Page 21 - G. Cell differentiation. division. and death

Establishment of canine primary myoblast culture

and myotubes differentiation

Feriel Yasmine Mahiddine , Jin Wook Kim, Min Jung Kim a,*
a

a Department of Theriogenology and Biotechnologies, College of Veterinary Medicine, Seoul National University, South Korea.

* Corresponding author: tinia19@snu.ac.kr

INTR ODUC TION R E S ULTS

Skeletal muscle cells originates from their

precursors, satellite cells or also called myogenic

stem cells, and are multinucleated and form striated

tissues. The use of skeletal muscle cells in vitro

became common in aging or muscle pathology

research; however, most studies use established cell

lines rather than primary isolated cells, which can

provide results closer to the in vivo state. In this

study, we tried to establish a protocol for myoblast

isolation, culture and differentiation in canine

species.

MATE R IALS AND ME THODS

1. Animal use

Animal experiments were done following a Figure 1. Canine myoblast primary culture and

standard procedure established by the Committee myotubes differentiation

for Accreditation of Laboratory Animal Care and the

Guideline for the Care and Use of Laboratory

Animals of Seoul National University.

2. Myoblast isolation, 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 (380 g/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 Figure 2. Canine myotubes and satellite cells

differentiated into myotubes by changing the media immunocytochemistry results using fluorescence

and serum (DMEM with 2% horse serum) microscope

3. Immunocytochemistry (ICC) Results show multinucleated structures expressing

Myotubes confirmation was proceeded through MyoD and Pax7 in the satellite cells, confirming the

immunocytochemistry (ICC) using muscle specified presence of myotubes and satellite cells.

anti-bodies: Myo-D and Pax7 for satellite cells C ONC LUS ION

identification. Briefly, cells were fixed with 4% PFA, In conclusion, the technique used in this study was

permeabilized with 0.3% Triton X-100, blocked with successful in dogs muscle cells and skeletal muscle

2% BSA, incubated with primary antibodies overnight cell differentiation was conducted successfully. These

at 4°C and then with secondary anti-body (Alexa- results prove that the protocol exposed here can be

488). Cells were washed 3 times with PBS between applied in canine species for muscle aging or

each step and DAPI was used for nuclei staining.

pathology research.

This study was supported by RDA (CCAR, #2018R1C1B6009536), Research Institute for Veterinary Science.