ITSE Animal Free™ has equivalent performance to animal-derived ITSE

Abstract

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.

Introduction

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 [2]. 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 [4]. 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 [9] in cell culture applications.

Selenium is required for the activity of glutathione peroxidase, thioredoxin reductase, and other antioxidant enzymes [10]. Ethanolamine is a precursor for phospholipid synthesis [11]. Some cell types are able to synthesize adequate levels of ethanolamine without supplementation [12]. Murine hybridoma cells require ethanolamine supplementation for serum-free growth [2]. 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 [2].

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.

Results

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.

Comparison of ITSE supplements to support the proliferation of cells
Figure 1. Comparison of ITSE supplements to support the proliferation of cells.
Yellow, medium without ITSE; red, animal derived ITSE; blue, ITSE Animal-Free™. Error bars denote the standard deviation between triplicate cultures

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 [15]. CCD , also known as the Integral of the Viable Cell Concentration (IVC, IVCN), is the calculated area under a multi-day growth curve [15].

Figure 2 shows that both ITSE supplements resulted in similar 6-fold increases in CCD.

Comparison of ITSE supplements to increase cumulative cell density
Figure 2. Comparison of ITSE supplements to increase cumulative cell density.
Yellow, medium without ITSE; red, animal derived ITSE; blue, ITSE Animal-Free™. Error bars denote the standard deviation between triplicate cultures.

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.

Comparison of ITSE supplements for the production of antibody from hybridoma cells
Figure 3. Comparison of ITSE supplements for the production of antibody from hybridoma cells.
Yellow, medium without ITSE; red, animal-derived ITSE; blue, ITSE Animal-Free™. Error bars denote the standard deviation between triplicate cultures.

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).

Comparison of hybridoma cell growth kinetics in batch culture with ITSE supplements.
Figure 4. Comparison of hybridoma cell growth kinetics in batch culture with ITSE supplements
Shown are the growth kinetics of hybridoma cells in serum-free medium supplemented with either ITSE Animal-Free™ (blue) or animal-derived ITSE (red) in a 6 day batch culture. Yellow, medium without ITSE. Error bars denote the standard deviation between triplicate cultures.

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.

References

  1. Breitman, T.R., S.J. Collins, and B.R. Keene, Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60. 1980. (6988226) Exp Cell Res. 126 (2): p. 494-8.
  2. Murakami, H., H. Masui, G.H. Sato, N. Sueoka, et al., Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. 1982. (7041116) Proc Natl Acad Sci U S A. 79 (4): p. 1158-62
  3. Ozturk, S.S. and B.O. Palsson, Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production. 1990. (1366938) J Biotechnol. 16 (3-4): p. 259-78.
  4. Straus, D.S., Growth-stimulatory actions of insulin in vitro and in vivo. 1984. (6376081) Endocr Rev. 5 (2): p. 356-69.
  5. Anderson, G.J., D.M. Frazer, and G.D. McLaren, Iron absorption and metabolism. 2009. (19528880) Curr Opin Gastroenterol. 25 (2): p. 129-35.
  6. Johnson, M.B. and C.A. Enns, Diferric transferrin regulates transferrin receptor 2 protein stability. 2004. (15319290) Blood. 104 (13): p. 4287-93.
  7. Eisenstein, R.S. and K.P. Blemings, Iron regulatory proteins, iron responsive elements and iron homeostasis. 1998. (9868172) J Nutr. 128 (12): p. 2295-8.
  8. Tanaka, H. and S.L. Teitelbaum, Vitamin D regulates transferrin receptor expression by bone marrow macrophage precursors. 1990. (2246328) J Cell Physiol. 145 (2): p. 303-9.
  9. Young, S.P. and C. Garner, Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro. 1990. (2302189) Biochem J. 265 (2): p. 587-91.
  10. Saito, Y., Y. Yoshida, T. Akazawa, K. Takahashi, et al., Cell death caused by selenium deficiency and protective effect of antioxidants. 2003. (12888577) J Biol Chem. 278 (41): p. 39428-34.
  11. Arthur, G. and L. Page, Synthesis of phosphatidylethanolamine and ethanolamine plasmalogen by the CDP-ethanolamine and decarboxylase pathways in rat heart, kidney and liver. 1991. (1989575) Biochem J. 273(Pt 1): p. 121-5.
  12. Voelker, D.R., Phosphatidylserine functions as the major precursor of phosphatidylethanolamine in cultured BHK-21 cells. 1984. (6425837) Proc Natl Acad Sci U S A. 81 (9): p. 2669-73.
  13. Kim, E.J., N.S. Kim, and G.M. Lee, Development of a serum-free medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44) using a statistical design: beneficial effect of weaning of cells. 1999. (10478796) In Vitro Cell Dev Biol Anim. 35 (4): p. 178-82.
  14. Rourou, S., A. van der Ark, T. van der Velden, and H. Kallel, Development of an animal-component free medium for vero cells culture. 2009. (19768803) Biotechnol Prog. 25 (6): p. 1752-61.
  15. Renard, J., R. Spagnoli, C. Mazier, M. Sales, et al., Evidence that the monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. 1988. Biotechnol Let. 10 (2): p. 91-96.