Abstract
According to the DIN EN ISO 10993-5 which contains recommendations on the biological
evaluation of medical devices cellular release of enzymes is a sufficient parameter to
determine cytotoxicity. Generally, there are two possibilities for cells in culture to react
towards cytotoxic substrates. They can stop growing or dividing, or they can die through
necrosis or apoptosis. Necrosis is accompanied by mitochondrial swelling and increased
plasma membrane permeability, while apoptosis involves an articulated breakdown of the cell
into membrane-bound apoptotic bodies. In this way necrosis and apoptosis result in a
compromised or damaged cell membrane, and lactate dehydrogenase (LDH), a soluble
enzyme of the cytoplasm, becomes released into the extracellular space. Since this only
happens when cell membrane integrity is compromised, the content of this enzyme in the
supernatants from cells can be used as an indicator of damaged cell membrane integrity and
serve as a general means to assess cell viability by measuring plasma membrane permeability.
However, cell culture media, which are used to maintain and to support cell growth
commonly need serum supplementation, and the serum content varies depending on the
cellular needs. Additionally, sera already contain various LDH amounts, which may increase
background absorbance in LDH release assays. For this reasons we tested the effect of
different serum loads (inactivated fetal bovine serum [FBS]; 1-10 vol%) on the results of a
commercially available LDH release assay (Cytotoxicity detection Kit, Roche) using 3T3
cells. An FBS concentration of 1 vol% was found to provide the best compromise between
cell growth support and variability of the results. Test with primary cells will be performed in
the future to confirm these results.
