Fetal Bovine Serum (FBS) is a supplement used in cell culture laboratories worldwide. Derived from the liquid fraction of blood from a bovine fetus, it is rich in proteins and growth factors that support cell growth in an artificial environment. A specialized variant is extracellular vesicle-depleted FBS, from which naturally abundant extracellular vesicles (EVs) have been removed. This purified serum is produced for scientific studies where the presence of these vesicles would interfere with experimental outcomes.
The Confounding Variable in Standard FBS
Extracellular vesicles are membrane-bound particles released by cells into their environment. These vesicles act as messengers, carrying proteins, lipids, and nucleic acids like RNA from one cell to another, influencing the recipient cell’s behavior. Standard FBS is naturally filled with a high concentration of EVs originating from the bovine fetus. This creates a problem for researchers studying the EVs produced by the specific cells they are growing in the lab.
Cultured cells can internalize bovine EVs from the serum, introducing a confounding variable where observed effects could be from the bovine vesicles instead of the cells of interest. When scientists attempt to isolate vesicles secreted by their cultured cells, the large quantity of bovine EVs in standard FBS can co-purify with them. This contamination compromises the integrity of the results.
Imagine trying to study messages sent by one cell type, but the environment is flooded with messages from another source, making it difficult to distinguish the desired signals from background noise. This is the challenge in studies analyzing the molecular cargo of EVs. Researchers looking for specific protein or RNA biomarkers, or investigating the function of cell-secreted vesicles, find their work obscured by the presence of these bovine contaminants.
Methods of Extracellular Vesicle Depletion
Scientists employ several methods to remove contaminating vesicles from FBS, with ultracentrifugation being a well-established technique. This process involves subjecting the serum to high gravitational forces, often exceeding 100,000 times the force of gravity, for periods as long as 18 hours. This force pellets the vesicles, allowing the EV-depleted supernatant to be collected. While effective, ultracentrifugation can also co-precipitate other large protein complexes, which can alter the serum’s composition and affect its ability to support cell growth.
Other methods include ultrafiltration, which uses membranes with specific pore sizes to separate molecules. The serum is passed through a filter with pores small enough to block EVs, while allowing smaller serum proteins and nutrients to pass through. This method can be less disruptive to the serum’s components. Size exclusion chromatography (SEC) is another technique that separates particles by size, allowing smaller serum components to enter porous beads while larger EVs are excluded and flow through more quickly.
Commercially available precipitation-based kits also provide a method for EV depletion. These kits use polymers, such as polyethylene glycol (PEG), that reduce the solubility of the vesicles, causing them to precipitate from the solution. The mixture is then subjected to low-speed centrifugation to pellet the EV-polymer complexes. Each method presents a trade-off between depletion efficiency, cost, and the potential for altering the serum’s growth-promoting properties.
Critical Applications in Cell Biology Research
The use of EV-depleted FBS is important in several areas of cell biology research. One primary application is in functional studies designed to understand the specific roles of EVs secreted by a particular cell type. For example, researchers investigating how cancer cells communicate with their environment use the depleted serum to ensure observed effects are caused only by cancer cell-derived EVs. This allows them to isolate the cancer EVs and study their direct impact on other cells.
This serum is also used in biomarker discovery. Scientists search for proteins, RNA, and other molecules within EVs that can serve as early indicators of diseases like cancer, neurodegenerative disorders, or cardiovascular conditions. The goal is to develop non-invasive diagnostic tests from patient blood samples. For these studies to be accurate, the analyzed EVs must originate solely from the human cells being studied, free from contaminating bovine EVs.
Research into the therapeutic potential of EVs also relies on this purified reagent. Scientists are exploring the use of EVs from stem cells as drug delivery vehicles or as agents to promote tissue repair. Developing these therapies requires a precise characterization of the EVs produced by the therapeutic cell line. Using EV-depleted FBS ensures the isolated vesicles are pure so their therapeutic efficacy can be accurately assessed.
Verification and Performance Considerations
After preparing EV-depleted FBS, researchers verify the extent of vesicle removal. Several quality control techniques are used to confirm a batch of serum is depleted. Nanoparticle Tracking Analysis (NTA) is a method used to quantify the concentration and size distribution of particles. A successful depletion is indicated by a reduction in particles within the EV size range of 30 to 150 nanometers, providing a quantitative measure of efficiency.
Another verification method is molecular analysis. Western blotting can detect common EV marker proteins, such as CD9, CD63, or TSG101, and a decrease in their signal indicates successful removal. For visual confirmation, Transmission Electron Microscopy (TEM) can be employed. This imaging technique allows researchers to directly see the vesicle-like structures, and images of depleted serum should show an absence of these particles compared to untreated serum.
Beyond verifying depletion, it is important to assess the performance of the treated serum. The processes used to remove EVs can also strip the serum of factors that support cell growth. Researchers test the EV-depleted FBS on their specific cell line before launching large-scale experiments. By culturing cells in both standard and depleted FBS, they can monitor for changes in cell proliferation or viability to ensure the depleted serum can still support their experimental system.