title: Simultaneous isolation of enriched myoblasts and fibroblasts for migration analysis within a novel co-culture assay.
abstract: Skeletal muscle injury elicits the activation of satellite cells and their migration to the wound area for subsequent terminal differentiation and tissue integration. However, interstitial fibroblasts recruited to the site of injury promote deposition of fibrotic tissue, which hampers myoblast-mediated muscle regeneration. Currently, analysis of myoblast migration in vitro can be accomplished using chemotactic, cell-exclusion, or wound healing assays. Yet, to investigate cell motility following skeletal muscle damage more accurately, migration assays need to better simulate the repair process. Here we present a protocol for the simultaneous isolation of myoblasts and fibroblasts from the same muscle tissue, ensuring the consistent generation of enriched, purified, and matched cell populations at a low passage number. We then describe a wound assay that uses a novel approach to the co-culture of myoblasts and fibroblasts to mimic the injured environment more closely than other established methods. Using this assay, we demonstrate that fibroblasts are able to increase myoblast migration significantly, validating our new in vitro method. As the observed effect on migration is most likely mediated by secreted factors, our assay could easily be extended to include antibody-based protein analysis of secreted factors in animal or human systems.
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Scottsville, South Africa.
authors.forename: Kyle Peter
authors.initials: KP
authors.lastname: Goetsch
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Scottsville, South Africa.
authors.forename: Celia
authors.initials: C
authors.lastname: Snyman
authors.affiliation: Department of Physiological Sciences, University of Stellenbosch, Matieland, South Africa.
authors.forename: Kathryn Helen
authors.initials: KH
authors.lastname: Myburgh
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Scottsville, South Africa.
authors.forename: Carola Ulrike
authors.initials: CU
authors.lastname: Niesler
mesh_terms: D000818:Animals; D002465:Cell Movement; D018920:Coculture Techniques; D005347:Fibroblasts / Q000166:cytology*; D008807:Mice, Inbred BALB C; D032446:Myoblasts / Q000166:cytology*
keywords: co-culture; fibroblast; injury; isolation; myoblast; primary culture; satellite cell