifugation EV-depleted FBS (UC-dFBS), ultrafiltration EV depleted FBS (UF-dFBS), commercial depleted FBS (S (...truncated)