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Biol Open
2019 Mar 05;83:. doi: 10.1242/bio.039289.
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Sea urchin Paracentrotus lividus immune cells in culture: formulation of the appropriate harvesting and culture media and maintenance conditions.
Pinsino A
,
Alijagic A
.
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The sea urchin is an emergent model system for studying basic and translational immunology. Here we report a new method for the harvesting and maintenance of primary immune cells isolated from adult Paracentrotus lividus, a common Mediterranean sea urchin species. This optimised method uses coelomocyte culture medium, containing a high-affinity Ca2+ chelator, as the ideal harvesting and anti-clotting vehicle and short-term culture medium (≤48 h), and artificial seawater as the master medium that maintains cell survival and in vitro-ex vivo physiological homeostasis over 2 weeks. Gradually reducing the amount of anticoagulant solution in the medium and regularly replacing the medium led to improved culture viability. Access to a robust and straightforward in vitro-ex vivo system will expedite our understanding of deuterostome immunity as well as underscore the potential of sea urchin with respect to biomedicine and regulatory testing.This article has an associated First Person interview with the first author of the paper.
Fig. 1. Adult sea urchin and immune cells in culture under the microscope. (A) Graphic representation of the three major immune cell types (phagocytes, amoebocytes and vibratile cells), in percent. A violet adult P. lividus maintained in an aquarium under controlled conditions. Scale bar: 1 cm. Immune cells were inspected under a microscope just after collection (B–E) and after 24 h in culture (F–H). Arrowheads with different colours indicate the major cell types of freely circulating P. lividus immune cells: red amoebocytes (red arrowheads), white/colourless amoebocytes (white arrowheads), phagocytes (black arrowheads), and vibratile cells (blue arrowheads). Live immune cells were collected in (B) CCM, (C) ISO-EDTA, (D) CF without an additional solution, and (E) ASW. (F) Immune cells cultured for 24 h in CCM show extensive spreading of the phagocytes and ovoid amoebocytes. (G) Immune cells collected in CCM and cultured for 24 h in CCM and ASW (1:1) supplemented with P/S display ovoid amoebocytes and long cytoplasmic processes of phagocytes that form an elaborated network. (H) Immune cells cultured for 24 h in ISO-EDTA show poor adhesion and morphological alterations. Scale bars: (B–H) 10 µm; white frames in B–H indicate inset images shown at a higher magnification.
Fig. 3. Long-term maintenance of primary immune cell culture in CCM-ASW-based medium plus P/S. (A) Graph showing the percentage of the Trypan Blue-negative cells and percentage of immune cells with the stable lysosomal membrane labelled with Neutral Red. The data represent mean±s.d. of at least three independent experiments. (B–I) Representative images of long-term immune cell cultures maintained with daily replacement of ASW. The culture show stable, well-adhered immune cells over the course of 2 weeks. Scale bar: 10 µm. White frames indicate the higher-magnification images shown in each inset. (B–I) Primary immune cells after (B) 1 h, (C) 6 h, (D) 1 day, (E) 2 days, (F) 3 days, (G) 4 days, (H) 6 days and (I) 14 days in culture.
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