A convenient embryotoxicity screening service for in vitro developmental and reproductive toxicity (DART) testing
An automated EST for convenient, affordable embryotoxicity screening
The embryonic stem cell test (EST) is a powerful method to assess the effects of any new compound on cell differentiation and viability. Introduced in 1997, the EST quickly became recognized as the best available method for in vitro embryotoxicity testing as an alternative to prenatal developmental toxicity studies using animals.
However, the classical EST protocol (outlined as ICCVAM/ECVAM INVITTOX No. 113) is laborious and complex, significantly hampering its adoption. InSphero's patented and automation-compatible GravityPLUS™ Hanging Drop System irons out the practical issues with the formation of mouse ESD3 cell-derived embryoid bodies (EB) used in the classical EST protocol.
This method is now offered as the easyEST™ Embryonic Stem Cell Test Service. InSphero's experienced scientific team will test your compounds and rapidly return an easy-to-understand final report. By outsourcing your EST activities to InSphero you'll not only gain important information on your new compounds, as InSphero can offer this service at a fraction of classical EST testing cost – in a range similar to standard toxicology testing.
Seiler AE and Spielmann H. (2011). "The validated embryonic stem cell test to predict embryotoxicity in vitro." Nat Protocols, 6:961-78.
Confocal image of mouse ES cells derived cardiomyocytes. Sarcomeric alpha-actinin (green) actin cytoskeleton (red) and nuclei (blue). Scale bar 15 μm.
What makes easyEST™ so efficient?
The petri dish hanging drop method used in the classical EST protocol to form EBs requires substantial hands-on work, making the process expensive and subject to user-dependent variability. The easyEST™ Embryonic Stem Cell Test Service gives you access to a breakthrough in efficiency and scalability:
- Excellent size uniformity of embryoid bodies (<5%) for better differentiation efficiency
- Consumes less than 50 mg of your valuable compounds (we accommodate liquid stocks or dry compounds)
- Tests the solubility and assay compatibility of your compound
- Automated and efficient EB generation and medium change
- Direct, automated transfer into flat-bottom adherent plate
All relevant aspects of the INVITTOX 113 protocol remain unchanged to allow for a straightforward migration to the easyEST™ Embryonic Stem Cell Test Service.
How do I take this forward?
1. Tell us how many compounds you want to test: 4, 8, 16 or more (volume discounts are available for larger screening projects)
2. InSphero will test as follows using the automated GravityPLUS™ Hanging Drop System:
- Compatibility of compounds with assay
- Range finding (n=6) for both ID50 and IC50
- Embryonic stem cell testing
3. You receive:
- A summary report including IC50 data for 3T3 and ESD3 cells (n=6) and ID50 data for ESD3 cells (n=24)
- Embryotoxic classification with linear discriminants according to ECVAM INVITTOX Protocol No. 113
- A web-based presentation and discussion
Catalog # Description Price in USD SP-03-001-00 easyEST™ Embryonic Stem Cell Test Service Pack – 4 compounds 22,047 SP-03-002-00 easyEST™ Embryonic Stem Cell Test Service Pack – 8 compounds 40,486 SP-03-003-00 easyEST™ Embryonic Stem Cell Test Service Pack – 16 compounds 73,415
Please inquire for different numbers of compounds. Volume discounts for larger testing programs are available.
- Q: What is the rating of the EST in the field of reproductive toxicity?
A: The EST is one of the three rodent based embryotoxicity / teratogenic tests accepted and validated by the ECVAM. It has a predicitivity of up to 80%, similar to the other rodent based assay. Because the EST is cell- based, the associated cost are lower and it's ethically most accepted.
Q: What are the differences between the InSphero protocol and the original protocol?
A: InSphero's easyESTTM procedure only differs in the use of the technical cultivation platforms and culture medium volumes in the differentiation assay. The differences are listed in our product brochure.
Q: Is the InSphero easyESTTM test completely in accord with the guidelines despite the implemented InSphero automated technology?
A: Yes, we established the protocol for the easyESTTM according to the INVITTOX 113 protocol. The INVITTOX protocol was validated by ECVAM and has several key quality criteria which are also met by the easyESTTM. These quality criteria include tested serum for differentiation of EBs, tested metabolic activity during the MTT assay, sensitivity of cell metabolism of the ESD3 and Balb/c3T3 cells to 5-Fluorouracil, beating efficiency of EBs, Calculation methods for embryonic toxicity, and many more. The required standard from the INVITTOX 113 protocol is met by the easyESTTM service.
Q: Is the easyESTTM performed under GLP at InSphero?
A: The easyESTTM is not performed under GLP, however InSphero is ISO certified since 2011 and all processes are controlled under standard operating procedures and a test protocol is available.
Q: How many compounds can be tested in one EST run?
A: We test eight compounds together in one run.
Q: Can multiple ESTs be performed in parallel?
A: We can run multiple ESTs in parallel per week. We can also do runs in staggered manner.
Q: Alternative endpoints in the EST like transcriptional and proteomic analyses are proposed in several scientific publications. Can InSphero also provide service in these regards?
A: InSphero can work out specific service projects in the field of investigative embryotoxicology. Endpoints such as RNA extractions or transcriptional analyses from embryoid bodies are possible. Please contact us for detailed options in these regards.
Q: What are benefits from the modified ESTs looking at alternative endpoints (for example, RNA or protein)?
A: There are several alternative endpoints proposed for the EST which are looking at RNA regulation and/or protein expression together with reporter assays. They can provide information of dysregulated pathways involved the development of the embryo. The traditional endpoint assesses stem cells differentiating into beating cardiomyocytes and therefore is a functional endpoint. Functional endpoints like the beating readout, provide a benchmark and are indispensable. Endpoints (e.g. RNA and protein analysis) can provide a tool for in-depth mechanistic analyses for specific compounds and optimally complement the classic readout.
Documents and downloads