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)

Human serum C4-depleted
C415-DS-1 1 ml
EUR 489.6

Human Serum (CRP Depleted)
90R-105 100 ml
EUR 888
Description: C-reactive protein depleted normal human serum

Human Complement C5 depleted serum
C5512-DS 1 ml
EUR 489.6

Fibrinogen (plasminogen depleted), Human Plasma
7692-1
EUR 352.8

Chimpanzee control serum, (IgG's-depleted)
NCMS-DG-1 1 ml
EUR 270

Psoriasis Exosome
P141-PS - Ask for price

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 352.8

Monkey (Cynomolgus) control serum (IgG's-depleted)
NMCS-DG-1 1 ml
EUR 270

Human Comp IgG/IgM Depleted Pooled (mL)
34010-0 mL
EUR 78

Human Comp IgG/IgM Depleted Pooled (1mL)
34010-1 1mL
EUR 123.67

Human Comp IgG/IgM Depleted Pooled (10mL)
34010-10 10mL
EUR 534.76

Human Comp IgG/IgM Depleted Pooled (100mL)
34010-100 100mL
EUR 4645.5

Human Comp IgG/IgM Depleted Pooled (2mL)
34010-2 2mL
EUR 169.36

Human Comp IgG/IgM Depleted Pooled (3mL)
34010-3 3mL
EUR 215.03

Human Comp IgG/IgM Depleted Pooled (5mL)
34010-5 5mL
EUR 306.37

Fibrinogen (Pg, Fn & vWF depleted), Human Plasma
7694-250
EUR 216

Lyophilized Exosome Standards
ExoA375 100 μg
EUR 364.2

Lyophilized Exosome Standards
ExoHT29 100 μg
EUR 364.2

Lyophilized Exosome Standards
ExoMCF7 100 μg
EUR 364.2

Lyophilized Exosome Standards
ExoPC3 100 μg
EUR 364.2

Lyophilized Exosome Standards
ExoPLASMA 100 μg
EUR 364.2

Lyophilized Exosome Standards
ExoUrine 100 μg
EUR 364.2

Exosome precipitation Solution
EPStep 12 ml
EUR 333

Leukemia - ALL Exosome
P141-ALL - Ask for price

Leukemia - AML Exosome
P141-AML - Ask for price

Bladder Cancer Exosome
P141-BL - Ask for price

Breast Cancer Exosome
P141-BR - Ask for price

Leukemia - CLL Exosome
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Leukemia - CML Exosome
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Colorectal Cancer Exosome
P141-CO - Ask for price

Crohn's Disease Exosome
P141-CR - Ask for price

Kidney Cancer Exosome
P141-KN - Ask for price

Lung Cancer Exosome
P141-LG - Ask for price

Liver Cancer Exosome
P141-LV - Ask for price

Nasopharyngeal Cancer Exosome
P141-NA - Ask for price

Prostate Cancer Exosome
P141-PR - Ask for price

Rheumatoid Arthritis Exosome
P141-RA - Ask for price