Brains and spinal cords were dissected from canine fetuses between E32–37. Briefly, the tissue was incubated in pre-warmed TrypLE Express (Gibco) with 10 mg/ml DNase-1 (A&A Biotechnology, Gdansk, Poland) for 10–12 mins, gently triturated and incubated in 37 °C for 10 min. Next, 5 ml of GRP medium (Gibco) was added and the suspension was centrifuged at 1000 rpm for 5 mins. Obtained pellet of cells was resuspended in 10 ml of GRP medium with 10 mg/ml of DNase, incubated at 37 °C in humified atmosphere with 5% CO2 for 10 mins. Then, the pellet was triturated again and centrifuged at 1000 rpm for 5 mins, resuspended in 10 ml of GRP medium and plated on coated PLL/Laminin 25 ml flasks in 37 °C in humified incubator with 5% CO2. Cells were cultured for 5–10 days (1–2 passages) in GRP medium with bFGF, harvested with TrypLE Express (Gibco), cryopreserved in ATCC medium (LGC Standards, Teddington, UK), and stored in vapor phase liquid nitrogen until transplantation.
Malysz-Cymborska I., Golubczyk D., Kalkowski L., Burczyk A., Janowski M., Holak P., Olbrych K., Sanford J., Stachowiak K., Milewska K., Gorecki P., Adamiak Z., Maksymowicz W, & Walczak P. (2018). MRI-guided intrathecal transplantation of hydrogel-embedded glial progenitors in large animals. Scientific Reports, 8, 16490.
+ Open protocol