Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation

International audienceIn Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons

Establishment of functional primary cultures of heart cells from the clam Ruditapes decussatus

Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (IK slow) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (IK fast) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (IKCa) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (ICa) inhibited by verapamil at 5 × 10−4 M, T-type Ca2+ current, rapidly activated and inactivated, and sodium current (INa) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 μM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2′-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology

Development of an in vitro culture method for cells and tissues from the zebra mussel (Dreissena polymorpha)

Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability >80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates