Hello everyone,

I have been trying to maintain neural stem cells isolated from embryonic mice brains but without much success. My eventual goal was to induce differentiation in-vitro after culture grew, but after 5 days of initial seeding they haven’t really proliferated.

Currently cells look like attached pictures after 5 days from initial seeding, after two half media change every 2-3 days. This cell culture has some attached cells, but most other look like dead, small dot-looking cells. Other strange thing that I noticed was after 2-3 days, cells/tissue like structure “rolled” up like carpet and was moving in suspension, while others formed neurosphere.

I have been using Thermo Fisher protocol for media composition, and StemCell company’s protocol for collection of cells from embryonic brain (page 13 on pdf)

https://www.thermofisher.com/us/en/home/references/protocols/neurobiology/neurobiology-protocols/differentiating-neural-stem-cells-into-neurons-and-glial-cells.html

https://cdn.stemcell.com/media/files/manual/MA28704-In_Vitro_Proliferation_Differentiation_Mouse_Neural_Stem_Cells_Progenitor_Cells_Using_NeuroCult.pdf

The changes I had to make were:

1) Use E15.5 mouse brain without cerebellum, meninges, and olfactory bulb

2) Use 2 mL 0.1% gelatin for coating solution on T-25 flask, 1 hr incubation in 37 °C, PBS wash. I know PLO/Laminin or Matrigel is more suitable, but it wasn’t available option for me.

3) The media had KO DMEM/F-12, L-glutamine, EGF, antibiotic/antimycotic. No bFGF and StemPro neural supplement.

4) Trypsin-EDTA 0.25% instead of Accutase

5) One brain per flask due to different genotype of each embryo

I initially thought these deaths were due to low density of cells, but these phenomena have been happening throughout my culture regardless of cell density.

Any advice or comments are welcomed.

Thank you!