Print this page / Imprima esta página
ALTEX :: Alternatives to Animal Experiments
2001, VOLUME 1
Biomaterials in vitro: Monitoring Cell Lines from Primary Cultures using Multilocus-DNA-Fingerprinting
Erwin Falkner, Wolfgang Ficke, Barbara Kapeller, Heidrun Eberl, Karin Macfelda, Udo Losert
Center for Biomedical Research, University of Vienna
Cell lines originating from primary cultures are innovative tools for e.g. biocompatibility testing of biomaterials in vitro. In our studies we used fibroblast/endothelial cell/chondrocyte cultures of human origin and of the test animal species most common for this purpose in vivo. Verification of the identy of these cells is obligatory for reproducibility of the tests and valid interpretation of the results. Cultured cells have to be checked for identity, contaminations of various origins and also for genomic mutations occuring during prolonged cultivation in vitro or due to exposition to biomaterials. Furthermore, the risk of genetic cross-contamination with other cells increases with the number of cell cultures passaged parallel in the same laboratory. Therefore, we generated reference fingerprints of the cultures in varying passages for comparative monitoring of cells purposed for in vitro tests.
Minisattelite DNA polymorhism resulting in reproducible individual DNA fingerprints is very discriminatory and can be used for cell culture monitoring. The patterns are stable over several passages, although sudden changes did happen in some cases, i.e. loss/gain of bands or changes in band-intensity, indicating massive genomic mutations of the cultures in vitro. Influences of biomaterials on the prints could not be detected.
Impure genomic DNA resulting from problems during isolation due to influences of still adhering biomaterial-particles led to troublesome band-detecting with ImageMaster™ standard-software. These could be solved in most cases optimizing the scans with MSTM Corel PHOTOPAINT 7.
Several tasks can be followed at the same time: detection of contaminant cells, identification of these cells of primary culture origin used for in vitro testing and finally, monitoring for eventual genomic mutations due to prolonged cultivation or contact to biomaterials. Inconclusive results in just one of these aspects should lead to the disqualification of the monitored cultures from usage in vitro.