Abstract

Extracellular vesicles (EVs), including nano-sized exosomes, have the ability to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement the cell culture medium as a nutrient, but it is important to know that FBS also contains significant amounts of EV. The objective of the present study was to determine whether EV-FBS can influence the phenotype of cultured cells and, secondly, to determine the efficacy of EV-FBS removal protocols.

First, FBS that had not been depleted of EV induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated EV FBS alone. The FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18 hour centrifugation period removes approximately 95% of the RNA-containing EV FBS, while a 1.5-hour protocol is insufficient. In conclusion, this study shows that EV FBS substantially influence the behaviour of cultured cells, but also that they can be virtually eliminated by an 18-hour ultracentrifugation protocol.

Keywords: depletion protocol, exosomes, extracellular vesicles, fetal bovine serum, RNA

Material and methods

• Cell cultures

The human lung epithelial cell line, A549 (ATCC), was grown in DMEM / F12 (1: 1) medium (HyClone Laboratories, Inc., Logan, UT), the human mast cell, HMC-1 (Dr Joseph Butterfield, Mayo Clinic, Rochester, MN, and a kind gift from Professor Gunnar Nilsson of the Karolinska Institute, Stockholm, Sweden), was grown on IMDM (Sigma Aldrich, St. Louis, MO) and the mouse fibroblast cell line, NIH3T3 ( ATCC), was cultured in DMEM (with glutamine; HyClone Laboratories, Inc.).

All media were supplemented with 10% FBS, 100 units/ml penicillin and 100 µg / ml streptomycin (HyClone Laboratories, Inc.). More 2 mM l-glutamine (HyClone Laboratories, Inc.) and 1.2 mM alpha-thioglycerol (Sigma Aldrich) were added to the HMC-1 cell medium. All FBS supplements are reduced from EVs by ultracentrifugation for 18 hours at 120,000 × g (Type 45 Ti rotor, 38,800 rpm, 178.6 k-factor, Beckman blade, Brea, CA) unless specifically mentioned as no impoverished. All cells were cultured at 37 ° C and 5% CO2.

• Isolation of FBS-derived extracellular vesicles for characterization

The FBS was purchased from Sigma Aldrich and had been filtered by the manufacturer (0.1 µm) prior to purchase. The FBS was subjected to centrifugation for 18 hours (overnight) at 120,000 xg (Type 45 Ti rotor, Beckman Coulter). The pellet was washed by adding PBS and placing the sample under gentle agitation until the pellet dissolved, before filtering (0.2 µm) and re-pelleting with ultracentrifugation at 120,000 xg for 70 minutes.

• Analysis of FBS vesicle depletion protocols

To test EV exhaustion protocols, FBS was kept undiluted or diluted with PBS in a ratio of 3: 7 (FBS: PBS). These preparations were then subjected to centrifugation for 1.5 hours or 18 hours (overnight) at 120,000 xg (Type 45 Ti rotor, Beckman Coulter). The supernatants were then used to prepare a medium containing 10% FBS that was never in contact with the cells.

The medium containing 10% FBS was subsequently subjected to a standard exosome isolation protocol using differential centrifugation described previously (14). Briefly, the medium containing FBS was sequentially centrifuged at 300 xg (10 minutes) to remove debris and at 16,500 xg (20 minutes) to remove large vesicles. In addition, the supernatant was filtered with a 0.2 µm filter before centrifuging the samples at 120,000 xg (70 minutes) to pellet the exosomes.

• Reverse migration test

To assess the migratory phenotype of lung epithelial cells (A549), a reverse migration assay was performed with a 46-well Boyden chamber (Neuroprobe Inc., Gaithersburg, MD) as reported (15). Briefly, 20,000 cells / well were added to the lower chamber and allowed to adhere to the gelatin-coated polycarbonate membrane (0.1%) by inverting the entire chamber upside down for 3 hours.

The chamber was then placed in the correct orientation and incubated for 12 hours with various doses of FBS-EV in the upper chamber, which is the opposite side of the membrane in relation to the cells. The membrane was then carefully removed and the cells on the migrated side were fixed in ethanol and stained with Giemsa (Histolab, Gothenburg, Sweden). Cells on the non-migrated side were completely removed before imaging with a light microscope (Zeiss Axioplan, Gottingen, Germany).

• RNA isolation and detection

RNA was isolated from the different sediments described in the sections “Isolation of extracellular vesicles derived from FBS for characterization” and “Analysis of FBS vesicle depletion protocols”, using miRCURY ™ RNA Isolation Kit for Cell and Plant (Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol and as previously described (16). A microliter of isolated RNA was analyzed for quality, yield, and nucleotide length with capillary electrophoresis using an Agilent RNA 6000 Pico chip on an Agilent 2100 Bioanalyzer® (Agilent Technologies, Santa Clara, CA).

• Proteinase and RNase Protection Assay

For this assay, washed sediments obtained according to the method section “Isolation of extracellular vesicles derived from FBS for characterization” were used. The isolated vesicles were incubated with proteinase K (0.05 µg / µl; Sigma Aldrich) for 10 minutes at 37 ° C, before adding 5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Aldrich) for 10 minutes at room temperature to inhibit the Proteinase K activity. Proteinase K activity was further inhibited by incubating the solution at 90 ° C for 5 minutes.