Development of a mouse embryo limb bud cell culture system for the estimation of chemical teratogenic potential

Abstract

High-density cultures of mouse embryo limb bud cells differentiate and synthesize an extracellular matrix containing sulfated proteoglycans. Since these in vitro events are related to those that occur during fetal development, we have investigated the use of cultured limb bud cells for the analysis of the activities of chemical teratogens. We have established the conditions for the use of the radiochemicals 35SO=4 and 3H-thymidine for the assessment of chemical effects on sulfated proteoglycan and DNA synthesis, respectively, in mouse limb bud cells. By performing double-labeling experiments in the presence of test chemicals, the preferential inhibition of either proteoglycan synthesis or DNA synthesis could be demonstrated for 19 of 22 known mouse teratogens tested. The five nonteratogens tested did not cause significant differences in the inhibition of either macromolecular class. The overall predictive accuracy of the system was approximately 89%, and the false-negative rate was approximately 14.8%. No false positives were observed. These data showed that this cell culture system differentiated between the activities of a limited set of selected mouse teratogens and nonteratogens, suggesting that cultured mouse embryo limb bud cells may constitute the basis for the development of a powerful tool for analyses of chemical teratogenic potential.

The Validation and Use of In Vitro Teratogenicity Tests

“…High density cultures of mouse or chick limb bud cells have been proposed as teratogenicity screening systems by Wilk et al (1980); Hassell and Horigan (1982) and Guntakatta et al (1984). Flint and Orton (1984) and Kistler (1985) used cells from rat limb buds and Renault et al (1993) modified the system to be used with rabbit limb bud cells.…”

“…The cells proliferate and undergo differentiation into chondrocytes after 5 to 6 days in culture. The rate of proliferation and differentiation into cartilaginous nodules can easily be measured quantitatively by simple staining methods with subsequent spectrophotometry and fully automated image analysis (Renault et al 1989) or by radiochemistry (Guntakatta et al 1984). The test compound is added to the cultures in different concentrations.…”

Scientific Opinion on Chloramphenicol in food and feed

“…The maximum concentration, having no inhibitory effect for chloramphenicol, was 60 µg/mL. Chloramphenicol caused a statistically significant reduction in 35 SO 4 2-incorporation when tested in the mouse limb bud cell spot culture assay (Guntakatta et al, 1984).…”

Teratological research using in vitro systems. IV. Cells in culture.

“…Controls are treated in the same manner as experimental compounds. After the test agent has been added, the cultures are placed in 25°C incubators and are scored for cell and tissue differentiation 24 hr later. During this period, several types of cells differentiate from their stem cells, as observed by phase contrast microscopy.…”

“…Trosko and co-workers (7) found that a variety of chemicals have the ability to increase the recovery of mutant 6-TGr cells when co-cultured with wild-type 6 24 hr, at which time the medium is replaced with or without the test compound. The HEPM cells are then maintained in culture for an additional 72 hr.…”