Page 22 - G. Cell differentiation. division. and death

[G. Cell differentiation, division, and death-16]

Establishment of canine primary myoblast culture and

myotubes differentiation

Feriel Yasmine Mahiddine¹, Jin Wook Kim¹, Min Jung Kim¹

¹Department of Theriogenology and biotechnologies, Seoul National University, Seoul 08826, South Korea

The use of primary isolated cells can provide results closer to the in vivo state, however, there is no defined method
for primary myoblast culture and differentiation in dogs. Here, we tried to establish a simplified protocol for canine
primary myoblast culture and differentiation. Muscle tissue samples were collected by biopsy from healthy beagle

dogs. Samples were dissected, digested, filtered and cultured in Ham’s F-10 media,10% FBS according to Hindi et

al. 2017 protocol with changes in the centrifugation conditions (380Xg/8min) and digestion incubation times (2
hours, 37°C, 5% CO2). Primary myoblast cells were seen after 3 days of incubation and were identified as bipolar
spindle-shaped cells. They were purified by sub-culture up until passage-3 and differentiated into myotubes by

changing the media and serum (DMEM with 2% horse serum). Myotubes confirmation was proceeded through
immunocytochemistry (ICC) using muscle specified anti-bodies (Myo-D, Pax7). ICC results showed multinucleated

structures expressing MyoD and Pax7 in the differentiated cells, confirming the presence of myotubes. In conclusion,
the technique used in this study was effective in dogs. This study was supported by RDA (CCAR,

#2018R1C1B6009536), Research Institute for Veterinary Science.