1. ¹Volkswagen-Foundation Junior Group, University of Regensburg, and Center of Gynecological Endocrinology, Regensburg, Germany
2. ²Department of Neurology, University of Regensburg, Reproductive Medicine and Human Genetics, Regensburg, Germany
3. ³Center of Gynecological Endocrinology, Reproductive Medicine and Human Genetics, Regensburg, Germany
4. ⁴Institute of Neuropathology, University of Frankfurt, Frankfurt, Germany
5. ⁵Waisman Center and Department of Anatomy, University of Wisconsin, Madison, Wisconsin

Correspondence: Dr. L. Aigner, Department of Neurology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. E-mail: ludwig.aigner@klinik.uni-regensburg.de

⁶F-PW and SC-D contributed equally to the present study.

Received 12 March 2003.

Abstract

Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs.