An Evaluation of a Novel Chick Cardiomyocyte Micromass Culture Assay with Two Teratogens/Embryotoxins Associated with Heart Defects

Abstract

This study was aimed at determining whether the chick cardiomyocyte micromass (MM) system could be employed to predict the teratogenicity/embryotoxicity of exogenous chemicals. Two documented teratogens/embryotoxins, sodium valproate (the sodium salt of valproic acid; VPA) and all -trans retinoic acid (tRA), were used in the initial phase of the study. White Leghorn 5-day-old embryo hearts were dissociated to produce a cardiomyocyte suspension in Dulbecco's Modified Eagle's Medium. Cultures were incubated at 37°C in 5% CO2 in air, and observations were made every 24 hours over 5 days, for the detection of beating. Culture viability was assessed by using the resazurin reduction assay for determining culture activity and the kenacid blue assay for determining cell number. It was found that tRA significantly reduced cell activity and beating, whilst not affecting total cell number. VPA up to 500μM induced no cytotoxicity in the MM cardiomyocyte cultures, whilst all the VPA concentrations tested reduced beating. The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter functionality, which may result in a teratogenic outcome, whilst not causing cytotoxicity (direct embryotoxicity). This could form part of a screen for developmental toxicity related to cardiac function, whilst limb cultures and brain cultures based on the same system could be relevant to teratogenic effects on those tissues.

Regulation of the chondrogenic phenotype in culture

“…A micromass culture is a three‐dimensional, high‐density cell culture that, for the purposes of this review, is comprised of precursor cells characterized by the potential to differentiate into chondrocytes (Ahrens et al, 1977). Nevertheless, micromass cultures have been employed to study both the differentiation of and the effects of exogenous agents on a variety of different cell types (Flint, 1983; Archer et al, 1990; Hurst et al, 2007). Within the chondrogenic micromass culture, the high seeding density stimulates the precartilage cells to mimic the condensation and differentiation events that normally occur during embryonic hyaline cartilage formation in vivo.…”

The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system

“…The embryonic chick cardiomyocyte MM culture system used here as a model system is relatively novel and is an attractive method for developmental cardiotoxicity screening, as the endpoint of cardiomyocyte beating can be seen as a measure of functional integrity and re‐differentiation. Previously it has been used for the screening of potential human teratogens for developmental cardiotoxicity testing (Ahir and Pratten, , ; Hurst et al , , ; Memon and Pratten, ; Pratten et al , ). Indeed, MM culture systems generally have already been used to investigate a number of teratogens, including mycotoxins, herbicides, pesticides and xenobiotic compounds (Flint and Orton, ; Johnson et al , ; L'Huillier et al , ; Parsons et al , ).…”

“…There is not much evidence of work being performed on embryonic chick heart cells (cardiomyocytes) as a MM culture system. However, the system may be useful for studying embryonic developmental mechanisms and the perturbation of heart development (Ahir and Pratten, , ; Hurst et al , ; Memon and Pratten, ; Pratten et al , ), while limb buds and neural tissue (midbrain MM culture) may be used for effects on chondrogenesis and neurogenesis by teratogens (Atterwill et al , ; Wiger et al , ).…”

Cytotoxicity as a Fundamental Response to Xenobiotics

“…Generally, the cells are exposed to the product for evaluation for a time period of 72 h, and the product is then removed and the cells are exposed to the dye, which then binds to the cellular proteins. Finally, the amount of kenacid blue retained by the cells is determined and the percent of inhibition of cell growth is quantified [51,52].…”