Primary Mouse Neuron Progenitor Cells
Commonly used acronym: Mouse NPCs
Scope of the method
- Animal health
- Basic Research
- In vitro - Ex vivo
- Animal derived cells / tissues / organs
- Neuron progenitor cells
- mouse brain
- neuron differentiation
- neurodevelopmental disorders
- cellular proliferation
We have developed a protocol for culturing primary neuron progenitor cells (NPCs) derived from mouse embryos at embryonic day 15 (E15). These cells are highly proliferative, can be subcultured and cryopreserved and can be differentiated to neurons or astrocytes. Hence, this method can greatly reduce the number of laboratory animals needed for the culture of primary neuronal cultures. They can be grown as adherent monolayers or as neurospheres.
The protocol consist of the following steps: Dissection of brain from mouse fetus at E15, separation of dorsal forebrain, dissociation into single cell suspension, culture onto poly-D-lysin coated vessels (for adherent cultures), subculturing, cryopreservation of cells, thawing of frozen cells, differentiation to neurons, maturation of differentiated neurons. The protocol is partially based on the protocol first described by Steven Pollard, Methods Mol Biol 2013.
- - Dissection material
- - Stereotactic microscope
- - Horizontal flow cabinet
- - Laminar flow cabinet
- - Incubator
- Still in development
- History of use
- Internally validated
Pros, cons & Future potential
Possibility of subculturing and cryopreservation constitutes an almost unlimited source of primary cells that can be used for differentiation into neurons, thereby dramatically reducing the need for animals to generate primary neuron cultures. The cells are highly suitable for research on neurodevelopmental diseases which most often underlie defects in neuron progenitors, rather than neurons. The cells are also very suitable for genetic interference, e.g. via siRNA, CRISPR-Cas9 or lentiviral vectors.
The cells need specific growth media, which regularly needs to be prepared freshly and the medium should be changed every two days.
We have started from the method as described by Pollard, Methods Mol Biol 2013 which did not work for us. We have gradually tweaked this method and are still trying to improve it by making small modifications, although our current protocol is very robust.
We currently have different cryopreserved cell lines from transgenic animals (e.g. Trp53 knockout) and cell lines that stably overexpress some of our genes of interest. Our research focuses on radiation-induced microcephaly but these cells could be applied as well for other neurodevelopmental disorders.
References, associated documents and other information
Mfossa et al., bioRxiv. 2020. doi: https://doi.org/10.1101/2020.06.26.171132. Not yet peer-reviewed.
Contact personRoel Quintens
OrganisationsBelgian Nuclear Research Centre