Survival and process regrowth of purified chick retinal ganglion cells cultured in a growth factor lacking medium at low density. Modulation by extracellular matrix proteins.
Panning-purified retinal ganglion cells (RGCs) were cultured at low density in a chemical-defined growth factor (GF)-lacking medium on substrate of different extracellular matrix (ECM) proteins. The process regrowth under these severe conditions were evaluated by morphometric measurements and by cell ELISA (CELISA) performed for neurofilaments regardless of their phosphorylated state (NF-CELISA), or for phosphorylated neurofilaments (PNF-CELISA), to respectively assess process regrowth or axonal development. The development obtained in cultures performed on laminin was taken as standard to refer the other substrata. The cellular content of Thy-1 required for panning purification as well as the gangliotetraosylganglioside (GTOG) expression and the lack of the immunolabeling of the RA4 antigen strongly suggest that the purified RGCs were mature neurons. About 80% of the 7-day-old embryo (E7)-RGCs survived 4 days in culture on any substrate, including polylysine. Conversely, E10-RGCs in about 75% of cultures on polylysine did not survive for 4 days. E7-RGCs developed better on thrombospondin and vitronectin. E10-RGCs cultured on vitronectin grew better than on laminin; on thrombospondin and collagen, E10-RGCs grew like on laminin and on fibronectin they had a poor development. The values of PNF-CELISA obtained on vitronectin, collagen and fibronectin on E7-RGC cultures were significantly higher than on laminin, which are in agreement with the longer processes observed. The flavoridin disintegrin caused a dose-response inhibition on E7-RGC cultures on thrombospondin but not on laminin, suggesting on process regrowth, the integrin-thrombospondin interaction(s) are significantly involved, while on laminin, it is the non-integrin receptor(s) which are significant involved.
Brocco
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MA
Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC-CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000, Córdoba, Argentina.
Panzetta
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P
D000818:Animals; D000831:Animals, Newborn / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002645:Chickens; D003435:Crotalid Venoms; D003470:Culture Media / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin / Q000494:pharmacology; D009416:Nerve Regeneration / Q000187:drug effects / Q000502:physiology*; D010455:Peptides / Q000494:pharmacology; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000502:physiology*; D019699:Thrombospondins / Q000494:pharmacology
D003435:Crotalid Venoms; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; D010455:Peptides; D019699:Thrombospondins; C069044:flavoridin protein, Trimeresurus