title: Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle.
abstract: Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved (1). In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation (2, 3). The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions (3-6). The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation (7). Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles (8, 9) so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium (1-3). This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process (1-3). The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).
authors.affiliation: Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pasut
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Jones
authors.forename: Michael A
authors.initials: MA
authors.lastname: Rudnicki
mesh_terms: D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002642:Chick Embryo; D006736:Horses; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology*; D018482:Muscle, Skeletal / Q000166:cytology*; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology*
references.citation: Stem Cells. 2009 Oct;27(10):2527-38
references.pmid: 19609936
references.citation: Methods Mol Biol. 2005;290:281-304
references.pmid: 15361669
references.citation: J Cell Biol. 2004 Aug 2;166(3):347-57
references.pmid: 15277541
references.citation: J Histochem Cytochem. 2003 Nov;51(11):1437-45
references.pmid: 14566016
references.citation: Hum Gene Ther. 1997 Mar 1;8(4):371-80
references.pmid: 9054512
references.citation: J Cell Biol. 2004 Sep 13;166(6):877-87
references.pmid: 15364962
references.citation: In Vitro Cell Dev Biol Anim. 1995 Nov;31(10):773-9
references.pmid: 8564066
references.citation: Skelet Muscle. 2011 Feb 02;1(1):7
references.pmid: 21798086
references.citation: Tissue Eng Part B Rev. 2008 Dec;14(4):419-31
references.pmid: 18817477
references.citation: Cell. 2007 Jun 1;129(5):999-1010
references.pmid: 17540178
references.citation: BMC Dev Biol. 2010 Feb 22;10:21
references.pmid: 20175910
references.citation: Cell. 2000 Sep 15;102(6):777-86
references.pmid: 11030621
references.citation: Methods Mol Biol. 2012;798:53-64
references.pmid: 22130830
references.citation: J Histochem Cytochem. 2011 Jan;59(1):60-7
references.pmid: 20876523
references.citation: Methods Mol Biol. 2012;798:85-102
references.pmid: 22130833
references.citation: J Virol. 1996 Nov;70(11):8117-23
references.pmid: 8892937
references.citation: Dev Biol. 1986 May;115(1):129-39
references.pmid: 3516758
references.citation: Biochem Cell Biol. 2005 Oct;83(5):674-6
references.pmid: 16234857