Primary neural precursor cell expansion, differentiation and cytosolic Ca(2+) response in three-dimensional collagen gel.
To investigate the ability to culture neural precursor cells in a three-dimensional (3D) collagen gel, neuroepithelial cells were isolated from embryonic day 13 rat cortex, dispersed within type I collagen and maintained for up to 30 days in vitro. Cultured in Neuorobasal medium supplemented with B27 containing basic fibroblast growth factor, the collagen-entrapped precursor cells actively expanded and formed clone-like clusters. Many cells in the center of the cluster were proliferating as revealed by 5-bromo-2'-deoxyuridine uptake. Some cells began to migrate away from the center at 5 days and were labeled by either neuronal marker neuron-specific beta-tubulin (TuJ1) or astrocytic marker glial fibrillary acidic protein. The differentiated neurons (TuJ1(+)) exhibited characteristic cytosolic Ca(2+) oscillations in response to excitatory neurotransmitter glutamate. These findings suggest the suitability of the 3D culture system for the proliferation and differentiation of neural precursor cells.
O'Connor
S M
SM
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
Stenger
D A
DA
Shaffer
K M
KM
Maric
D
D
Barker
J L
JL
Ma
W
W
D000818:Animals; D001253:Astrocytes / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D003094:Collagen; D016895:Culture Media, Serum-Free; D003600:Cytosol / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005782:Gels; D018698:Glutamic Acid / Q000494:pharmacology; D009474:Neurons / Q000166:cytology; D009991:Oscillometry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*
D016895:Culture Media, Serum-Free; D005782:Gels; D016222:Fibroblast Growth Factor 2; D018698:Glutamic Acid; D003094:Collagen; D002118:Calcium