Cell Cultures of Coelomocytes New Tools to Detect Marine Pollution

As already mentioned, the development of in vitro cultures of coelomocytes has been considered important and recommended in order to better analyze the mechanisms involved in defence/stress response and to overcome the stress of manipulation. Media for keeping sea urchin coelomocytes outside of the animal, for a reasonably sufficient period of time, generally involve the use of anti-coagulant reagents, such as the chelating agents EDTA and EGTA. In our studies, P.lividus coelomocytes were kept and separated into subpopulations by gradients in an EDTA-containing buffer (ISO-EDTA). This medium was good enough to show, for the first time, that coelomocytes respond to physical and chemical stresses by elevating the hsp70 expression levels (Matranga et al. 2000). However, it was not preserving the morphological features of freshly collected cells and it was not usable at all for in vitro exposure to chemicals or other stresses. A medium involving EGTA, although utilized with success for the American species Lythechinus variegatus (Koros 1993), was, in our hands, not working.We then used successfully a medium modified from that originally described by Henson et al. (1992). We were able, under these conditions, to keep cells for short-term cultures in vitro, but not longer than 4-6 h (Matranga et al. 2002). In fact, by Hoechst staining, we observed that the number of apoptotic cells increased after prolonged time culture: from 5-6% for culture times up to 6 h, gradually increasing to 10-12% between 8 and 12 h, and above 20 % after 14 h. The number of apoptotic cells reached 25 and 44 % after 16 and 18 h, respectively (not shown). Therefore, for studies on chronic effects of pollutants on sea urchin coelomocytes, it was necessary to define a culture medium for long-term cultures. For this purpose we took advantage of the technique described for primary cultures of epithelial cells and myocytes obtained from the heart of the mollusc Pecten maximus (Le Marrec-Croq et al. 1999), which utilizes a simple medium based on sterile seawater. In fact, mollusc cultures were maintained viable in vitro for at least 2 months and used as a sensitive test to study the effects of pollutants at the cellular level (Pennec et al. 2002).

The medium has been adapted to the P. lividus coelomocytes osmolarity, thanks to collaboration with G. Dorange within the framework of the UVTOX European project. Total cells were collected by bleeding with a sterile syringe introduced through the peristomal membrane and plated on tissue culture-treated 24-well plates. Half of the medium was refreshed every other day. Under these conditions, cells attached to the plastic-ware without the need of any special adhesive factor. The number of attaching cells grad-

Fig. 3a-d. Surface antigens localized on sea urchin coelomo-cytes attached to the substrate. Bright field and immunofluores-cence images of whole mounts decorated with WGA-FITC. Bar 10 |m

Fig. 3a-d. Surface antigens localized on sea urchin coelomo-cytes attached to the substrate. Bright field and immunofluores-cence images of whole mounts decorated with WGA-FITC. Bar 10 |m

ually increased with time, and within 1 h virtually all cells plated were bound to the substratum. The vitality of cells was confirmed by the expression of surface antigens, as assessed by WGA-FITC fluorescence on whole mount specimen (Fig. 3).

When maintained in culture for longer periods of time, cells have the tendency to form bundles and fibres, although maintaining their individuality (Fig. 4); petaloid-shaped phagocytes were no longer visible and possibly philopodial phagocytes were converted into a fibroblastoid-like morphology. Interestingly, both red and colourless amoebocytes, as well as vibratile cells, continued to be observable, constituting an easy criterion for assessing the viability of the culture.

An attempt to keep cells in culture for more than 8 days, which in some cases lasted up to 20 days (Fig. 5), resulted in the formation of a meshwork of cell clumps interconnected by macrofibres made of cellular protrusions (Fig. 5a). Even after such a long time it was possible to distinguish red amoe-bocytes. Possibly, colourless amoebocytes were passively trapped or adhered to the large phagocyte aggregates present in the culture (Fig. 5b,c), in agreement with earlier reports on hanging drops cultures (Johnson 1969).

The culture media tested, namely the Henson and the Le Marrec-Croq modified coelomocyte media, respectively, referred to as HMCC and LMCC below, were used for the next experiments and constitute good starting points to define an appropriate coelomocyte culture medium for establishing continuous cell cultures.

Fig. 4. Sea urchin coelomocyte cultures. Cells, maintained in culture in vitro for 4 (a) and 8 (b) days tend to form bundles and fibres. Bar 10 |m

Fig. 5. Sea urchin coelomocyte long-term cultures. Cells were maintained for 20 days in culture after plating. Bar: a 158 |m; b 100 |m; c 50 |m

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