Optimization and standardization of the rat whole embryo culture (WEC) as an in vitro embryotoxicity test through the development of a new culture medium

摘要

According to the REACH Program of the new European chemical politics 30 000 of the “existing substances” need to be investigated to determine their toxic potential. For this purpose, valid alternative methods to animal testing should be preferred. Therefore, the WEC is a candidate in reproductive toxicology. The recommended culture medium in the WEC, which consists of 100% rat serum, has two disadvantages: 1. Rats have to be sacrificed to gain blood for the culture medium. 2. The charge variability of the rat serum biases the comparability of the obtained results. Therefore, it was the aim of this thesis to establish a culture medium which would be superior to rat serum.So far commercially available bovine serum (fetal bovine serum [FBS], donor bovine serum [DBS] and adult bovine serum [ABS]) were not suitable to support embryonic development in culture. The investigation of both these sera as culture media and of possible variables in the serum production influencing the development-promoting potencies of sera resulted in two new culture media. They are based either on a combination of FBS and DBS or donor pregnant bovine serum (DPBS), which is a new subtype of industrially produced DBS, especially developed for the WEC. Both were supplemented with 10% rat serum and supported the development of rat embryos comparable to 100% rat serum.Furthermore, the significance of the growth factors (insulin-like growth factor [IGF-1], epidermal growth factor [EGF] and vascular endothelial growth factor [VEGF]) was investigated as an alternative to the 10% rat serum supplementation of heterologous sera. RT-PCR analyses of the growth factor receptors between in vivo and in vitro and the investigation of bovine serum supplemented with growth factors individually in the WEC was performed. It was revealed that VEGF is involved in the effect observable after a supplementation of 10% rat serum to heterologous sera.The possibility of changed sensitivity of embryos to test substances caused by the new culture media could be excluded through the investigation of penicillin G, ethanol, valproic acid, all-trans retinoic acid and 5-fluorouracil. The concentration-effect relationships and the specific adverse effects using the new established culture media were comparable to the effects observed previously in WEC studies or in vivo.The new culture media provide the possibility to reduce the needed number of animals to produce a culture medium for the WEC by 90 % and to increase the batch size of the culture medium from 50-80 ml up to 20 000-50 000 ml. To ensure the availability of the new culture media they were incorporated into the commercially available product assortment of the cooperating company (Biochrom AG, Berlin).Two new culture media could be established for the WEC, thus enabling the demanded widespread standardization of the WEC. They ensure the reproducibility of all evaluation parameters, and will dramatically reduce the sacrificing of animals for the WEC.