A Simple Method for the Isolation and in vitro Expansion of Highly Pure Mouse and Human Satellite Cells

Anna Benedetti; Gianluca Cera; Daniele De Meo; Ciro Villani; Marina Bouche; Biliana Lozanoska-Ochser

doi:10.21769/BioProtoc.4238

A Simple Method for the Isolation and in vitro Expansion of Highly Pure Mouse and Human Satellite Cells

Bio Protoc. 2021 Dec 5;11(23):e4238. doi: 10.21769/BioProtoc.4238.

Authors

Anna Benedetti¹, Gianluca Cera^{2

3}, Daniele De Meo^{2

3}, Ciro Villani^{2

3}, Marina Bouche¹, Biliana Lozanoska-Ochser¹

Affiliations

¹ Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Section of Histology and Embryology, Sapienza University of Rome, Rome, Italy.
² Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Section of Orthopedics, Sapienza University of Rome, Rome, Italy.
³ Department of Orthopaedics and Traumatology, Policlinico Umberto I, Rome, Italy.

Abstract

Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs, which retain myogenic properties after serial passages in vitro. Here, we describe a protocol for the isolation and in vitro expansion of highly pure mouse and human SCs based on ice-cold treatment (ICT). The ICT is carried out by briefly incubating the dish containing a heterogeneous mix of adherent muscle mononuclear cells on ice for 15-30 min, which leads to the detachment only of the SCs, and gives rise to SC cultures with 95-100% purity. This approach can also be used to passage the cells, allowing SC expansion over extended periods of time without compromising their proliferation or differentiation potential. Overall, the ICT method is cost-effective, accessible, technically simple, reproducible, and highly efficient. Graphic abstract: Figure 1.Satellite cell isolation using the ice-cold treatment method.

Keywords: In vitro expansion; MyoD; Pax7; Satellite cell isolation; Skeletal muscle.