Carlos E. Semino

Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

Center for Learning and Memory, Department of Brain and Cognitive Science

RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts

Center for Biomedical Engineering

Isolation and expansion of self-renewing neural cells ex vivo are required for neural tissue repair in regenerative medicine. Neurogenesis occurs in restricted areas of postnatal mammalian brain including dentate gyrus and subventricular zone. We developed a simple method to entrap migrating neural cells (potential neuroprogenitors) from postnatal hippocampal organotypic cultures in three-dimensional (3-D) peptide nanofiber scaffolds. A few hours after placing the hippocampal slices in culture, cell proliferation activity at the "interface zone" between the tissue slice and the membrane culture surface was observed. Pulse-chase experiments using 5-bromodeoxyuridine (BrdU), which measures mitotic activity, showed that a number of cells incorporated BrdU at the interface zone. The number of BrdU⁺ cells increased exponentially during the first 3 days of exposure to the label. The BrdU⁺ cells also stained positive for glial fibrillary acidic protein (2.2 ± 0.5%), a marker for astroglia; and for III tubulin (7.3 ± 2.8%) and nestin (2.7 ± 0.9%), markers for neural progenitors. When hippocampal slices were cultured on a peptide nanofiber scaffold layer (~500 m thick), a more extended interface zone between each tissue slice and the scaffold was formed. Moreover, the migrating BrdU⁺ cell population entrapped in the 3-D peptide scaffold was readily isolated by mechanically disrupting the scaffold and then used for conventional 2-D culture systems for further studies. This simple method may be useful not only in developing technology for neural progenitor cell isolation and enrichment in vitro, but also for expanding cells for cell-based therapies of regenerative medicine.