Long-term growth comparison studies of FBS and FBS alternatives in six head and neck cell lines

<div><p>Fetal bovine serum (FBS) is depended upon by investigators as an indispensable supplement in cell and tissue culture systems. Due to increased demand and limited availability, the price of FBS has increased by greater than 300% in the past few years. In addition, there are ethical and scientific controversies about the collection and use of FBS in culture systems. In response to the shortage of FBS, many FBS alternative serum products have been developed. Although many have claimed comparable performance to FBS, their support of long-term cell growth and effects on cell phenotype have not been revealed. In this study, we examined the performances of six bovine calf serum-based FBS alternatives in six head and neck cell lines and compared them with FBS. The results indicate that some of these sera had growth promoting capabilities comparable or superior to that of FBS. Additionally, these alternative sera supported long-term (30 passages) growth of tested cells and exhibited plating efficiencies comparable to that of FBS. Cells cultured in alternative sera also exhibited comparable anchorage-independent growth and similar drug inhibition responses in FBS. Still, caution should be taken in choosing suitable sera given that changes in cell morphology and variations in chemotactic responses were noted for cells maintained in certain sera. These FBS alternatives are more readily available, cost less, and are associated with less ethical concerns, thus making them attractive alternatives to FBS in cell culture systems.</p></div

Morphology analysis of FaDu cells cultured in alternative sera.

<p>The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.</p

Plating efficiency of cells cultured in alternative sera.

<p>Plating efficiency was determined in cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) serum. Data indicate the average value of duplicates (mean ± SD). *:p<0.05 compared with cells cultured in FBS. N.D.: not determined because subculture terminated at P10 in OECM-1 cells.</p

The transcription factor profile of TW01 cells cultured in FBS and FC3.

<p>(A) The scatter plots of the transcription factor profile in TW01 P1 cells. (B) The scatter plots of the transcription factor profile in TW01 P30 cells. The central line indicates unchanged gene expression; dotted lines represent the two-fold regulation cut-off. Genes with fold change > 2 indicated. (C) Western blot analysis of CTNNB1, ELK1, SMAD4, and c-Fos expression in TW01 and HONE-1 P30 cells cultured in different sera. GAPDH is detected as a loading control.</p

Additional file 3: Figure S2. of Secretory RAB GTPase 3C modulates IL6-STAT3 pathway to promote colon cancer metastasis and is associated with poor prognosis

RAB3C overexpression increases exocytosis of colon cancer cells and promotes metastasis through IL-6 secretion. (A) The significant effect of RAB3C overexpressing cell-conditioned medium on the migration ability of parental colon cancer cells indicate that the metastasis-promoting role of RAB3C is exocytosis dependent. (B) Relative IL-6 activity in conditioned medium of CX-1 cells and SW48 cells with or without the exogenous RAB3C gene. The data were the average of three independent experiments and are presented as the mean ¹ SEM. The significance of the difference was analyzed using the nonparametric Mann-Whitney U test. (TIFF 28902 kb

Newborn calf serum did not provide favorable conditions for the growth of head and neck cell lines.

<p>The proliferation of cells cultured in NBCS and CS at 72 h was presented as the relative proliferation ratio of cells in FBS (1.0-fold). Data indicate the average value of triplicates (mean ± SD). *:p<0.001 compared with cells cultured in FBS. NBCS-G: Gibco NBCS; NBCS-H: HyClone NBCS.</p

Anchorage-independent growth of cells cultured in alternative sera.

<p>Soft-agar colony formation assays were performed in TW01 and HONE-1 cells. Colony numbers were counted after three weeks of growth. Data indicate the average value of triplicates (mean ± SD). *:p = 0.004 compared with cells cultured in FBS.</p

Morphology analysis of SCC25 cells cultured in alternative sera.

<p>The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.</p

Morphology analysis of HONE-1 cells cultured in alternative sera.

<p>The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.</p

Long-term proliferation comparison of cells in FBS and alternative sera.

<p>The proliferation profiles of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, 20, and 30. Cells cultured in FBS were adjusted as the baseline (1.0-fold), and the relative proliferation ratio of cells in other sera was determined accordingly. Data indicate the average value of triplicates (mean ± SD). *:p<0.01 compared with cells cultured in FBS at the same passage. N.D.: not determined for OECM-1 cells cultured in CS due to subculture termination at passage 10.</p