Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey (Macaca fascicularis) fetuses.
We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl-D-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage-colony stimulating factor (M-CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.
Negishi
Takayuki
T
Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. taka-u@yayoi.club.ne.jp
Ishii
Yoshiyuki
Y
Kyuwa
Shigeru
S
Kuroda
Yoichiro
Y
Yoshikawa
Yasuhiro
Y
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods*; D016923:Cell Death / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003563:Cyclic CMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005333:Fetus / Q000166:cytology*; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008252:Macaca fascicularis; D017628:Microglia / Q000187:drug effects / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D016895:Culture Media, Serum-Free; D003563:Cyclic CMP; D003561:Cytarabine; D016291:Dizocilpine Maleate; D004815:Epidermal Growth Factor; D018691:Excitatory Amino Acid Antagonists; D005346:Fibroblast Growth Factors; D005904:Glial Fibrillary Acidic Protein; D018698:Glutamic Acid; D007166:Immunosuppressive Agents; D008869:Microtubule-Associated Proteins; D004676:Myelin Basic Protein; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C053441:dibutyryl cyclic-3',5'-cytidine monophosphate