Background

The exceptional regenerative ability of skeletal muscle is primarily due to a resident population of stem cells called satellite cells (SCs) (Mauro, 1961; Chang and Rudnicki, 2014; Wang et al., 2014). The study of their functional potential depends on the availability of methods for the isolation and expansion of highly pure SCs with preserved myogenic properties after serial passages in vitro (Danoviz and Yablonka-Reuveni, 2012; Keire et al., 2013; Syverud et al., 2014).

Presently, there are three main methods commonly used for the isolation of SCs: pre-plating, fluorescence activated cell sorting (FACS), and magnetic bead isolation.

The pre-plating method is based on the differing adhesive properties of muscle cells, with SCs being the least adherent (Gharaibeh et al., 2008; Danoviz and Yablonka-Reuveni, 2012; Keire et al., 2013; Syverud et al., 2014). Although cheap and straightforward to perform, this method’s main disadvantages are that it is time consuming and gives rise to cultures of variable purity, often with fibroblast contamination and overgrowth by day 7 of culture (Keire et al., 2013).

The FACS sorting method sorts muscle mononuclear cells pre-labeled with SC specific antibodies (Fukada et al., 2004; Sherwood et al., 2004; Montarras et al., 2005; Pasut et al., 2012; Chapman et al., 2013; Liu et al., 2015). At present, FACS sorting is the gold standard for the isolation and study of SCs. Nevertheless, there are several disadvantages to this method, including high cost and the requirement for a FACS sorter instrument. Moreover, this method is time consuming, requires expertise to perform, and cell purity can be variable. The cell labeling step that precedes the sorting procedure can potentially stress or damage the cells, decrease their viability, or alter their functional properties in vitro (Syverud et al., 2014).

Finally, the third method is based on magnetic cell separation (MACS) and uses magnetic columns and SC specific magnetic bead kits (Blanco-Bose et al., 2001). Because this method assumes that all the other cell types are successfully removed from the muscle cell preparation, it is less precise than the FACS sorting method. This method is expensive to perform, time consuming, and stressful for the cells. As for the other two methods, cell purity is variable, and often the SC cultures become overgrown by fibroblasts by day 7 (Keire et al., 2013; Syverud et al., 2014).

The ideal SC isolation technique would permit isolation of pure SCs with minimal manipulation, producing cells that could be expanded ex vivo without losing their stemness and regenerative capacity. Here, we describe a protocol for ice-cold treatment (ICT); this is a simple, inexpensive, and efficient method for the isolation and long-term expansion of highly pure mouse and human SCs that preserves their myogenic potential (Benedetti et al., 2020). In terms of purity of the isolated cell population, the ICT method outperforms others, such as pre-plating or magnetic bead isolation. Furthermore, it is fast and easy to perform—apart from the time required for enzymatic digestion (1.5 h), it involves minimal manipulation of the cells. Another major advantage of the ICT method is that it doubles up as a very gentle passaging technique, allowing long-term serial expansion of SCs ex vivo without altering their proliferation and differentiation properties. In turn, this drastically reduces the number of mice or muscle biopsies required to obtain a sufficient number of cells (Benedetti et al., 2021). The ICT method permits growing mouse and human SCs to be passaged at least 10 times, expanding their number 150- and 300-fold, respectively (Benedetti et al., 2021). This represents a clear advantage over the most commonly used passaging reagent (trypsin), which typically accelerates the differentiation of passaged SCs after only two passages (Danoviz and Yablonka-Reuveni, 2012; Benedetti et al., 2020 and 2021; Fiore et al., 2020).

Overall, the cost-effectiveness, accessibility, and technical simplicity of this protocol, as well as its remarkable efficiency, represent major improvements over existing protocols. The next step will be to test this protocol for the isolation and expansion of stem cells from tissues other than muscle.