Cell viability slow decline could be due to apoptosis induced by the reduction in the number of the non-adherent cells (rare immune cells: vibratile cells and amoebocytes) after each medium change. cell lines that all relate to a single theme, Rabbit Polyclonal to ALS2CR11 such as the marine invitrome (Bols et al., 2017) C have the disadvantage of having altered or lost specific cell functions because of mutations. In contrast, primary cell culture represents much more accurately the biological microenvironment in which cells reside in tissues, as cellCcell signalling remains preserved; thus, primary cultures are a more appropriate tool Solenopsin for biotechnological applications and pathological investigations (Bols et al., 2017). The principles reduce, refine and replace (3Rs) (Russell et Solenopsin al., 1959) have developed into imperative considerations in the design of scientific experiments that use animal models. Importantly, new and more sustainable methods, which minimise animal usage, have resulted in the development of novel methods specifically to address and limit the use of mammals. Only a few studies have addressed the development Solenopsin of marine invertebrate primary cultures (and these have focused on cells) derived from different tissues of a few species used for basic biological studies (response to pathogens, toxins, etc.) (Majeske et al., 2013; Vandepas et al., 2017; Maselli et al., 2018), even though primary cultures represent a rich source of cell and tissue types (Rinkevich, 2011). This limited understanding of marine primary cell cultures includes the absence of an appropriate medium formulation and a shortage of cell proliferation assessments (Cai and Zhang, 2014). Sea urchins are marine deuterostome invertebrates and as Nobel legacy model organisms have been highly exploited for biological studies. In addition, the sea urchin has been nominated for inclusion on the list of alternative animal models presented by the EPAA (European Partnership for Alternative Approaches to Animal Testing). The full sequence release of the sea urchin genome (purple sea urchin) revealed the close genetic relationship between sea urchins and humans, an exceptional example of immune system complexity and sensing capacity (Sea Urchin Genome Sequencing Consortium, 2006), thus further reinforcing the relevance of this model organism. Immune cells function as the central sensing and effector components of the sea urchin (phagocytes, amoebocytes and vibratile cells) reside within the coelomic cavity as well as in all other tissues, and orchestrate key innate immune functions, which consist of complement and cytokine secretion, chemotaxis, opsonisation, complement activation, phagocytosis and cytotoxic/cytolytic response. Immune cells produce and secrete specific regulatory biomolecules into the coelomic fluid (CF) (a ?uid with functions similar to the blood and the lymph of vertebrates), to maintain functional homeostasis and intercellular crosstalk (Smith et al., 2018; Pinsino and Matranga, 2015; Pinsino et al., 2015). The establishment Solenopsin of suitable harvesting methods, a well-defined medium and long-term cultivation protocol, to result in a stable long-term system, is still needed. Previous sea urchin primary immune cultures, based on both simple and complex media, have not maintained satisfactory cellular viability over long periods (Johnson, 1969; Bertheussen and Seljelid, 1978; Dan-Sohkawa et al., 1993; Matranga et al., 2002; Matranga et al., 2006; Majeske et al., 2013). Here we formulated a physiological-like medium and developed successful methodologies to culture immune cells, and have accomplished the following aims: (i) we developed a long-term, easy and reliable cultivation protocol and (ii) we compared different cell culture media already in use for sea urchin species Solenopsin for cell adherence, survival and growth. Our findings should further support the development of a new, powerful proxy for human immunology and toxicology studies. RESULTS AND DISCUSSION Quality control of freshly harvested sea urchin immune cells and related primary short-term cell cultures The three major cell types of freely circulating immune cells have been described in (phagocytes, amoebocytes and vibratile cells) (Pinsino and Matranga, 2015). Thus, a morphological analysis of the harvested cells in coelomocyte culture medium (CCM), ISO-EDTA and ASW as collected from sea urchins maintained under controlled conditions (Fig.?1ACE) was performed in a Fast-Read chamber.