3D InSight™ Embryonic Stem Cell Test (EST)

An efficient, animal-free in vitro embryotoxicity screening service for developmental and reproductive toxicity (DART) testing

  • Secure protocol parallels ECVAM Invittox 113
  • Efficient embryoid body (EB) production results in faster, cost-effective assay
  • Only small amounts of your valuable compounds required

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Service at a Glance

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  • To get started, tell us the number of compounds you would like to test, and arrange delivery to our testing facility. The assay consumes <100 mg of your compound (liquid or powder equivalent). You will 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

    For pricing information and shipping costs, please request a quote, indicating the desired product, number of compounds, and the preferred shipment date.


    Catalog # Description
    SP-03-001-00 3D InSight™ Embryonic Stem Cell Test (EST) Pack – 4 compounds
    SP-03-002-00 3D InSight™ Embryonic Stem Cell Test (EST) Pack – 8 compounds
    SP-03-003-00 3D InSight™ Embryonic Stem Cell Test (EST) Pack – 16 compounds


    Please inquire for different numbers of compounds. Volume discounts for larger testing programs are available.

    Customize your 3D InSight™ EST endpoint

    The classic EST procedure determines effects of compounds on cell viability and on the functional differentiation of ES cells into beating cardiomyocytes. However, because the classification into non-, weak-, or strong-embryotoxic compound is based on mathematical modeling, it does not provide in-depth information about the mechanism of embryotoxicity.

    Several scientific publications describe methods which look at alternative gene expression, transcriptomic, and proteomic endpoints to give additional mechanistic insight beyond differentiation. Transcriptional endpoint ESTs2 are proposed to characterize potential chemical effects on osteogenic, chondrogenic and neural differentiation. Likewise, characterization of marker proteins3 which are critical in embryonic development can provide additional mechanistic insights. 


  • An efficient 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 embryotoxicity testing that avoids the use of animals.

    However, the classical EST protocol (ICCVAM/ECVAM INVITTOX No. 113) is laborious and complex, which has significantly hampered its adoption in industry. The use of InSphero's patented GravityPLUS™ Hanging Drop System in the ECVAM protocol improves EST efficiency and reproducibility, eliminating the practical issues with the classical EST protocol. 

    This method is now offered as the 3D InSight™ Embryonic Stem Cell Test Service. InSphero's experienced scientific team will test your compounds and rapidly return a detailed final report with compound classification, allowing you to gain important information about your new compounds at a fraction of classical EST testing cost. 

    Embryonic Stem Cell Test Assay Endpoints

    Technical overview of the classical EST protocol

    Embryonic Stem Cell Test Protocol

    Robustness of the 3D InSight™ EST assay

    To validate adaptation of the classical EST protocol, the 3D InSight™ EST was conducted on the test compound 5-Fluorouracil in three independent runs, and results were compared to corresponding values from the ECVAM validation study by ZEBET/BgVV (far right column - black values). The resulting average values for cytotoxicity (far right - purple, red) and differentiation inhibition (green) endpoints display impressive reproducibility across multiple runs indicating the robust nature of the 3D InSight™ EST protocol, and results in accordance with the classical protocol.

    Embryonic Stem Cell Test Robustness

    Reduced variability. Improved efficiency.

    Incorporating InSphero's GravityPLUSTM technology into the 3D InSight™ EST protocol improves assay efficiency by reducing handling time associated with formation and processing of EBs. The excellent size uniformity provided in the hanging drop system improves differentiation efficiency and reduces user-dependent variability. Thus, the test concentration range of both the ID50 and IC50 assays are defined in a single range-finding assay, eliminating the need to perform a repeat o f the Main EST test, and reducing total assay time by >30%.

    Embryonic Stem Cell Test Efficiency

    How are compounds classified?

    To classify compounds, the results of the three endpoints are entered into a series of three linear discriminant functions1 (see below). The resulting values of each function are then compared relative to one another, and compounds are classified as non-, weak-, or strong-embryotoxicants.

    Embryonic Stem Cell Test Classification Formulas


    Seiler AE and Spielmann H. (2011).  "The validated embryonic stem cell test to predict embryotoxicity in vitro." Nat Protocols, 6:961-78.

    Nieden N.I., et al. (2004). Toxicol Appl Pharmacol. 194(3):257-69.

    Groebe K., et al. (2010). J Proteome Research. 9(11):5727-38.

  • 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 3D InSight™ EST 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 EST completely in accord with the guidelines despite the implemented InSphero automated technology?

    A: Yes, we established the protocol for the 3D InSight™ EST 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 3D InSight™ EST. 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 3D InSight™ EST.

    Q: Is the 3D InSight™ EST performed under GLP at InSphero?

    A: The 3D InSight™ EST 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.


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  • Support

    Please send your inquiry to This email address is being protected from spambots. You need JavaScript enabled to view it. or contact This email address is being protected from spambots. You need JavaScript enabled to view it. for pricing/ quotation matters.

    Documents and downloads

    Product brochure

    Webinar:  Streamlining 3D hepatotoxicity & developmental toxicity testing with 3D InSight services

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