Efficient ultrafiltration‐based protocol to deplete extracellular vesicles from fetal bovine serum

Abstract

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.

Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles

“…Briefly, cells were left to adhere for 24 h in MM and osteogenic differentiation was induced using osteogenic media (MM) supplemented with 50 µM L-ascorbic acid 2-phosphate, 10 mM β-glycerophosphate disodium salt hydrate and 5 nM dexamethasone (all Sigma-Aldrich). Early osteogenic response was assessed by quantitative alkaline phosphatase activity (qALP) assays and mineralization was assessed using Alizarin Red staining (ARS) as described previously [26] after 3 weeks of osteogenic induction [27].…”

“…The EVs were analyzed by NTA to determine vesicle concentration and size distribution using (0.1 μm) DPBS to obtain the optimal detection concentration of 10 6 -10 9 particles/ml, and three 60 s videos were recorded using camera level 13. The data was analyzed using NTA software 3.0 with the detection threshold 5 [27].…”

“…EV samples were prepared for EM and imaged as described previously [27,28]. Briefly, samples from 2-3 biological replicates were viewed with transmission EM using Tecnai 12 (FEI Company) operating at 80 kV.…”

Mesenchymal stromal cell-derived extracellular vesicles for regenerative therapy and immune modulation: Progress and challenges toward clinical application

“…28 One important consideration for cell culture conditions is the potential contamination of bovine EVs from serum in growth media. 29 Hence, the use of serum-free conditions when isolating cell-derived EVs are likely necessary to align manufacturing methods with the ever-increasing stringency of regulatory requirements for accreditation standards.…”

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

“…Therefore, we contend that it remains impossible to confidently assert that using existing methodologies for FBS EV-depletion (i.e. ultracentrifugation [3], chemicalbased precipitation, size-exclusion chromatography, ultrafiltration [13]) can reduce the number of FBS EVs present within the resultant supernatant used to supplement culture media to non-confoundable levels.…”