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A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

Babu H, Claasen JH, Kannan S, Rünker AE, Palmer T, Kempermann G - Front Neurosci (2011)

Bottom Line: The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27.Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart.We also highlight potential modifications to the protocol.

View Article: PubMed Central - PubMed

Affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Stanford, CA, USA.

ABSTRACT
In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.

No MeSH data available.


Workflow diagram for the experimental procedure.
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Figure 1: Workflow diagram for the experimental procedure.

Mentions: The workflow is depicted in Figure 1. After separation of the hippocampus from the rest of the brain, the dentate gyrus is further isolated by micro-dissection from the adjacent walls of the lateral ventricles and cornu ammonis of the hippocampus to avoid a commixture of hippocampal neural precursor cells with other precursor cell populations. The excised tissue containing the neurogenic subgranular zone is then further dissociated into a single-cell suspension using an enzymatic solution supported by mechanical trituration steps with fire-polished Pasteur pipettes. In the ensuing step, neural precursor cells are separated from differentiated cells, myelin, and extracellular matrix using density gradient separation by a centrifugation step with 22% Percoll. Pelleted cells are separated from supernatant, washed, resuspended in chemically defined culture medium (Neurobasal A with supplements B27 and Glutamax-1 and growth factors EGF and FGF2) and plated on coated culture dishes. Two days after isolation neural precursor cells that have not yet attached to the surface are collected, centrifuged, and replated into the same culture dish with fresh medium. This enrichment step is necessary to ensure an optimal density for further cell expansion. From day 4 after cell extraction onwards, 75% of the culture medium is substituted every second day. In about 9–12 days 70–80% of the culture dish surface is populated and the culture is ready for the first passage.


A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

Babu H, Claasen JH, Kannan S, Rünker AE, Palmer T, Kempermann G - Front Neurosci (2011)

Workflow diagram for the experimental procedure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3140691&req=5

Figure 1: Workflow diagram for the experimental procedure.
Mentions: The workflow is depicted in Figure 1. After separation of the hippocampus from the rest of the brain, the dentate gyrus is further isolated by micro-dissection from the adjacent walls of the lateral ventricles and cornu ammonis of the hippocampus to avoid a commixture of hippocampal neural precursor cells with other precursor cell populations. The excised tissue containing the neurogenic subgranular zone is then further dissociated into a single-cell suspension using an enzymatic solution supported by mechanical trituration steps with fire-polished Pasteur pipettes. In the ensuing step, neural precursor cells are separated from differentiated cells, myelin, and extracellular matrix using density gradient separation by a centrifugation step with 22% Percoll. Pelleted cells are separated from supernatant, washed, resuspended in chemically defined culture medium (Neurobasal A with supplements B27 and Glutamax-1 and growth factors EGF and FGF2) and plated on coated culture dishes. Two days after isolation neural precursor cells that have not yet attached to the surface are collected, centrifuged, and replated into the same culture dish with fresh medium. This enrichment step is necessary to ensure an optimal density for further cell expansion. From day 4 after cell extraction onwards, 75% of the culture medium is substituted every second day. In about 9–12 days 70–80% of the culture dish surface is populated and the culture is ready for the first passage.

Bottom Line: The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27.Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart.We also highlight potential modifications to the protocol.

View Article: PubMed Central - PubMed

Affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Stanford, CA, USA.

ABSTRACT
In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.

No MeSH data available.