Embryoid Body Test: A Simple and Reliable Alternative Developmental Toxicity Test

Abstract

The increasing emphasis on animal welfare and ethics, as well as the considerable time and cost involved with animal testing, have prompted the replacement of many aspects of animal testing with alternative methods. In the area of developmental toxicity, the embryonic stem cell test (EST) has played a significant role. The EST evaluates toxicity using mouse embryonic stem cells and somatic cells and observes the changes in heartbeat after cardiac differentiation. Nevertheless, the EST is a relatively complex testing process, and an in vitro test requires a long duration. Several attempts have been made to develop a more straightforward testing method than the EST, with improved reproducibility and accuracy, leading to the development of the embryoid body test (EBT). Unlike the EST, which involves cardiac differentiation stages, the EBT verifies toxicity by measuring the changes in the area of the embryoid body. Despite its short testing period and simple procedure, the EBT offers high accuracy and reproducibility and is fully validated through two rounds of validation, making it ready for practical application. The EBT is expected to play a crucial role in the rapidly increasing demand for alternative methods to animal testing, particularly for screening early developmental toxicity.

Keywords: alternative test; developmental toxicity; embryoid body test (EBT).

Figure 1

Method for measuring embryoid body (EB) size. 1. Collect EBs from hanging drops. 2. Capture 7~8 images of each group, ensuring that at least 5 EBs are included. 3. Convert the image to black and white. 4. Count the number of black pixels in the images. 5. Calculate the relative area per EB. 6. Plot the graph and determine the ID50.

Figure 2

Experimental setup and timeline of the EST, Hand1-Luc EST, and EBT. The EST requires a 10-day testing period, making it relatively time-consuming and less reproducible, but it is the most extensively studied method. The Hand1-Luc EST offers the advantage of simplifying the detection of differentiation toxicity using luciferase; however, it requires luciferase measurement equipment, genetic modification for Hand1-Luc introduction, and maintenance of the cell line, which are notable drawbacks. The EBT, on the other hand, is the fastest and simplest method, offering higher reproducibility and accuracy. Nevertheless, it has the limitation of not providing direct post-differentiation results (e.g., beating activity).

Figure 3

Test composition and features of the EBT.