Neurosphere Culture from Embryonic Mouse Forebrain

We sacrificed pregnant mice at gestational day 14.5 (E14.5) and removed the embryos [28 (link), 29 (link)]. Forebrains (cerebellar cortex or ganglionic eminence) were dissected from the embryonic brains and triturated with a P1000 micropipette for dispersal into single cells. The dispersed cells (1 x10⁶ cells) were seeded into 60-mm low-attachment PrimeSurface (Sumilon) dishes and cultured in neural stem cell growing medium consisting of NeuroBasal medium, 2% B-27 without vitamin A, 2 mM glutamine, 20 ng/mL rhEGF (Pepro Tech), and 10 ng/mL rhFGF2 (Pepro Tech) in a humidified incubator containing 5% CO₂. After 4–6 days, the cells aggregated, proliferated, and formed neurospheres. The neurospheres were collected and used for further experiments.

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Kuroda R., Tominaga K., Kasashima K., Kuroiwa K., Sakashita E., Hayakawa H., Kouki T., Ohno N., Kawai K, & Endo H. (2021). Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo. PLoS ONE, 16(7), e0255355.

Publication 2021

NeuroBasal medium rhEGF rhFGF2

Brains Cells Cerebellar cortex Dishes Embryonic Forebrains Ganglionic Gestational Glutamine Mice Neural stem cell Vitamin b 2

Corresponding Organization : National Institute for Physiological Sciences

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Variable analysis

independent variables

Gestational day (E14.5) of sacrificed pregnant mice

dependent variables

Characteristics of the dispersed single cells from forebrains (cerebellar cortex or ganglionic eminence)
Formation and properties of neurospheres after 4-6 days of culture

control variables

Cell culture conditions: NeuroBasal medium, 2% B-27 without vitamin A, 2 mM glutamine, 20 ng/mL rhEGF, 10 ng/mL rhFGF2, 5% CO2 humidified incubator
Cell seeding density: 1 x 10^6 cells per 60-mm low-attachment PrimeSurface (Sumilon) dish
Cell culture duration: 4-6 days

controls

Positive control: Not mentioned
Negative control: Not mentioned

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