Submitted to: European Association of Fish Pathologists
Publication Type: Abstract Only
Publication Acceptance Date: 5/22/2001
Publication Date: 9/9/2001
Citation: KLESIUS, P.H., XU, D., EVANS, J.J., SHOEMAKER, C.A. DEVELOPMENT AND CHARACTERISTICS OF AN ESTABLISHED MICROGLIA CULTURE FROM BRAIN OF TILAPIA. TENTH INTERNATIONAL CONFERENCE OF THE EUROPEAN ASSOCIATION OF FISH PATHOLOGISTS "DISEASES OF FISH AND SHELLFISH". 2001.
Technical Abstract: The microglia is the intrinsic immune cell of the central nervous system (CNS) and is of the monocyte/macrophage lineage. Little or no information is available on the characteristics of microglia of fish. The brains of exsanguinated tilapia were aseptically removed, trimmed and blotted to remove connective tissues and blood vessels, passed through a 100m mesh tissue screen, digested with DNase, trypsin and collagenase, triturated and semi-purified by percoll fractionation to obtain a single brain cell suspension in Leibovitz's L-15 medium supplemented with 5% fetal bovine serum and antibiotics. Cell viability in trypan blue solution was greater than 95% and 5 * 10**6 cells were cultured in each well of a 6- well tissue culture plate at an atmosphere of 5% CO2 and 95% air at 28 deg C. The majority of the attached cells stained non-specific esterase positive, rosetted opsoninized sheep red blood cells and phagocytized both latex beads and opsoninized Streptococcus iniae cells. Flow cytometric and microscopic analysis of the attached cells treated with isolectin B4, mammalian microglia marker, revealed positively staining cells. Additional phenotypic characteristics of glia cells were analyzed by immunostaining using anti-mouse antibodies to CD11b, CD95, GFAP and galactocerebroside. Microglia cultures had a purity of >90% and were maintained in culture for over 50 days. These results demonstrate it is possible to maintain microglial cultures in vitro for future studies on the determination of the role of microglia in CNS pathology, immune and vaccine responses in fish.