Cell culture supplements containing insulin and transferrin are used to reduce or eliminate the need for serum in cell culture media. One widely used supplement is a mixture of insulin, transferrin, selenium and ethanolamine (ITSE). In this study, the performance of an animal-free version of ITSE supplement (ITSE Animal-Free™, InVitria) was compared to an ITSE supplement containing animal-derived components. Three cellular performance parameters were compared: log-phase cell prolifeation, cumulative cell density (CCD), and the production of antibody. InVitria’s ITSE Animal-Free™ was found to have equivalent activity to animal-derived ITSE supplement.
Classical media formulations (such as DMEM, RPMI 1640, and MEM) were designed for use with serum. Serum contains the factors necessary for cells to grow and maintain their phenotypic properties. Many of the serum factors required for the growth of cells have been identified. Among these are insulin, transferrin, and selenium [1-3]. Depending on the cell type, ethanolamine may be required . When added to media, these components can reduce or eliminate the need for serum in cell culture.
Insulin is a growth factor that promotes the uptake and utilization of glucose and amino acids . Transferrin delivers iron into cells in a regulated manner and avoids the toxic oxidative effects of free iron compounds [5-8]. Due to its increase potency in human and murine cell cultures, human transferrin is typically preferred over bovine transferrin  in cell culture applications.
Selenium is required for the activity of glutathione peroxidase, thioredoxin reductase, and other antioxidant enzymes . Ethanolamine is a precursor for phospholipid synthesis . Some cell types are able to synthesize adequate levels of ethanolamine without supplementation . Murine hybridoma cells require ethanolamine supplementation for serum-free growth . Other cell types, such as Vero and CHO, benefit from ethanolamine supplementation of the medium [13, 14].
Insulin-transferrin supplements on the market are typically denoted by the first letters of the ingredients. ITS supplement contains insulin, transferrin, and selenium. ITSE, also known as SITE, (insulin, transferrin, selenium, ethanolamine) is a commonly used formulation .
One disadvantage to the use of commercial Insulin-transferrin supplements is the inclusion of blood-derived proteins in the formulation. Blood-derived components present a risk of contamination from blood-borne pathogens.
InVitria manufactures the only animal-free insulin-transferrin supplement available on the market, ITSE Animal-Free™. In this study, we compared the performance of ITSE Animal-Free™ to a commercially available animal-derived ITSE supplement.
Materials and Methods
ITSE Animal-Free™ is a product of InVitria. Animal-derived ITSE was obtained from Invitrogen (cat # 51500). While both products supplement media with 10 mg/L human recombinant insulin, 0.0067 mg/L sodium selenite, and 2 mg/L ethanolamine, ITSE Animal-Free™ contains 5.5 mg/L Optiferrin™ recombinant human transferrin, (InVitria) as a substitute for the 5.5 mg/L of blood derived human transferrin in the animal-derived ITSE.
SP2/0 M:M hybridoma cells were maintained in DMEM/F12 medium supplemented with 10% FBS. Cell based assays were performed using a serum-free medium composed of DMEM/ F12 medium supplemented with 1 g/L Cellastim™ recombinant human albumin (InVitria) and the respective ITSE supplement.
Cells were washed three times in DMEM/F12 medium without serum and seeded at 0.5 x 105 cells/ml in triplicate 4 ml cultures in a 6-well plate (on day 0). The concentration of viable cells was determined daily for 6 days by a flow cytometer (Millipore). The concentration of secreted IgG antibody in the medium after 6 days of culture was determined by a proprietary florescence-based ELISA.
The comparison of ITSE supplements to support cell proliferation.
The two ITSE supplements were compared for the ability to support cell proliferation.
The concentration of viable cells was determined during log phase growth, after 3 days of culture. Figure 1 shows that ITSE Animal-Free™ had equivalent activity to the animal-derived ITSE to support cell proliferation.
The comparison of ITSE supplements for increasing Cumulative Cell Density.
Cumulative cell density (CCD) is a cell culture performance metric that measures of the ability of a cell culture medium to support sustained high density cell growth . CCD , also known as the Integral of the Viable Cell Concentration (IVC, IVCN), is the calculated area under a multi-day growth curve .
Figure 2 shows that both ITSE supplements resulted in similar 6-fold increases in CCD.
The comparison of ITSE supplements for the production of antibody.
Figure 3 shows that both ITSE supplements resulted in similar increases in antibody production. The antibody concentration in the medium was > 3 fold increased in the ITSE supplemented medium.
Discussion and conclusion
We found that ITSE Animal-Free™ has equivalent activity to animal-derived ITSE supplement in 3 different measure of cell performance. Moreover, the growth curves of cells in medium supplemented with either of the two ITSE supplements were identical (Figure 4).
In summary, these data show that ITSE Animal-Free™ has the same performance as animal-derived ITSE supplement and is an effective animal-free substitute. Moreover, ITSE Animal-Free#8482; is a cost effective alternative to commercial animal-derived versions.
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