Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Summary

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Abstract

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Introduction

While often cited as an indication of overall metabolic health, multiple studies have shown that body mass index (BMI) in older adults is not consistently associated with higher risk of mortality. To date, the only factor shown to be consistent with reduced mortality in this population is increased muscle mass¹. Muscle tissue represents one of the largest sources of insulin-sensitive cells in the body, and is therefore critical in the maintenance of overall metabolic homeostasis². Activation of skeletal muscle tissue via exercise is associated with increases in both local insulin sensitivity and overall metabolic health<....

Protocol

This protocol follows the animal care guidelines of Scripps Research.

1. Collection and Processing of Muscle Tissue Explants

1. The day prior to dissection, sterilize all dissection equipment (forceps, razor blades, and scissors) and prepare all required media: phosphate buffered saline (PBS), MB Plating media (12.5 mL of DMEM, 12.5 mL of HAMS F12, 20 mL of heat-inactivated fetal bovine serum (FBS), 5 mL of amniotic fluid medium supplement), and Coating Solution (24 mL of DMEM, 24 mL .......

Discussion

Skeletal muscle is vital for the establishment and maintenance of metabolic homeostasis¹¹. The study of muscle physiology is complicated by interindividual variability, as well as difficulty in obtaining samples, particularly in the case of human studies. Cultured primary myotubes have been shown to recapitulate many features of muscle physiology, including calcium homeostasis¹², regeneration of damaged muscle tissue⁵, metabolic alterations in respon.......

Materials

Name	Company	Catalog Number	Comments
Coating Solution:
DMEM	Gibco	10569010	Always add gentamicin (1:1000 by volume) prior to use; 24 mL
HAMS F12	Lonza	12-615F	Always add gentamicin (1:1000 by volume) prior to use; 24 mL
Collagen	Life Technologies	A1064401	1.7 mL
Matrigel	Fisher	CB40234A	1 mL
Plating Media:
DMEM	Gibco	10569010	Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL
HAMS F12	Lonza	12-615F	Always add gentamicin (1:1000 by volume) prior to use; 12.5 mL
Heat Inactivated FBS	Life Technologies	16000044	20 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 °C water bath for 20 minutes
Amniomax	Life Technologies	12556023	5 mL
Myoblast Media:
DMEM	Gibco	10569010	Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL
HAMS F12	Lonza	12-615F	Always add gentamicin (1:1000 by volume) prior to use; 17.5 mL
Heat Inactivated FBS	Life Technologies	16000044	10 mL; can be purchased as regular FBS and heat-inactivated by placing in a 40 °C water bath for 20 minutes
Amniomax	Life Technologies	12556023	5 mL
Differentiation Media:
DMEM	Gibco	10569010	Always add gentamicin (1:1000 by volume) prior to use; 24 mL
HAMS F12	Lonza	12-615F	Always add gentamicin (1:1000 by volume) prior to use; 24 mL
Heat Inactivated Horse Serum	Sigma	H1138	1.5 mL
Insulin-Selenium-Transferrin	Life Technologies	41400045	0.5 mL
Other Materials:
PBS	Gibco	14040133
Gentamicin	Sigma	G1397
TrypLE	Gibco	12604013
DMSO	Sigma	472301	Prepare as 10% DMSO in Myoblast Media for freezing cells
Forceps	Any
Razor Blades	Any
Scissors	Any
Whatman paper	VWR	21427-648
60 mm plate	VWR	734-2318
10 cm plate	VWR	25382-428 (CS)
T25 Flasks	ThermoFisher	156367
T75 Flasks	ThermoFisher	156499
Centrifuge Tubes (15mL)	BioPioneer	CNT-15
Oxygen Consumption Rates:
Seahorse XFe96 Analyzer	Agilent	Seahorse XFe96 Analyzer	Instrument used to measure oxygen consumption rates read out by acidification of the extracellular media
Seahorse XFe96 FluxPak	Agilent	102416-100	96-well plates for use in XFe96 Analyzer
Seahorse XF Cell Mito Stress Test Kit	Agilent	103015-100	components may be purchased from other suppliers once assay is established; some recommendations are listed below
Seahorse XF Palmitate-BSA FAO substrate	Agilent	102720-100	components may be purchased from other suppliers once assay is established; some recommendations are listed below
Palmitic acid	Sigma	P5585-10G	for measurement of fatty acid oxidation
carnitine	Sigma	C0283-5G	for measurement of fatty acid oxidation
Etomoxir	Sigma	E1905	for measurement of fatty acid oxidation
BSA	Sigma	A7030	used as control or in conjugation with palmitic acid for use in measurement of fatty acid oxidation