Cryopreservation and storage of mussel (Mytilus spp.) haemocytes for latent analysis by the Comet assay

Estuarine and coastal habitats are known to be polluted by a range of chemical contaminants from both industrial and domestic sources. Blue mussels (Mytilus spp.), which inhabit these areas, are widely used as bio-indicators in eco-toxicological studies, because of their sedentary nature and their ability to bio-accumulate contaminants. The analysis of DNA damage in mussel haemocytes is a valuable tool for biomonitoring but sampling issues related to storage, handling and transportation have often limited its application in large-scale monitoring programmes. This study uses a trial and error method to evaluate and validate a suitable protocol for cryopreservation of mussel haemocytes, thereby allowing material collected in the field to be analysed later under controlled laboratory conditions. Three different cell-culture media, i.e. Leibovitz-15, Hank's balanced salt solution and mussel physiological saline, along with four different cryoprotectants, i.e. dimethyl sulphoxide (10% and 20%), 1,2-propanediol (10%), ethylene glycol (10%) and glycerol (10%) were tested to assess their suitability for cryopreservation of mussel haemocytes for analysis in the comet assay. Experimental studies where mussel haemocytes were also exposed to UV radiation or benzo(a)pyrene were conducted in order to mimic environmental stresses and to verify the effectiveness of newly defined cryopreservation protocols. The comet assay was used to demonstrate that mussel haemocytes could be preserved at cryogenic temperatures for a month without altering levels of DNA damage, which could possibly be used for lab or field studies where time constraints or facilities do not allow instant analysis.