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The Euromed-N composition has been customized to fit the unique growth requirements of embryonic and adult neural precursor cells isolated from the mammalian central nervous system (CNS)
Euromed-N is a basal medium and, just like DMEM/F-12, does not contain any growth or trophic factors, hormones and L-glutamine.
Therefore, all existing protocols used to grow neural precursor cells in vitro can be employed without any modification except for the replacement of DMEM/F12 with the Euromed-N basal medium.
Its specific formulation meets the basic requirements for the culturing of embryonic and adult neural stem cells and in combination with other supplements (such as N2, NeuroMix ,G5 and NSS) this medium allows consistent growth and/or differentiation of neural cells.
Culture of neural stem cells form mouse embryonic neural tissue
To cultivate neural stem cells from mouse embryonic neural tissue, Euromed N (ECM0883L) must be supplemented with N2 (final concentration 1x), L-glutamine (ECB3000D) (final concentration 2mM), FGF (final concentration 20ng/ml) and EGF (final concentration 20ng/ml).
1. After the dissection of the desired brain region from the mouse embryos, transfer the tissue in a 15 ml conical tube containing complete Euromed N medium (without growth factors), which has been pre warmed at 37°C.
2. Dissociate the tissue by Pasteur pipetting until a homogenous single cell suspension is achieved. If undissociated tissue remains in suspension, allow the sample to settle for 1-2 minutes and transfer the supernatant containing single cells into a fresh tube.
Note: do not introduce air bubbles while pipetting into the cell suspension.
3. Add more complete Euromed N medium (without growth factors) to the undissociated tissue and continue to dissociate. Then transfer and pool the supernatant containing single cell suspension. Repeat this step if necessary.
4. Centrifuge the cells at 75 x g for 10 minutes, remove supernatant and resuspend the cells in 1-2 ml of complete Euromed N.
5. After counting, plate the cells at the desired concentration (the suggested density is about 5 x 10 4 / cm2) with complete Euromed N medium containing FGF (final concentration 20ng/ml) and EGF (final concentration 20ng/ml) (for T-25 flask approximately 6 ml).
6. Single cells will proliferate and form spheroid clusters of cells called “neurospheres” which will float in suspension (1-3 days). The neurospheres will be ready for subculturing after 3-7 days.
Culture and maintenance of neurospheres
1. Tap the culture flasks to detach the neurospheres from the plastic, transfer the cell culture to a sterile 15 ml centrifuge tube, rinse the flask with medium and transfer again, and centrifuge at 110xg for 10 minutes.
2. Remove the supernatant and leave some medium with the cell pellet (approx. 300 microlitres)
3. Dissociate the neurospheres accurately with a p200 pipette until a fine single cell suspension is achieved, (if necessary repeat the steps as described above).
4. Plate the cells as described above and dissociate again the neurospheres when they begin to detach from the bottom of the flask (generally after 3-7 days).
Shelf life: 18 months
Store at +4°C
For research use only