Development of alternatives to animal experiments with in vitro cultures of undifferentiated embryonic cells and organ primodia

化学物质对培养的胎儿器官的影响将第12.5天和第13.5天的胎鼠的腭部外植，体外暴露于0.1 ~ 50 mg 5-氟尿嘧啶(5-FU) /ml或5 ~ 76 mg羟基脲(HU) /ml中72小时。5-FU对体外12.5 d腭架生长和融合的抑制作用存在浓度依赖性。第13.5天的胎儿腭部对5-FU的敏感性明显低于第12.5天，第12.5天和第13.5天的胎儿腭部5-FU的最小毒性浓度（MTCs）分别为0.1和10 mg/ml。HU对12.5日龄胎儿腭架的体外生长和融合呈浓度依赖性，但对13.5日龄胎儿腭架的生长只有轻微的抑制作用。在第12.5天和第13.5天，HU的MTCs分别为19和76 mg/ml。第12.5天ICR胎鼠的腭可能比第13.5天的腭更适合体外致畸原筛选和正常和异常腭发生机制的研究。采用化学定义的无血清培养基培养12.5天ICR小鼠上颌区域胎儿，研究了15种化学致畸物对培养腭的发育毒性。将离体的上颚以不同浓度暴露于每种药物72小时。15种化合物中的所有化合物在体内都具有显著的体外毒性，体外发育毒性的可预测性为73%。根据腭融合率计算的IC_<50>（50%抑制浓度）值似乎是药物发育毒性的有用指标。此外，应通过比较人体临床条件下的最低毒性浓度与最高血浆浓度来评估人体风险。

Effects of chemicals on cultured fetal organsThe palates of day-12.5 and day-13.5 fetal mice were explanted and exposed in vitro for 72 hr to 0.1-50 mg 5-fluorouracil (5-FU) /ml or to 5-76 mg hydroxyurea (HU) /ml. 5-FU inhibited the growth and fusion of day-12.5 palatal shelves in vitro dependently on its concentrations. Day-13.5 palates were significantly less sensitive to 5-FU than day-12.5 palates, and the minimal toxic concentrations(MTCs)of 5-FU were 0.1 and 10 mg/ml for day-12.5 and day-13.5 fetal palates, respectively. HU inhibited the in vitro growth and fusion of day-12.5 fetal palatal shelves in a concentration dependent manner, but only slightly suppressed the growth of day-13.5 fetal palates. The MTCs of HU were 19 and 76 mg/ml for day-12.5 and day-13.5 fetal palates, respectively. The palates of day-12.5 ICR fetal mice may be more suitable than day-13.5 palates for in vitro teratogen screening and for the study of mechanisms of normal and abnormal palatogenesis.2.Usefulness of fetal organ cultures for teratogen screeningThe maxillary regions of day-12.5 ICR mouse fetuses were cultured in a chemicallydefined serumless medium, and the developmental toxicity of 15 chemical teratogens on cultured palates was studied. Explanted palates were exposed in vitro for 72 hr to each drug at various concentrations. A significant in vitro toxicity was shown for ll out of the 15 compunds which are teratogenic in vivo, and the in vitro predictability for developmental toxicity was 73%. The IC_<50>(50% inhibitory concentration)values calculatrd based on palatal fusion rates appeared to be a useful index of developmental toxicty of drugs. In addition, the human risk shall be assessed by comparing the minimal toxic concentrations with maximal plasma levels in human clinical conditions.