In vitro system for toxicological studies on the development of mammalian limb buds in a chemically defined medium

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Abstract
In organ culture systems using the Trowell setup, morphogenetic differentiation (which largely mimics the development reached in vivo within 2–3 days) can be obtained in limb buds of mouse embryos during a culture period of 6 days. We succeeded in improving the technique and in achieving good differentiation of the limb buds in a chemically defined culture medium from which all additions of the heterologous serum used in previous studies were omitted. With this technique of using a chemically defined medium, the following results were obtained: (1) A high degree of reproducibility can be obtained in the grade of differentiation if the experimental conditions are standardized and limb buds of the same developmental stage (somite stage) are used. (2) The technique is applicable to species other than mice, such as rats and rabbits, however, the results obtained so far are not so satisfactory as those acquired with mouse limb buds. (3) In an attempt to offer some colloid osmotic pressure, macromolecules like polyvinylpyrrolidone (Periston), polydextrans (Macrodex) or polypeptides (Haemaccel) were added to the culture medium. None of these macromolecules had a beneficial effect on the differentiation of the limb buds in vitro; in the case of Macrodex the differentiation was even impaired. (4) In comparison with the development in a serum-containing medium, the results attained with a chemically defined medium are just as good when judged from microscopical and some biochemical studies (total content of DNA, RNA, protein, and protein-bound hydroxyproline in the expiants). The applicability of the test system for the evaluation of embryotoxic effects is discussed.