title: Optimisation of the cell cultivation methods in the embryonic stem cell test results in an increased differentiation potential of the cells into strong beating myocard cells.
abstract: In order to support drug research in the selection process for non-embryotoxic pharmaceutical compounds, a screening method for embryotoxicity is needed. The murine embryonic stem cell test (EST) is a validated in vitro test based on two permanent mouse cell lines and delivering results in 10-days. Implementation of this test within our laboratory, revealed variability in the differentiation potential of the embryonic stem cells and, as a consequence, a lot of assays needed to be rejected due the fact the acceptance criteria were not reached. In order to gain a better yield of contracting myocardial cells, we used (1) a stringent control of the cell growth during subcultivation and a standardised hanging drop culture method and (2) a non-enzymatic cell harvest instead of a trypsin/EDTA cell harvest. Implementing of these cell culture modifications resulted in a decreased variability in the size of embryonic bodies, an increase of the number of acceptable tests and a significant increase of the differentiation potential of embryonic cells into strong beating myocardium, which made scoring less time consuming. Testing of 6 reference compounds in the optimized EST showed that the cell culture modifications did not changed the in vitro classification.
authors.affiliation: Johnson and Johnson Pharmaceutical Research and Development, A division of Janssen Pharmaceutica n.v., Turnhoutseweg 30, 2340 Beerse, Belgium. adsmedt1@prdbe.jnj.com
authors.forename: Ann
authors.initials: A
authors.lastname: De Smedt
authors.forename: Margino
authors.initials: M
authors.lastname: Steemans
authors.forename: Marlies
authors.initials: M
authors.lastname: De Boeck
authors.forename: Annelieke K
authors.initials: AK
authors.lastname: Peters
authors.forename: Bas-jan
authors.initials: BJ
authors.lastname: van der Leede
authors.forename: Freddy
authors.initials: F
authors.lastname: Van Goethem
authors.forename: Ann
authors.initials: A
authors.lastname: Lampo
authors.forename: Philippe
authors.initials: P
authors.lastname: Vanparys
mesh_terms: D000818:Animals; D041702:BALB 3T3 Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D004492:Edetic Acid / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000166:cytology; D018675:Toxicity Tests / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D004492:Edetic Acid; D014357:Trypsin