Specialty FBS: Fetal Bovine Serum for Specialized Applications

Ultra-low IgG fetal bovine serum has been chromatographically depleted of IgG, an important factor in isolating organisms that may be inhibited by pre-existing levels of IgG or for quantitative IgG immunoassays. Despite IgG depletion, the serum retains full biological activity.

Exosome-depleted FBS, also known as exosome-free FBS, is an ultrapure FBS that provides high levels of exosome depletion (≥90%) while maintaining cell culture performance. This specialty FBS is relied upon for exosome isolation assays and research involving exosome contents, such as miRNAs. By choosing exosome-depleted FBS, the inefficiencies that come with ultracentrifugation of serum are removed.

Figure 1. Growth rate of rat oligodendrocyte cells in exosome-depleted FBS. Rat oligodendrocyte cells were seeded at 1,000 cells per well in 96-well plates and grown in medium containing 2% or 10% by volume of one of the following supplements: Gibco Exosome-Depleted FBS, Gibco FBS, the source of the exosome-depleted version), or ultracentrifuged FBS. After 72 hours in culture, the live cells were stained with Invitrogen Hoechst 33342, and the plate was imaged and analyzed on a 96-well plate imaging instrument (Trophos Plate RUNNER HD). Results are presented as the total cell count as reported by the 96-well plate analysis. (The results were obtained from the laboratory of Dr. Jonathan Gilthorpe in the department of Pharmacology and Clinical Neuroscience at Umeå University, Sweden.)

Figure 2. Viability and viable cell density of cells grown in exosome-depleted FBS. Several cell lines were grown in basal medium (DMEM, high glucose, GlutaMAX Supplement) containing 10% exosome-depleted FBS or 10% source FBS and assayed for viable cell density (VCD) and cell viability by Vi-CELL instrument analysis. Results are presented as the viability or viable cell density that was achieved in exosome-depleted FBS as a percentage of that achieved in the source FBS (used to make the exosome-depleted FBS).

Figure 3. Comparison of exosome depletion methods for FBS. Three different lots of FBS were subjected to 3 different methods of exosome depletion. The first used ultracentrifugation at 110,000 X g for 3 hours (labeled as Ultracentrifuge 1). The second method used ultracentrifugation at 150,000 X g for 18 hours (labeled as Ultracentrifuge 2). The third method used our proprietary process for the depletion of exosomes from FBS (labeled as exosome-depleted FBS). Source FBS refers to the FBS prior to any exosome depletion process. After the exosome depletions, relative levels of remaining exosomes were measured by extracting exosomes from depleted sera using the Total Exosome Isolation Reagent (from serum), then staining the isolated exosomes with a lipophilic dye. The fluorescent signal is displayed as Relative Fluorescence Units (RFU). The data demonstrate the improved efficiency of exosome depletion using our proprietary method compared to ultracentrifugation.

Figure 4. Verification of FBS exosome depletion. The exosome depletion from FBS samples was verified by analysis on a NanoSight instrument (comparing the 30–150 nm count before and after exosome depletion) as well as via fluorescence-based assay. Briefly, this assay involves extracting exosomes from serum using the Total Exosome Isolation Reagent, and then staining the isolated exosomes with Invitrogen BODIPY TR Ceramide. The percent depletion is derived from comparing the fluorescent signal of the exosome-depleted FBS with the source FBS. The first two exosome-depleted lots shown were produced by our proprietary manufacturing method. Included in the same analysis was a sample of FBS that was ultracentrifuged to deplete exosomes and an exosome-depleted FBS product from a competitor.

Figure 5. Western blot analysis of exosome-depleted FBS. (A) Analysis of FBS by SDS-PAGE: Equal masses (10 µg) of FBS proteins were analyzed by SDS-PAGE. Exosome-depleted FBS looks very similar to the source FBS used in production. (B) CD63 content by western blot analysis: Exosomes were precipitated from FBS samples, run by SDS-PAGE, and probed by western blotting for CD63. Compared to all other FBS samples tested, exosome-depleted FBS shows no observable CD63, indicating excellent exosome depletion. MW = molecular weight marker.

Figure 6. miRNA detection in FBS sample-derived exosomes. Exosomes were isolated from FBS samples, and total RNA was isolated from the exosomes (using the Total Exosome RNA & Protein Isolation Kit). RNA was then analyzed by qRT-PCR for Let7e, miR16, miR21, miR23a, miR24, and miR122 using the Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit.

MSC-qualified FBS is specially tested to support the expansion and clonal enumeration (by colony forming unit-fibroblast assay) of mesenchymal stem cells (MSCs). MSC-qualified FBS has been carefully designed to eliminate the need for testing multiple FBS lots to identify the optimal one for MSC research.

Figure 7. Effect of FBS source on MSC clonal efficiency. Results from Gibco FBS (2 lots tested are indicated by red and blue bars) are shown along with the results from a competitor's FBS product (yellow bar). P<0.05 via student’s t-test.

Figure 8. Effect of FBS source on MSC expansion. Results from Gibco FBS (2 lots tested are indicated by red and blue bars) are shown along with the results from a competitor's FBS product (yellow bar). P<0.05 via student’s t-test.

ESC-qualified FBS, produced using an industry-leading qualification assay, is specifically designed for achieving high plating efficiencies and maintaining pluripotency of embryonic stem cells (ES cells). ES cells are derived from the inner cell mass of blastocysts and are used in a variety of studies regarding biological functioning. When culturing ES cells, it is critical that they maintain the ability to differentiate into the cell type of interest in order to test expression and other characteristics. Therefore, ESC-qualified FBS helps maintain proper growth conditions for ES cells.

Figure 10. Phase contrast and fluorescent images of mESC colonies. mESCs cultured in media supplemented with ESC-qualified FBS exhibit the morphological traits of well-defined refractive edges, tight colonies with small cells, and few differentiated cells. These cells also exhibit pluripotency via Alkaline Phosphatase (AP) Live Stain and the expression of Oct4 and Sox2.

Figure 11. mESCs grown in media supplemented with ESC-qualified FBS exhibit normal karyotypes. Normal karyotype report for PRX129/X1 mESC line. 18 of 20 cells tested exhibited a normal karyotype (Cell Line Genetics).

Figure 12. mESCs cultured in media supplemented with ESC-qualified FBS maintain LIF responsiveness. When LIF is removed from mESC media, normal mESC cultures respond with widespread differentiation. PRX129/X1 murine ES cells grown with and without LIF demonstrate this response.