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.
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.
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 health3. While in vivo models are essential for studying muscle physiology and the impact of muscle function on integrated metabolism, primary cultures of myotubes provide a tractable system that reduces the complexity of animal studies.
Myoblasts derived from post-natal muscles can be used to study the impact of numerous treatment and growth conditions in a highly reproducible manner. This has long been recognized and several methods for myoblast isolation and culture have been described4,5,6,7,8,9. Some of these methods use neonatal muscles and yield relatively low numbers of myoblasts5,8, requiring several animals for larger scale studies. Also, most widely used methods for culturing myoblasts use "pre-plating" to enrich for myoblasts, which are less adherent than other cell types. We have found the alternative enrichment method described here to be much more efficient and reproducible for enriching a highly proliferative myoblast population. In summary, this protocol enables the isolation of highly proliferative myoblasts from young adult muscle explants, via outgrowth into culture media. Myoblasts can be harvested repeatedly, over several days, rapidly expanded, and induced to differentiate into myotubes. This protocol reproducibly generates a large number of healthy myoblast cells that robustly differentiate into spontaneously twitching myotubes. It has enabled us to study metabolism and circadian rhythms in primary myotubes of mice of a variety of genotypes. Finally, we include methods for preparing myotubes for the study of oxidative metabolism, using measurements of oxygen consumption rates in 96-well plates.
This protocol follows the animal care guidelines of Scripps Research.
1. Collection and Processing of Muscle Tissue Explants
2. Harvesting Outgrowing Myoblasts
3. Expansion and Enrichment of Proliferating Myoblasts
NOTE: The P0 harvest will be heterogeneous (~60% myoblasts). The next 2 passages use PBS to selectively harvest myoblasts. Many of the more adherent cells will be left behind and the rapidly proliferating myoblasts will be ≥95% pure within 2 passages. Once myoblasts are established, they should be maintained at a low density to avoid spontaneous differentiation.
4. Differentiation of Primary Myoblasts to Myotubes
5. Measuring Oxygen Consumption Rate in Myoblasts or Myotubes in 96-well Plates
Following Section 1 of the provided protocol should yield primary cells emerging from the explants that will be visible under a standard light microscope (Figure 2). A heterogeneous cell population will be seen growing out of and surrounding each muscle tissue explant. Myoblasts will appear as small, round, bright spheres. Following Section 2 of the protocol will yield early harvests of myoblasts from tissue explants, which will contain few cells and will be heterogeneous (Figure 3). Section 3 of the protocol describes passaging early harvests with PBS (rather than trypsin), which will provide a relatively pure population of myoblasts for further culturing. Following Section 4 of the protocol will yield fully differentiated myotubes for further experimental manipulation. Differentiation of myoblasts typically takes 4-6 days, during which the morphology of the cells will change from single, round spheres to elongated, fused, long multinucleated fibers (Figure 4). Following section 5 of the protocol will produce differentiated myotubes in 96-well plates to enable a variety of metabolic characterizations based on the changes in oxygen consumption and extracellular acidification rates10 (Figure 5).
Figure 1: Dissection and processing of quadriceps muscle. (A) Quadriceps muscle that has been freshly dissected and rinsed with PBS prior to transfer to a 10 cm dish. 1 mL of plating media has been overlaid for processing. (B) Quadriceps muscle tissue pieces after transfer to pre-coated 6 cm plate. (C) 6 cm plates inside moist chamber prior to placement in the 37 °C incubator. Please click here to view a larger version of this figure.
Figure 2: Outgrowth of myoblasts. Outgrowth of myoblasts from quadriceps muscle explants. Please click here to view a larger version of this figure.
Figure 3: Early passage myoblasts. P0 myoblasts after transfer and attachment to T25 flask. Please click here to view a larger version of this figure.
Figure 4: Plating and differentiation of primary myotubes. (A) Myoblasts one day after initiating exposure to Differentiation Media. (B,C) Differentiated myotubes five (B) or six (C) days after initiating differentiation. Please click here to view a larger version of this figure.
Figure 5: Myotubes ready for measurement of oxygen consumption rates. Fully differentiated myotubes five days after plating 20,000 myoblasts in Differentiation Media in each well of a 96-well cell culture microplate. Please click here to view a larger version of this figure.
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 response to exercise13, and alterations to metabolism resulting from diseases such as diabetes14. Primary culture of myoblasts and myotubes from mice enables investigation of muscle cells harboring well defined genetic manipulations, and provides a complement to studies of myotubes derived from human muscle biopsies12,15,16. Therefore, methods for isolation and culture of mouse primary myoblasts and myotubes are essential to enable reproducible, high-throughput investigation of muscle cell function ex vivo. The protocol described here allows for the establishment and study of primary mouse myoblasts and myotubes under a variety of experimental manipulations.
While previous protocols have described the isolation of muscle stem cells from explant cultures, this protocol provides a method for the successful isolation of myoblasts from multiple different types of muscle tissue. In addition, this method yields a significantly larger population of stem cells for further experimental manipulation. Further, this method has been validated as yielding differentiated myotubes that express markers of mature muscle cells17, and exhibit normal physiology, such as circadian rhythms17 and mitogen activated protein kinase (MAPK) signal transduction18.
The critical steps in the protocol are the dissection and processing of the muscle tissue explants, as well as the avoidance of contamination between harvests. Care should be taken to avoid over-processing of the tissues. While smaller pieces of muscle yield larger numbers of myoblasts, excessive cutting of the muscle could prevent stem cell outgrowth. While it is important not to dislodge the explants once they are plated, careful washing of the plates with PBS/gentamicin is critical for reducing contamination. Harvested myoblasts may be frozen as P2 cells in cryovials using a 10% DMSO/90% Myoblast Media mixture. While myoblasts do not need to be maintained at a high density to facilitate growth, it is advised that cells are frozen at 40-50% confluence. Typically, one T75 flask yields 4 cryovials of cells.
The authors have nothing to disclose.
The authors are grateful to Dr. Matthew Watt at the University of Melbourne and Dr. Anastasia Kralli at Johns Hopkins University for assistance adopting this protocol based on the work of Mokbel et al.6. We also thank Dr. Sabine Jordan for assistance developing and adopting this protocol in our laboratory. This work was funded by the National Institutes of Health R01s DK097164 and DK112927 to K.A.L.
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 |