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

In 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.

Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia).

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.

Cryopreservation of pecten maximus heart cells

A dissociation protocol for Pecten maximus heart has been established that makes it possible to obtain functional primary cultures routinely (9, 10); freezing assays of isolated cells were performed with the aim of making it possible to cultivate these cells in vitro after thawing to provide a constant standardized source of cells for applied research. Various parameters such as the nature and the concentration of the cryoprotectant, the cooling rate, and the incubation time of cells with the cryoprotective agent were evaluated. Best results were obtained by freezing cells in 12% dimethyl sulfoxide (Me2SO) in Leibovitz L15 medium at a cooling rate of approximately 2-3 degreesC/min. Thawed cells in culture attached to the substrate and survived for at least 3 weeks. They exhibited similar morphology and synthesised proteins, DNA, and lipids in vitro at levels close to those observed in fresh cells. To our knowledge, it is the first time that cultures have been obtained from cryopreserved marine invertebrate cells. Copyright 1998 Academic Press.