A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment

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 with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15–30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

Key concepts in muscle regeneration: muscle “cellular ecology” integrates a gestalt of cellular cross-talk, motility, and activity to remodel structure and restore function

“…The tools for tracking the outcome of muscle regeneration have evolved tremendously since the early development of standardized instrumentation. Even a new, simple way to isolating primary satellite cells using ice-cold incubation, can advance research on regeneration (Benedetti et al 2021 ). Recent discovery of MYC-dependent satellite cell function, revealed through CRISPR/Cas9 editing of the MyoD locus in young growing muscle, opened new potential for understanding how genome topology mediates activation of satellite cells (He et al 2021 ).…”

Optimal Pre-Plating Method of Chicken Satellite Cells for Cultured Meat Production

“…However, purification can biochemically stimulate cells, so there are problems using FACS and MACS for food production processes. FACS, which can isolate cells with high purity, requires expensive equipment and skilled experts, and can also cause cell death by the high pressure and laser applied to the cell suspension during sorting ( Benedetti et al, 2021 ; Matsuyoshi et al, 2019 ; Yoshioka et al., 2020 ). MACS has the advantage of less apoptosis but results in less accurate purification than FACS by negatively selecting cells using labeling magnetic bead-conjugated cell antibodies ( Benedetti et al, 2021 ).…”

“…FACS, which can isolate cells with high purity, requires expensive equipment and skilled experts, and can also cause cell death by the high pressure and laser applied to the cell suspension during sorting ( Benedetti et al, 2021 ; Matsuyoshi et al, 2019 ; Yoshioka et al., 2020 ). MACS has the advantage of less apoptosis but results in less accurate purification than FACS by negatively selecting cells using labeling magnetic bead-conjugated cell antibodies ( Benedetti et al, 2021 ). In addition, FACS and MACS have a common drawback that studies on specific antibodies for cell sorting have not been conducted in various animal species except for cattle and pigs because of the risk that the cell-staining antibodies themselves might be toxic ( Choi et al, 2020 ; Ding et al, 2017 ; Ding et al, 2018 ; Liu et al, 1999 ; Yoshioka et al., 2020 ).…”

Development of a High-Yield Isolation Protocol Optimized for the Retrieval of Active Muscle Satellite Cells from Mouse Skeletal Muscle Tissue

“…MSCs constitute approximately 2∼5% of muscular tissue ( 22 , 23 ). A variety of protocols for the isolation of MSCs from muscle tissue have recently been introduced, including pre-plating ( 24 , 25 ), magnetic-activated cell sorting (MACS) ( 26 , 27 ), and fluorescence-activated cell sorting (FACS) ( 28 , 29 ). However, these protocols have one common limitation.…”

“…The purification efficiency is too low to provide a sufficient amount of cells ( 30 ). Furthermore, the pre-plating method does not provide certainty regarding the time point when cell populations, including high-yield MSCs, can be collected ( 24 , 25 ), and the isolation process using both the MACS and FACS methods is complicated and inconvenient ( 26 - 29 ). Therefore, a high-yield, simple, and convenient protocol for collecting MSCs from muscular tissues needs to be developed to facilitate basic or applied muscle-related research.…”