Rapid method for culturing embryonic neuron-glial cell cocultures.
A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system and central nervous system cultures was very robust.
Fex Svenningsen
Asa
A
Fishberg Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029, USA. asa.fex-senningsen@mssm.edu
Shan
Wei-Song
WS
Colman
David R
DR
Pedraza
Liliana
L
D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D064026:Calbindins; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects*; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D018920:Coculture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D017615:Enteric Nervous System / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D004676:Myelin Basic Protein / Q000378:metabolism; D009186:Myelin Sheath / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018992:Myelin-Associated Glycoprotein / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D064531:Peripherins; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D012583:Schwann Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D064026:Calbindins; D016895:Culture Media, Serum-Free; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D004676:Myelin Basic Protein; D018992:Myelin-Associated Glycoprotein; D009419:Nerve Tissue Proteins; D064531:Peripherins; D064030:S100 Calcium Binding Protein G; D014357:Trypsin