Optimized method for extraction of exosomes from human primary muscle cells

Abstract

Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 10 6 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation. Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml −1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons. Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.

Exosomes in Ageing and Motor Neurone Disease: Biogenesis, Uptake Mechanisms, Modifications in Disease and Uses in the Development of Biomarkers and Therapeutics

“…Skeletal muscle-derived exosomes are secreted by both myoblasts and myotubes [69,185] and may be involved in neuronal cell survival [186], myogenesis and muscle regeneration [73,187] as well as in myoblast differentiation [71,79] or during muscle ageing [188]. C 2 C 12 myotube-derived exosomes reduced myoblast proliferation and induced differentiation while negatively regulating Sirt1 expression in C 2 C 12 myoblasts, further supporting the existence of myoblast-myotube crosstalk mediated by exosomes [71,79].…”

The Neurotoxicity of Vesicles Secreted by ALS Patient Myotubes Is Specific to Exosome-Like and Not Larger Subtypes

“…For extraction of extracellular vesicles, myoblasts were plated at a density of 33,400 cells·cm −2 as described in [ 45 ] (7.5 × 10 6 myoblasts in 225 cm 2 flasks (Falcon™)) for 24 h before washing six times with supplement-free DMEM. The cells were then differentiated in DMEM to form myotubes, and conditioned culture medium was collected at 72 h. All cell cultures were regularly checked for mycoplasma infection.…”

“…EVs were extracted as previously described, at myoblast cell division counts of less than 20 in order to avoid potential artefacts due to pre-senescence [ 13 , 44 , 45 ]. Briefly, the conditioned cell culture media from differentiated myoblasts was centrifuged at 260 g for 10 min and the resulting supernatant centrifuged at 4000 g for 20 min.…”

“…The lmEVs were then pelleted by ultracentrifugation at 20,000 g for 70 min at 4 °C. The MuVs were extracted and washed from the remaining supernatant as previously described [ 45 ]. The lmEVs were washed thrice with PBS (20,000 g for 70 min at 4 °C) and resuspended in PBS for functional studies or lysed in NuPage/RIPA buffer for Western blot analysis.…”

Effect of Glioma Stem Cells-derived Extracellular Vesicles on Glucose Metabolism Reprogramming in Glioma via the miR-26a/KLF4/PI3K/Akt axis

“…Finally, the precipitates were resuspended with 50-100 µL PBS and stored at -80℃. The size of EVs was measured by Nanosight [15]. The EV marker proteins CD44 (1:1000, ab189524, Abcam) and CD63 (1:1000, ab134045, Abcam) were identi ed using Western blotting.…”