In vitro culture of tissue from the tunicateStyela clava

Abstract

Pharyngeal explants and circulatory hemocytes from the tunicate Styela clava were cultured in a medium containing tunicate plasma, artificial seawater, RPMI 1640, and antibiotics. Pharyngeal tissue remained viable and proliferated for up to 72 d in vitro. Proliferative activity maintained the pool of hemocytes within explants and facilitated the migration of pharyngeal hemocytes from explants into culture supernatants. The diversity of morphologically distinct cell types within the hemocyte pool of pharyngeal cultures indicated that cell division was followed by regulated differentiation. In contrast to pharyngeal cultures, suspensions of circulatory hemocytes did not survive for prolonged periods in vitro. Proliferative activity could not be detected in circulatory hemocyte cultures. These results are discussed in terms of the differentiation state of hemocytes and the efficacy of culture conditions.

Exocytosis of a complement component C3-like protein by tunicate hemocytes

“…Hemocytes were centrifuged (12,000g; 8 s) and resuspended at 4 £ 10 6 cells/ml in tunicate tissue culture medium (TTCM; 20 ml RPMI 1640 containing 20% (w/v) sodium chloride in 180 ml FSW pH 7.0) [19]. This cell density is equivalent to that of whole hemolymph.…”

The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury

“…Styela plicata hemocytes showed several crucial immune reactions including cytotoxic activity (Cammarata et al, 1995(Cammarata et al, , 1997Lipari et al, 1995), phagocytosis (Cammarata et al, 2007), allograft rejection (Raftos et al, 1990) and phenoloxidase-dependent cytotoxicity (Cammarata et al, 1997).…”

Invertebrate cytokines: tunicate cell proliferation stimulated by an interleukin 1-like molecule.

“…The methods used to quantify this stimulatory activity are well established. Pharyngeal explants from tunicates retain both cell viability and proliferative activity for up to 70 days in vitro (15). These cultures are amenable to experimental manipulation.…”

“…Tunicate tissue culture medium (T-RPMI) was prepared in sterile-filtered ASW and contained, per liter, 454 mg of RPMI 1640 powder (with L-glutamine, without sodium bicarbonate), 105 units of penicillin sulfate, and 100 mg of streptomycin sulfate (15). This medium was sterilized by filtration and stored at 4°C.…”

“…The stimulatory effects of tunicate hemolymph fractions were tested in cultures of pharyngeal tissue from S. clava. The procedure used to culture pharyngeal explants has been described in detail elsewhere (15). Pharyngeal tissue was diced into explants (3 x 3 x 1 mm) and rinsed three times in T-RPMI.…”