Fetal Bovine Serum (FBS): Past - Present - Future.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

Reconsidering pluripotency tests: do we still need teratoma assays?

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.

Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning.

Metagenome cloning has become a powerful tool to exploit the biocatalytic potential of microbial communities for the discovery of novel biocatalysts. In a novel variant of direct expression cloning, metagenomic DNA was isolated from compost by a modified direct lysis method, purified by size exclusion chromatography and cloned into an expression vector allowing bidirectional transcription. Transformation of Escherichia coli DH5alpha resulted in a metagenomic expression library with an average insert size of 3.2 kb. To estimate the functional diversity of the constructed library, it was screened by different approaches based on functional heterologous expression. A large number of active clones were identified, including lipolytic enzymes, amylases, phosphatases and dioxygenases. Molecular analysis of one important class of industrial biocatalysts, the lipolytic enzymes, confirmed the novelty and dissimilarity of all recovered genes, which exhibited only limited similarity to known enzymes. Equally, the novelty of another three genes encoding phosphatase or dioxygenase activity, respectively, was shown. These results demonstrate the suitability of this direct cloning approach, which comprised a dual-orientation expression vector and a simple one-step DNA purification method, for the efficient discovery of numerous active novel clones. By this means it provides an efficient way for the rapid generation of large libraries of hitherto unknown enzyme candidates which could be screened for different specific target reactions.

Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments.

DNA quantification of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quantification is impaired by humic acids (HA). Due to its lack of specificity and sensitivity, UV spectrophotometry cannot be applied. Consequently, fluorimetric assays applying Hoechst (H) 33258 or PicoGreen (PG) are used. Here, we investigated the SYBR Green I (SG) assay, which was also affected by HA, but was found to be 25- and 1.7-fold more sensitive compared to the H 33258 and PG assays, respectively. Spectrophotometric, fluorimetric and quenching studies as well as gel mobility shift assays suggested that the effect of HA on the SG assay was based on an inner filter effect, collisional quenching and binding of SG to HA. As to the latter finding, the standard 6250-fold dilution of the SG reagent was optimised to a 2000-fold dilution. Although the sensitivity of the optimised SG assay was reduced by a factor of 1.3, the interfering effect of HA could be reduced up to 22-fold. A significant reduction of HA interferences by lowering the pH of the assay was not observed. Finally, the performance of the modified SG assay and the corresponding evaluation methods were verified by the determination of DNA recoveries and concentrations of standards and environmental samples in comparison to the PG assay.