The Mouse Embryonic Stem Cell Test: Technical Challenges and Recent Advances

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In the Spotlight

The Mouse Embryonic Stem Cell Test: Technical Challenges and Recent Advances

Erica Dahl, Study Director, Institute for In Vitro Sciences

Published: April 21, 2010

Reprinted with permission from the newsletter IIVS Update, Volume 9, Issue 1

The practicality of in vivo tests for reproductive toxicity has been the subject of much recent discussion. Besides an interest in reducing animal use for ethical concerns, this discussion has been driven by the massive number of chemicals subject to REACH legislation and a general desire to develop a more predictive paradigm for toxicity testing that includes more in vitro models and computational approaches. Though no single in vitro test is ever expected to replace animal models for reproductive toxicity, in vitro models designed to model discrete events within the reproductive continuum are available. The mouse Embryonic Stem Cell Test (EST) is a formally validated1 test for early embryotoxicity that uses stable mouse cell lines, sidestepping the need to sacrifice pregnant animals. Mouse embryonic stem cells (D3) form contracting myocardiocytes that are easily observed microscopically after a 10 day differentiation program. The test article concentration that inhibits differentiation by 50% (ID50) is calculated relative to control cultures. Cytotoxicity assays using undifferentiated D3 cells and an adult mouse cell line (3T3) are performed in parallel to generate IC50 values. The values for the ID50, the IC50 3T3 and the IC50 D3 are then evaluated using a validated prediction model to classify the test article as a nonembryotoxin, moderate embryotoxin, or strong embryotoxin. If the EST is to become widely used, it must be readily transferable among laboratories throughout the world.

Issues affecting transfer of the assay to new laboratories

To induce differentiation, D3 cells are seeded onto the lid of a culture dish and grown for 3 days in a “hanging drop” culture, which allows the cells to aggregate and form embryoid bodies, which are then transferred to bacterial petri dishes to continue growing in suspension culture. They are then transferred to a 24 well cell culture plate and allowed to form attachment cultures, in which contracting myocardiocytes can be observed microscopically after several days. In transferring the assay to the Institute for In Vitro Sciences, we initially had difficulty consistently generating contracting myocardiocytes in the differentiation assay. We often found embryoid bodies attached to the surface of the petri dishes despite using the exact catalog number and supplier specified in the validated protocol. The attachment problem was resolved by specifying nonsterile untreated bacterial petri dishes, which had a different catalog number in the United States. We also found that using Fetal Calf Serum (FCS) at a concentration of 15%, rather than the 20% specified in the protocol, increased the number of cultures that developed contracting myocardiocytes. We also developed a serum qualification procedure to identify sources based in the United States that produce serum that works well in the differentiation assay. Prequalified lots of serum are stored at -20°C. Since introducing these modifications we have consistently been able to run differentiation assays that pass our quality control criteria.

Recent refinements of the EST

The currently validated assay is labor intensive, technically demanding and fairly low throughput. However, improvements to increase throughput and lower the labor costs associated with the assay have recently been published. These include adapting the differentiation assay to a 96 well plate format2 and using cell surface markers analyzed by flow cytometry3 to assess myocardiocyte differentiation. An adaptation of the differentiation assay to produce neural cells rather than cardiomyocytes has also been published.4 We recommend establishing a public online forum for researchers who are working with the EST to communicate about serum qualification efforts, technical difficulties laboratories may encounter when setting up the EST, and to discuss recent technological advancements or new data.
1Embryonic Stem Cell Test (EST) INVITTOX n° 113, ECVAM 2006.

1Embryonic Stem Cell Test (EST) INVITTOX n° 113, ECVAM 2006.
2Peters, A.K., et. al. Toxicol. Sci. 2008 Oct;105(2):342-50.
3Buesen, R., et. al. Toxicol. Sci. 2009 Apr;108(2):389-400.
4Stummann, T.C., et. al. Toxicology. 2007 Dec 5;242(1-3):130-43.