Anna Price

Optimization of chemically defined cell culture media - Replacing fetal bovine serum in mammalian in vitro methods

Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

Induction of Blood-Brain Barrier Properties in Cultured Brain Capillary Endothelial Cells: Comparison Between Primary Glial Cells and C6 Cell Line

The communication between glial cells and brain capillary endothelial cells is crucial for a well‐differentiated blood‐brain barrier (BBB). It has been suggested that in vitro primary glial cells (GCs) be replaced by the glial C6 cell line to standardise the model further. This study compares directly the structural and functional differentiation of bovine brain capillary endothelial cells (BBCECs) induced by co‐culture with rat primary GCs or C6 cells, for the first time. Trans‐endothelial electrical resistance (TEER) measurements showed that under no condition were C6 cells able to reproduce TEER values as high as in the presence of GCs. At the same time, permeability of the BBCECs to both radioactive sucrose and FITC‐inulin was 2.5‐fold higher when cells were co‐cultured with C6 than with GCs. Furthermore, immunocytochemistry studies showed different cell morphology and less developed tight junction pattern of BBCECs co‐cultured with C6 toward GCs. Additionally, studies on P‐glycoprotein (P‐gp) showed much lower P‐gp presence and activity in BBCECs co‐cultured with C6 than GCs. Both VEGF mRNA expression and protein content were dramatically increased when compared with GCs, suggesting that VEGF could be one of the factors responsible for higher permeability of BBB. Our results clearly indicate that, in the presence of the glial C6 cell line, BBCECs did not differentiate as well as in the co‐culture with primary GCs at both structural and functional levels.

The value of alternative testing for neurotoxicity in the context of regulatory needs

Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.