Procedure for sea urchin embryo cryopreservation and attached bioassay

New and innovative aspects

As far as we know there is no other patent regarding the cryopreservation of sea urchin embryos (Paracentrotus lividus or any other sea urchin species) or any other patent dealing with the use of cryopreserved echinoderms for ecotoxicology or aquaculture. The knowledge regarding sea urchin cryopreservation is extremely scarce, research has been focused on later development stages and the percentage of normal larvae was low. Our work allowed to obtain healthy embryos (8 hours old) that develop to healthy larvae and can be incubated up to settlement. We have developed a complete cryopreservation protocol for sea urchin embryos that provides high survival and high long term development percentage that can settle and become juveniles. We have developed a sea urchin cryopreserved embryo bioassay and compared results obtained with the standard sea urchin bioassay. We have tested the bioassay with organic and inorganic compounds and with environmental elutriate samples. The possibility of a sea urchin embryo biobank ensures not only the access to test organism embryos all year round but also access to these embryos in places where it was not possible before either for their distance to the ocean or for the lack of the necessary facilities to maintain reproductive adults in optimum conditions to breed. The development of the bioassay using cryopreserved sea urchin embryos will help overcome the seasonal constraints in application of bioassays to assess marine water quality.

Main advantages of its use

It is a novel process for this kind of organisms, with application in research, ecotoxicology, conservation and aquaculture as well as industrial applications in aquaculture and environmental monitoring. The cryopreserved embryo bioassay is a good alternative to the standard sea urchin bioassay which is widely use to assess water quality nowadays but that is seasonally restricted due to the lack of biological material all-year round. Despite this cryopreservation protocol has been specifically developed for an specific species and cellular type, studies suggest that the protolol could be applied as well to other organisms and cell types with few adaptations.

Specifications

Cryopreservation is the conservation of cells, embryos or tissues at very low temperatures, usually liquid nitrogen. Cells at -196°C are in life suspension, there are no metabolic or chemical reactions of any kind that means that it is a stable method of storage of biological material. Cryopreservation relies in the use of cryoprotective solutions that minimize internal cell ice formation during freezing which is a lethal event. The type and concentration of those compounds, as well as, the methodology of addition are important. Cryoprotecting solutions can be toxic at high concentrations, a well-studied balance between toxicity and cryoprotection needs to be met. In addition if the cryoprotecting agents are introduced to the cells abruptly, osmotic damage can occur. Once cells are frozen and thawed, they will continue to develop, more slowly than fresh controls at first but soon at the same rate than unfrozen cells, this is believed to be caused by a need of time for the cells to achieve full reactivation of their metabolism after thawing. The process starts with obtaining the gametes from the sea urchins and performing an in vitro fertilization. After 8 hours of development at 20 ºC we obtain healthy blastula embryos. Once healthy embryos are obtained the cryopreservation protocol starts: 1.- Add the cryoprotective solutions composed of Dimethyl sulfoxide and Trehalose in several controlled steps to avoid osmotic shock. 2.- Vials are introduced in a controlled rate freezer , starting at 4 ºC for 2 min, cooling at a rate of 1 ºC min-1 to -12 ºC. Samples are seeded at a 2 minute holding at -12 ºC. Continue cooling at 1 ºC min-1 to – 80 ºC. After a 2 minute hold, vials are quickly taken out from the freezer into liquid nitrogen for conservation. 3.- Samples are stored in liquid nitrogen at -196 ºC for as long as necessary. No detrimental effects of storage have been reported in scientific literature regarding conservation time. 4.- Thawing of the samples is done in a 17 ± 1 ºC water bath (1.5-2 min.). 5.-Once thawed, the cryoprotective solutions have to be diluted in several steps to avoid osmotic damage of the cells. Once the cryoprotecting solutions have been diluted, embryos are ready to be used, either for further culture with aquaculture and research or ecotoxicology to be used in the sea urchin cryopreserved embryo bioassay.

Applications

• Water quality assessment technological companies, research agencies, aquaculture farms are many of the possible sectors where the technology can be applied.

• Potential users would be research institutions, environmental monitoring companies, environmental agencies, aquaculture farms.