Early developments in serum-free media led to a variety of formulations in which components normally provided in serum and required for growth (insulin, transferrin, lipid supplements, trace elements) and poorly defined components (extracts, hydrolysates) were added to defined basal media. These additives were mostly animal-derived. Given recent concerns about TSEs (transmissible spongiform encephalopathies) and other adventitious agents, the drive in media formulations must be towards elimination of animal-origin materials while maintaining cell line productivity. The progress made towards removing animal-derived components and the use of recombinant proteins in serum-free media for mammalian cells is reviewed.

DeltaFerrin TM , a yeast-derived recombinant human transferrin produced by Delta Biotechnology Ltd. (Nottingham UK), was found to be a suitable replacement for holo human transferrin in serum-free culture media of the MDCK cell line (chosen because of its transferrin dependence) in short-term screening assays. Long-term subculture was achieved with DeltaFerrin TM supporting growth equivalent to that of holo human transferrin. DeltaFerrin TM and a selection of chemical iron chelators were found in short-term assays to be equivalent to holo human transferrin in supporting growth of MDCK, BHK-21-PPI-C16 and Vero-PPI. In long-term subcultures, however, only DeltaFerrin TM was found to support cell growth in a manner essentially equivalent to holo human transferrin in all three cell lines. For both BHK and Vero variants tested, recombinant preproinsulin production was unaltered by replacing holo human transferrin with DeltaFerrin TM . As such, this is the first report of a recombinant human transferrin produced under animal-free conditions that can act as a universal iron chelator for cells grown in serum-free media (SFM).

Serum-derived albumin has for a long time been used in cell culture media, but the exact role of albumin and/or impurities bound to albumin has not been precisely defined. In this study, recombinant human albumin was evaluated for its growth-promoting activity on two cell lines, NRK and SCC-9. For NRK cells, the recombinant human albumin was found to exert an inhibitory effect. The fact that fatty acid free HSA was also inhibitory while HSA fraction V was stimulatory suggested a role for fatty acids or some other bound moieties in growth stimulation by HSA fraction V. Addition of oleic acid, cholesterol, phosphatidylcholine, phosphatidylserine or a combination of these lipids, however, did not significantly improve the growth stimulating activity of either fatty acid free HSA or the recombinant human albumin. For SCC-9 cells, both recombinant human albumin and fatty acid free HSA showed slight stimulation (although they were not as active as HSA fraction V), suggesting that in some cell systems, the albumin molecule per se may promote cell growth and survival.

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Sodium thiosulfate (STS) is currently in a phase III clinical trial to investigate its efficacy in reducing ototoxicity in patients receiving cisplatin for standard risk hepatoblastoma. In animal studies STS has shown protection against ototoxicity, although the mechanism through which this occurs is unknown. It is unclear to what extent STS enters cells, but there is evidence linking uptake with the sulfate transporters. Intracellular STS has the potential to directly affect platination of DNA. The aim of this study was to test the hypothesis that STS can alter the types of DNA adducts formed by cisplatin and to compare its effects on cisplatin sensitivity and adduct levels in cancer cell lines. Purified DNA was reacted with cisplatin alone or in combination with STS (2mg/ml) and enzymatically hydrolysed to nucleotides. Equimolar (1mM) deoxyguanosine monophosphate (dGMP) and cisplatin were reacted for 24h followed by reaction with STS for a further 24h. DNA extracted from human tumour cell lines (833K, A2780, LoVo and MorCPR) was hydrolysed in nitric acid. Anion-exchange chromatography was used in conjunction with atomic absorption spectrometry for dGMP reactions and inductively-coupled plasma mass spectrometry (ICP-MS) for calf thymus and cellular DNA to detect Pt species. Growth inhibition was measured using the sulphorhodamine B assay. MonoQ chromatographic analysis of adducts formed on purified DNA confirmed the formation of the major 1, 2-intrastrand cross-links (ApG and GpG). Total adduct levels were decreased approximately 10-fold in the presence of STS. In addition a novel Pt-containing product was detected, eluting later than the previously characterized adducts and accounting for 25% of total adduct formation. To test the hypothesis that the product was a DNA-Pt-thiosulfate cross-link, cis-Pt [(NH3)2Cl.dGMP)] was reacted with STS. A major Pt-containing product with the same chromatographic properties as the product identified in DNA was detected. Cisplatin, alone or in combination with STS (2mg/ml) was added to the cell lines and removed after 6h. In additional experiments STS was added immediately after or 6h after cisplatin removal. Total incubation time was 72h. Co-incubation of cisplatin and STS led to increases in IC50 of 1.4 to 50.3µM, 2.2 to 45.5µM, 2.7 to 60.2µM and 29.4 to >500µM in 833K, A2780, LoVo and MorCPR cells respectively, with associated 10-15 fold decreases in adduct levels observed. Delayed additions of STS had no significant effect on growth inhibition or overall adduct levels. Anion-exchange chromatography with ICP-MS demonstrated that STS has the potential to modify platination of DNA. However, analysis of four cell lines showed no evidence for STS affecting levels of preformed DNA adducts in cells, supporting the hypothesis that the mechanism of otoprotection is through covalent binding and inactivation of cisplatin, without affecting the platination of DNA in tumour cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3501.