Human AB serum as an alternative to fetal bovine serum for endothelial and cancer cell culture *

In vitro methods are widely used in biomedical research and industry, often reducing or replacing animal experiments. However, cell cultures are generally supplemented with fetal bovine serum (FBS), also known as fetal calf serum (FCS), to optimize cell growth. Concerns have been raised regarding both the method used to collect the serum from the donor fetus and the possibility of viral contamination of serum (van der Valk et al., 2004, 2010; Gstraunthaler, 2003; toldbod et al., 2003; Warncke et al., 2006; Brunner et al., 2010). thus, the replacement of FBS has become a desirable objective in the field of cell and tissue culture. Current approaches include the replacement of FBS by human platelet lysates, bovine milk growth factors, human umbilical cord blood, serum and plant extracts, among others (van der Valk et al., 2004; Bruserud et al., 2005; Clavreul et al., 2009; Kocaoemer et al., 2007; Rauch et al., 2011; tekkatte et al., 2011; Pazos et al., 2004; Gstraunthaler, 2003). Some of these approaches require special formulations to be developed for each cell type. A promising alternative for culture of human cells is the substitution of FBS with converted human AB serum (HABS), which is routinely tested for viral contamination. HABS supports the propagation of human osteoblasts, chondrocytes, and bone marrow cells (Munirah et al., 2008; Hankey et al., 2001; Yamamoto et al., 2003; Yamaguchi et al., 2002), as well as glioma and melanoma cancer cell lines (Clavreul et al., 2009; Pandolfino et al., 2010). these studies suggest that HABS may be a good candidate to replace FBS as a supplement for cell culture. We tested this using colorectal (Ht29), cervical (Hela-229), and breast (MCF 7) cancer cell lines routinely used in cancer research, as well as primary human umbilical vein endothelial cells (HUVeC) and human dermal microvascular endothelial cells (HuDMeC).


Human AB serum as an alternative to fetal bovine serum for endothelial and cancer cell culture*
In vitro methods are widely used in biomedical research and industry, often reducing or replacing animal experiments.However, cell cultures are generally supplemented with fetal bovine serum (FBS), also known as fetal calf serum (FCS), to optimize cell growth.Concerns have been raised regarding both the method used to collect the serum from the donor fetus and the possibility of viral contamination of serum (van der Valk et al., 2004(van der Valk et al., , 2010;;Gstraunthaler, 2003;toldbod et al., 2003;Warncke et al., 2006;Brunner et al., 2010).thus, the replacement of FBS has become a desirable objective in the field of cell and tissue culture.
A promising alternative for culture of human cells is the substitution of FBS with converted human AB serum (HABS), which is routinely tested for viral contamination.HABS supports the propagation of human osteoblasts, chondrocytes, and bone marrow cells (Munirah et al., 2008;Hankey et al., 2001;Yamamoto et al., 2003;Yamaguchi et al., 2002), as well as glioma and melanoma cancer cell lines (Clavreul et al., 2009;Pandolfino et al., 2010).these studies suggest that HABS may be a good candidate to replace FBS as a supplement for cell culture.
We tested this using colorectal (Ht-29), cervical (Hela-229), and breast (MCF 7) cancer cell lines routinely used in cancer research, as well as primary human umbilical vein endothelial cells (HUVeC) and human dermal microvascular endothelial cells (HuDMeC).
Ht-29 and Hela 229 were from the european Collection of Cell Culture (Salisbury, UK); MCF 7 was a gift from the Institute for Cancer Studies (School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK).Media were from Gibco (UK).Cancer cell lines were maintained in McCoys (Ht-29), DMeM with sodium pyruvate (Hela), and DMeM without sodium pyruvate (MCF 7) medium supplemented either with 50 ml of human AB converted serum (HABS; PAA laboratories, UK) or FBS (Invitrogen, UK).
Cells were diluted to 2 x 10 3 cells per 200 µl full growth medium per well and placed in quadruplicates on uncoated, collagen IV (primary cells, HABS group) or gelatin pre-coated (primary cells, FBS group) 96-well plates.Medium was changed after 4 h and cells were incubated at 37°C in 5% CO 2 .Proliferation was assessed by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Mtt) colorimetric assay (Sieuwerts et al., 1995) after 1, 2, 3, 4, and 5 days.Absorbance was measured at 540 nm using a MRx II microtiter plate reader using revelation software (Dynex, UK). the means were calculated for each quadruplicate, and the overall mean and standard deviation (SD) were calculated for each data point.Calibration curves were constructed by plating increasing densities of cells in triplicates and measuring their Mtt metabolism, Letter * This work was funded by The Lord Dowding Fund, UK.
as above, on day one.Mean absorbance values were calculated for each triplicate, and the overall mean and standard deviation (SD) were calculated for each data point.
We found that HABS supported the growth of all primary cultures of endothelial cells and cancer cell lines tested.Increasing numbers of cells correlated with increasing absorbance levels in all cells studied (shown exemplarily in Fig. 1, left panel).Over a 5-day period, numbers of endothelial cells and cancer cells increased in parallel in HABS-supplemented and FBS-supplemented cultures, as evidenced by increasing formazan metabolism (shown exemplarily in Fig. 1, right panel).No statistically significant differences were observed between HABS-and FBS-supplementation over this time period for any of the cell types.
A drawback of using HABS for cell culture is the cost.Commercial human serum can be up to 4-5 times more expensive than FBS.Here, we used serum concentrations of 10% and 5% for cancer and endothelial cells.However, it may be worthwhile to explore whether lower human serum levels in media would be able to support cell cultures without compromising cell growth.
In conclusion, human serum supported the growth of primary endothelial cells and cancer cell line cultures, and it seems to be a good candidate to replace FBS as a medium supplement in cell culture.Brunner, D., Frank, J., Appl, H., et al. (2010)

Fig. 1 :
Fig. 1: Relation between cell number and formazan production and comparison of growth with FBS and HABS shown exemplarily for HuDMEC and HT-29 cells Left panel: Increasing cell concentrations were plated (in triplicate) and cultured either with FBS-(solid bar) or HABS-(open bar) supplemented medium.Formazan production was measured after 24 h.Each data point represents the average of three (n=3) separate experiments ±SD.All n.s., Mann-Whitney U-test.Right panel: 2,000 cells/well were plated (in quadruplicate) and formazan production was measured on days 1, 2, 3, 4 and 5.Each data point represents the average of three (n=3) separate experiments ±SD.All n.s., Mann-Whitney U-test.Comparable observations were obtained for HUVEC, HeLa 229, and MCF 7 cultures (not shown).