Primary neural precursor cell expansion, differentiation and cytosolic Ca(2+) response in three-dimensional collagen gel

• Published in: Journal of neuroscience methods Vol. 102; no. 2; pp. 187 - 195
• Main Authors: O'Connor, S M, Stenger, D A, Shaffer, K M, Maric, D, Barker, J L, Ma, W
• Format: Journal Article
• Language: English
• Published: Netherlands 30-10-2000
• Subjects: Animals; Astrocytes - cytology; Calcium - metabolism; Cell Differentiation; Cell Division - drug effects; Cell Survival; Cells, Cultured; Cerebral Cortex - cytology; Cerebral Cortex - drug effects; Cerebral Cortex - physiology; Collagen; Culture Media, Serum-Free; Cytosol - metabolism; Fibroblast Growth Factor 2 - pharmacology; Gels; Glutamic Acid - pharmacology; Neurons - cytology; Oscillometry; Rats; Rats, Sprague-Dawley; Stem Cells - cytology; Stem Cells - drug effects; Stem Cells - physiology
• ISSN: 0165-0270
• DOI: 10.1016/S0165-0270(00)00303-4

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.