Preparation of mixed glial primary cultures from newborn murine brains

CABRERA A; PISTAN, M; SEGOVIA ESPINDOLA, L; TEIBLER, P.G.; BUSTILLO S; CHOLICH, L

Mar del Plata

Congreso; Reunión Anual de SOCIEDADES DE BIOCIENCIA; 2019

Hotel 9 de Julio

Primary glial cell cultures is the most commonly used in vitro model for neurobiological studies. The aim of this work was to develop a protocol in our lab that allows to obtain glial cells for the toxicological study of North Argentina plants that cause disorders in the Central Nervous System. The method here described has undergone minor modifications from the protocol published by Saura et al., 2003. Briefly, Poly-d-lisine-coating solution was applied to culture plates for 24 h at 37°C. Wells were then washed 4 times with PBS before cell seeding. Primary glial cell cultures were prepared from CF-1 mouse (1 to 3 days old). Brains (five) were removed aseptically, blood vessels and meninges were discarded. The brain tissues were mechanically dissociated by pipetting. Subsequently, cells were transferred to a precooled 50 ml sterile tube and centrifuged at 1,000 rpm for 5 min at 4°C and the resulting pellet triturated before seeded in a micro-full media: DMEM-F12 supplemented with FBS (10%), MEM NEAA (100X-1%), L-Glutamine (1%), Gentamicine (10 µg/ml) and Penicillin?streptomycin (1%). Culture was incubated at 37°C and 5% CO2, medium was replaced every 4?5 days and confluency was achieved after 21 days in vitro (DIV). Cells purity was examined by immunohistochemistry using specific antibodies. Morphological observation evidenced refringent amoeboid microglia and polygonal shape astrocytes. These results were confirmed by immunohistochemistry, using Iba-1 and GFAP, which are well-established markers of microglia and astrocytes, respectively. In conclusion, the Poly-d-lisine-coating used, the maintenance of the culture in a complete medium and only the mechanical dissociation of the tissue resulted in astroglial cultures with higher microglial proportions.