Impact of serum on lactate dehydrogenase (LDH) rlease determination in vitro


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.
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