Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Abstract

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

Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. 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 eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol.

Exosomes are small extracellular vesicles (EVs) that are 40–100 nm in size, and are considered to be released by most cells via an intracellular origin involving multivesicular bodies (1,2). However, several other subgroups of EVs also exist, including larger microvesicles thought to be released directly from the cell surface (3). EVs are thought to take part in cell-to-cell communication, as they can shuttle proteins, lipids, and RNA between cells (4–6), and the status of the producing cells determines the signal conveyed (6,7).

EVs are also found in all body fluids, such as serum, saliva, breast milk, and urine (8,9). As cell culture experiments often involve the presence of fetal bovine serum (FBS), it is possible that vesicles present in the FBS can influence experimental results. Indeed, it has been suggested that FBS-derived EVs may influence results in cell biology, such as growth of breast cancer cell lines (10). Also FBS-derived EVs are a major cause of concern as these vesicles could contaminate EVs derived from cell cultures (11,12). Therefore, EVs are often removed from the FBS prior to use in cell culture experiments, by the use of different depletion protocols (13,14).

exosome depleted fbs media supplement
exosome depleted fbs media supplement

The aims of this study were firstly to determine whether EVs present in FBS can influence cultured cells phenotype such as the migration of an airway epithelial cancer cells, and secondly to determine the efficiency of elimination of EVs by different ultracentrifugation protocols.

Bovine exosome-depleted FBS, for isolating exosomes from cultured cells without the complication of bovine exosomes that are present in conventional FBS.
  • Exosome-sized vesicles removed
  • Very low levels of CD63-positive bovine exosomes
  • Undetectable levels of bovine miRNAs
  • Comparable growth rates as standard FBS
  • Interchangeable with standard FBS (add 10% in DMEM or RPMI)

Exosome-depleted FBS Media Supplement Heat Inactivated - USA Certified      

EXO-FBSHI-50A-1 50 ml
EUR 255
  • Category: Exosomes

Human Serum (CRP Depleted)

90R-105 100 ml
EUR 740
Description: C-reactive protein depleted normal human serum

Human serum C4-depleted

C415-DS-1 1 ml
EUR 408

Human serum C3-depleted

C316-DS-1 1 ml
EUR 408

Human C1q depleted sera

C1Q17-DS-1 1 ml
EUR 408

Fibrinogen (plasminogen depleted), Human Plasma

7692-1
EUR 294

Human Complement C5 depleted serum

C5512-DS 1 ml
EUR 408

Chimpanzee control serum, (IgG's-depleted)

NCMS-DG-1 1 ml
EUR 225

Human Ferritin depleted/stripped serum (Ferritin

1810-FDS-10 10 ml Ask for price

Human Ferritin depleted/stripped serum (Ferritin

1810-FDS-1000 1000 ml Ask for price

Fibrinogen (Pg & vWF depleted), Human Plasma

7693-500
EUR 294

Monkey (Cynomolgus) control serum (IgG's-depleted)

NMCS-DG-1 1 ml
EUR 225

Psoriasis Exosome

P141-PS NULL
EUR 0

Fibrinogen (Pg, Fn & vWF depleted), Human Plasma

7694-250
EUR 180

Lyophilized Exosome Standards

ExoA375 100 μg
EUR 303.5

Lyophilized Exosome Standards

ExoHT29 100 μg
EUR 303.5

Lyophilized Exosome Standards

ExoMCF7 100 μg
EUR 303.5

Lyophilized Exosome Standards

ExoPC3 100 μg
EUR 303.5

Lyophilized Exosome Standards

ExoPLASMA 100 μg
EUR 303.5

Lyophilized Exosome Standards

ExoUrine 100 μg
EUR 303.5

Exosome precipitation Solution

EPStep 12 ml
EUR 277.5

Leukemia - ALL Exosome

P141-ALL NULL
EUR 0

Leukemia - AML Exosome

P141-AML NULL
EUR 0

Bladder Cancer Exosome

P141-BL NULL
EUR 0

Breast Cancer Exosome

P141-BR NULL
EUR 0

Leukemia - CLL Exosome

P141-CLL NULL
EUR 0

Leukemia - CML Exosome

P141-CML NULL
EUR 0

Colorectal Cancer Exosome

P141-CO NULL
EUR 0

Crohn's Disease Exosome

P141-CR NULL
EUR 0

Kidney Cancer Exosome

P141-KN NULL
EUR 0

Lung Cancer Exosome

P141-LG NULL
EUR 0

Liver Cancer Exosome

P141-LV NULL
EUR 0

Nasopharyngeal Cancer Exosome

P141-NA NULL
EUR 0

Prostate Cancer Exosome

P141-PR NULL
EUR 0

Rheumatoid Arthritis Exosome

P141-RA NULL
EUR 0

Thyroid Cancer Exosome

P141-TH NULL
EUR 0

Psoriasis Exosome RNA

P241-PS NULL
EUR 0

Fetal Bovine Serum,IgG Depleted, < 2 ug/ml

F0720-050 500 ml
EUR 1198

Exosome Isolation kit (for serum & plasma -Exosome-RNA-NGS)

P101NGS NULL
EUR 0