Culture and Differentiation of Rat Neural Stem/Progenitor Cells in a Three-Dimensional Collagen Scaffold

A stable and fast method for constructing a neural-like tissue from rat neural stem/progenitor cells (rNS/PCs) based on three-dimensional (3D) collagen gel is described. First step, the collagen-embedded rNS/PCs expanded with the medium consisting of DMEM/F12/RPMI1640 (1:1:1) supplemented with EGF and bFGF was used to expand the cells in gel in 96-well plates until the average diameter of cell clusters was about 50–100 μm with the cell density higher than 10⁷ cells/mL. In the second step, the initial medium was replaced with NB/B-27 supplemented with bFGF and BDNF. The results show that cells in collagen presented neural-like morphology and maintained live cell rate around 82 % in neural network pattern at least for 42 days under static conditions. The cell–collagen constructs were detected by immunofluorescence and immunohistochemistry test after 42 days of culture, part of cells still maintained the character of rNS/PCs, and others differentiated into neurons, astrocytes, and oligodendrocytes. Our 3D neural-like tissue construct was similar to the neural tissue in morphology and cell compositions. They thus have a potential to be used for drug screening, detection of environment toxins, and replacement therapy.