Developmental processes in gastrulating rat embryos were investigated by using an original, serum-free, chemically defined model system. 9.5-day-old rat embryos, without extraembryonic membranes, were cultivated at the air–liquid interface in a serum-free medium, with and without a protein supplement, for 2 weeks. A teratogenic, demethylating agent, 5-azacytidine, was added to serum-free and protein-free culture medium and to serum-free medium supplemented with human transferrin. A single dose of 5-azacytidine impaired the survival, growth and differentiation of embryos in protein-free medium and serum-free medium with transferrin. In contrast, repeated exposure to 5-azacytidine was required to impair growth in serum-supplemented medium. It was concluded that the activity of 5-azacytidine was easier to detect in a simple, chemically defined medium than in a serum-supplemented medium. This serum-free in vitro method could be useful in screening for teratogenic or embryotoxic substances during gastrulation, the most critical stage of mammalian development.
Three-dimensional tissue constructs have been proposed as in vitro screening models for ocular irritancy. Based on our previous studies, in which a full-thickness corneal model based exclusively on SV40-immortalised cell lines was generated, we have currently evaluated the effects of a range of commercially-available cell culture media on several cellular parameters in cultures of a human corneal epithelial (HCE) cell line. This cell line was used in an attempt to establish a rational basis for the development of serum-free culture media for the assembly and long-term tissue culture of full-thickness corneal models. Briefly, we investigated the impact of serum-free culture on the proliferation, morphology, barrier function and cytokine expression of HCE cells. The number of cell layers and the epithelial differentiation were evaluated by histology. Barrier properties were characterised via the determination of transepithelial electrical resistance (TEER), fluorescein permeation, and the expression of the tight junctionrelated protein, zona occludin 1 (ZO-1). The cytokine expression pattern in response to serum-free culture was measured by using an antibody array system. Our results revealed that both the morphology and the barrier function of the epithelial constructs were comparable to those of human donor corneas, when serum-free media were supplemented with ascorbic acid, calcium, hydrocortisone and retinoic acid. Under these conditions, the artificial epithelium based on serum-free HCE cultures represented a valid model for the natural ocular surface.
Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current ‘whole animal’ approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.