The development of a medium for the in vitro expansion of mammalian neural stem cells

Authors

  • Arindom Sen,

    1. Pharmaceutical Production Research Facility (PPRF), Faculty of Engineering, University of Calgary, Calgary, AB T2N 1N4, Canada
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  • Leo A. Behie

    Corresponding author
    1. Pharmaceutical Production Research Facility (PPRF), Faculty of Engineering, University of Calgary, Calgary, AB T2N 1N4, Canada
    • Pharmaceutical Production Research Facility (PPRF), Faculty of Engineering, University of Calgary, Calgary, AB T2N 1N4, Canada
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Abstract

A new medium, PPRF-m2, has been developed for the in vitro expansion of murine neural stem cells. Neural stem cells (NSC) are primitive cells that have the capability to give rise to all of the cells that comprise the central nervous system (CNS). PPRF-m2 was developed in part by examining key aspects of a commercial NSC medium used in our laboratory, and includes the basal media combination RPMI/DMEM/F12 in a 1:1:1 volumetric ratio supplemented with a hormone mixture, sodium bicarbonate (21.4 g/L), Hepes (4.3 g/L), glucose (1.75 g/L), glutamine (0.14 g/L), epidermal growth factor (20 μg/L) and basic fibroblast growth factor (10 μg/L). Cells inoculated into PPRF-m2 effectively doubled 4 times in 5 days to achieve a maximum viable cell density of 1.2 × 106 cells/mL in stationary culture. This was approximately 650% higher than the maximum cell density achieved in the commercial NSC medium. In addition, the PPRF-m2 cultures were observed to contain less cellular debris, and the cells were less granular and exhibited a lower degree of attachment. Immunocytochemistry revealed that cells grown in PPRF-m2 retained the ability to generate neurons, astrocytes, and oligodendrocytes.

Abstract

Un nouveau milieu, le PPRF-m2, a ètè dèveloppè pour l'expansion in vitro de cellules de tiges neuronales de rongeurs. Les cellules de tiges neuronales (NSC) sont des cellules primitives qui ont la capacitè d'engendrer toutes les cellules qui comprennent le systeáme nerveux centrale (CNS). Le PPRF-m2 a ètè dèveloppè en partie en examinant les aspects essentiels d'un milieu de NSC commercial utilisè dans notre laboratoire, et il inclut la combinaison de milieux principaux RPMI/DMEM/F12 dans un rapport volumètrique 1:1:1 additionnèe d'un mèlange d'hormones, de bicarbonate de soude (21,4 g/L), d'Hepes (4,3 g/L), de glucose ( 1,75 g/L), de glutamine (0,14 g/L), de facteur de croissance èpidermique (20 ug/L) et de facteur de croissance de fibroblaste de base (10 ug/L). Les cellules inoculèes dans le PPRF-m2 ont effectivement doublè 4 fois en 5 jours pour atteindre une densitè de cellules viables maximun de 1,2′106 cellules/ mL en culture station-naire. Ce qui est approximativement 650% fois plus grand que la densitè de cellules maximum obtenue dans le milieu de NSC commercial. En outre, on a constatè que les cultures de PPRF-m2 contenaient moins de dèbris cellulaires et que les cellules ètaient moins granulaires et montraient un plus faible degrè de liaison. L'immunocytochimie rèveále que les cellules cultivèes dans PPRF-m2 retiennent la capacitè de produire des neurones, des astrocytes et des oligodendrocytes.

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