Chick Embryonic Cardiomyocyte Micromass System for Assessing Developmental Cardiotoxicity of Drugs

  • Wasay Mohiuddin Shaikh Qureshi
  • Margaret K. Pratten
Part of the Methods in Molecular Biology book series (MIMB, volume 1797)


Heart is the first mesodermal organ to develop and is sensitive to life-threatening toxic effects of drugs during development. A number of methods have been devised to study developmental cardiotoxic effects of drugs including micromass system. The micromass system involves the culture of primary embryonic cells and reestablishment of tissue system in vitro. In chick embryonic cardiomyocyte micromass system the chick heart cells are cultured in a small volume at a very high cell density. These cells form synchronized contracting foci. Addition of drugs to this system allows us to study the developmental cardiotoxic effects at molecular level. Using appropriate end points and molecular marker or adopting high-throughput screening, this method can further help to identify and avoid the use of cardiotoxic compounds during development.

Key words

Developmental toxicology Micromass Cardiomyocytes Teratogens Chick Reactive oxygen species Connexin43 


  1. 1.
    Brown N, Fabro S (1983) The value of animal teratogenicity testing for predicting human risk. Clin Obstet Gynecol 26(2):467–477CrossRefPubMedGoogle Scholar
  2. 2.
    Piersma AH (2004) Validation of alternative methods for developmental toxicity testing. Toxicol Lett 149(1–3):147–153. CrossRefPubMedGoogle Scholar
  3. 3.
    Spielmann H, Liebsch M (2001) Lessons learned from validation of in vitro toxicity test: from failure to acceptance into regulatory practice. Toxicol In Vitro 15(4–5):585–590. CrossRefPubMedGoogle Scholar
  4. 4.
    Lilienblum W, Dekant W, Foth H et al (2008) Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European chemicals legislation (REACH). Arch Toxicol 82(4):211–236. CrossRefPubMedGoogle Scholar
  5. 5.
    Scialli AR (2008) The challenge of reproductive and developmental toxicology under REACH. Regul Toxicol Pharmacol 51(2):244–250. CrossRefPubMedGoogle Scholar
  6. 6.
    Bacon WJ, Duffy PA, Jones K (1990) Studies on variability of the micromass teratogen test. Toxicol In Vitro 4(4–5):577–581. CrossRefPubMedGoogle Scholar
  7. 7.
    Seiler A, Visan A, Buesen R et al (2004) Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test. Reprod Toxicol 18(2):231–240. CrossRefPubMedGoogle Scholar
  8. 8.
    Umansky R (1966) The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dev Biol 13(1):31–56CrossRefPubMedGoogle Scholar
  9. 9.
    Flint OP (1983) A micromass culture method for rat embryonic neural cells. J Cell Sci 61:247–262PubMedGoogle Scholar
  10. 10.
    Spielmann H, Genschow E, Scholz G et al (2001) Preliminary results of the ECVAM validation study on three in vitro embryotoxicity tests. Altern Lab Anim 29(3):301–303PubMedGoogle Scholar
  11. 11.
    Flint OP, Orton TC (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol Appl Pharmacol 76(2):383–395CrossRefPubMedGoogle Scholar
  12. 12.
    L'Huillier N, Pratten MK, Clothier RH (2002) The relative embryotoxicity of 1,3-dichloro-2-propanol on primary chick embryonic cells. Toxicol In Vitro 16(4):433–442CrossRefPubMedGoogle Scholar
  13. 13.
    Hurst H, Clothier RH, Pratten M (2009) An evaluation of the chick cardiomyocyte micromass system for identification of teratogens in a blind trial. Reprod Toxicol 28(4):503–510CrossRefPubMedGoogle Scholar
  14. 14.
    Atterwill C, Johnston H, Thomas SM (1992) Models for the in vitro assessment of neurotoxicity in the nervous system in relation to xenobiotic and neurotrophic factor-mediated events. Neurotoxicol Teratol 13(1):39–53Google Scholar
  15. 15.
    Fuscoe JC (2007) Impact of systems toxicology on the 3 Rs. AATEX 14(special issue):629–632Google Scholar
  16. 16.
    Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. Dev Dyn 195(4):231–272. CrossRefPubMedGoogle Scholar
  17. 17.
    Chandrashekhar Y, Prahash AJ, Sen S et al (1999) Cardiomyocytes from hearts with left ventricular dysfunction after ischemia-reperfusion do not manifest contractile abnormalities. J Am Coll Cardiol 34(2):594–602. CrossRefPubMedGoogle Scholar
  18. 18.
    Bueno C, Villegas ML, Bertolotti SG et al (2002) The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions. Photochem Photobiol 76(4):385–390CrossRefPubMedGoogle Scholar
  19. 19.
    Nakayama GR, Caton MC, Nova MP, Parandoosh Z (1997) Assessment of the Alamar blue assay for cellular growth and viability in vitro. J Immunol Methods 204(2):205–208CrossRefPubMedGoogle Scholar
  20. 20.
    Anoopkumar-Dukie S, Carey JB, Conere T et al (2005) Resazurin assay of radiation response in cultured cells. Br J Radiol 78(934):945–947CrossRefPubMedGoogle Scholar
  21. 21.
    Memon S, Pratten MK (2009) Developmental toxicity of ethanol in chick heart in ovo and in micromass culture can be prevented by addition of vitamin C and folic acid. Reprod Toxicol 28(2):262–269CrossRefPubMedGoogle Scholar
  22. 22.
    Qureshi WM, Memon S, Latif ML et al (2014) Carbamazepine toxic effects in chick cardiomyocyte micromass culture and embryonic stem cell derived cardiomyocyte systems-possible protective role of antioxidants. Reprod Toxicol 50:49–59. CrossRefPubMedGoogle Scholar
  23. 23.
    Clothier R, Starzec G, Pradel L et al (2002) The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar blue (resazurin) assay. Altern Lab Anim 30(5):493–504PubMedGoogle Scholar
  24. 24.
    St D, Groth SF, Webster RG, Datyner A (1963) Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochim Biophys Acta 71(0):377–391. CrossRefGoogle Scholar
  25. 25.
    Knox P, Uphill PF, Fry JR et al (1986) The FRAME multicentre project on in vitro cytotoxicology. Food Chem Toxicol 24(6–7):457–463CrossRefPubMedGoogle Scholar
  26. 26.
    Kobayashi CI, Suda T (2012) Regulation of reactive oxygen species in stem cells and cancer stem cells. J Cell Physiol 227(2):421–430. CrossRefPubMedGoogle Scholar
  27. 27.
    Guo Y-L, Chakraborty S, Rajan SS et al (2010) Effects of oxidative stress on mouse embryonic stem cell proliferation, apoptosis, senescence, and self-renewal. Stem Cells Dev 19(9):1321–1331CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Lee M, Lee SH, Lee MY et al (2008) Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H2O2-induced oxidative stress. J Vet Sci 9(3):247–256CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Vink MJ, Suadicani SO, Vieira DM et al (2004) Alterations of intercellular communication in neonatal cardiac myocytes from connexin43 null mice. Cardiovasc Res 62(2):397–406. CrossRefPubMedGoogle Scholar
  30. 30.
    Shaikh Qureshi WM, Latif ML, Parker TL, Pratten MK (2014) Evaluation of bupropion hydrochloride developmental cardiotoxic effects in chick cardiomyocyte micromass culture and stem cell derived cardiomyocyte systems. Birth Defects Res B Dev Reprod Toxicol 101(5):371–378. CrossRefPubMedGoogle Scholar
  31. 31.
    Brown N, Wiger R (1992) Comparison of rat and chick limb bud micromass cultures for developmental toxicity screening. Toxicol In Vitro 6(2):101–107CrossRefPubMedGoogle Scholar
  32. 32.
    Slack JMW (2006) Essential developmental biology, 2nd edn. Blackwell Publishing, Hoboken, New JerseyGoogle Scholar
  33. 33.
    Shaikh Qureshi WM (2012) The chick cardiomyocyte micromass system and stem cell differentiation along specific pathways: prediction of embryotoxic effects and their mechanism. University of Nottingham, NottinghamGoogle Scholar
  34. 34.
    Garle MJ, Knight A, Downing AT et al (2000) Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: lack of involvement of the vanilloid receptor. Biochem Pharmacol 59(5):563–572CrossRefPubMedGoogle Scholar
  35. 35.
    Qureshi WM, Latif ML, Parker TL, Pratten MK (2014) Lithium carbonate teratogenic effects in chick cardiomyocyte micromass system and mouse embryonic stem cell derived cardiomyocyte--possible protective role of myo-inositol. Reprod Toxicol 46:106–114. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Wasay Mohiuddin Shaikh Qureshi
    • 1
    • 2
  • Margaret K. Pratten
    • 1
  1. 1.School of Life SciencesUniversity of Nottingham, Queen’s Medical CentreNottinghamUK
  2. 2.Cardiovascular Research Centre, Institute of Genetic MedicineNewcastle UniversityNewcastle upon TyneUK

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