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.
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.
De Smedt
Ann
A
Johnson and Johnson Pharmaceutical Research and Development, A division of Janssen Pharmaceutica n.v., Turnhoutseweg 30, 2340 Beerse, Belgium. adsmedt1@prdbe.jnj.com
Steemans
Margino
M
De Boeck
Marlies
M
Peters
Annelieke K
AK
van der Leede
Bas-jan
BJ
Van Goethem
Freddy
F
Lampo
Ann
A
Vanparys
Philippe
P
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
D004492:Edetic Acid; D014357:Trypsin