Multicellular spheroid formation and evolutionary conserved behaviors of apple snail hemocytes in culture |
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Institution: | 1. School of Engineering and Technology, China University of Geosciences (Beijing), Beijing 100083, People''s Republic of China;2. School of Aeronautics and Astronautics, Sun Yat-Sen University, Guangzhou 510006, People''s Republic of China;3. Division of Intelligent and Biomechanical Systems, State Key Laboratory of Tribology, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, People''s Republic of China |
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Abstract: | A hemocyte primary culture system for Pomacea canaliculata in a medium mimicking hemolymphatic plasma composition was developed. Hemocytes adhered and spread onto culture dish in the first few hours after seeding but later began forming aggregates. Time-lapse video microscopy showed the dynamics of the early aggregation, with cells both entering and leaving the aggregates. During this period phagocytosis occurs and was quantified. Later (>4 h), hemocytes formed large spheroidal aggregates that increased in size and also merged with adjacent spheroids (24–96 h). Large single spheroids and spheroid aggregates detach from the bottom surface and float freely in the medium. Correlative confocal, transmission electron and phase contrast microscopy showed a peculiar organization of the spheroids, with a compact core, an intermediate zone with large extracellular lacunae and an outer zone of flattened cells; also, numerous round cells emitting cytoplasmic extensions were seen attaching to the spheroids' smooth surface. Dual DAPI/propidium iodide staining revealed the coexistence of viable and non-viable cells within aggregates, in varying proportions. DNA concentration increased during the first 24 h of culture and stabilized afterward. BrdU incorporation also indicated proliferation. Spontaneous spheroid formation in culture bears interesting parallels with spheroidal hemocyte aggregates found in vivo in P. canaliculata, and also with spheroids formed by tumoral or non-tumoral mammalian cells in vitro. |
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