For the generation of 3D hSkM cultures, hSkM were dissociated using Trypsin (Trypsin-EDTA, 0.25%, phenol red; Life Technologies) and resuspended in Geltrex™ (Life Technologies) at a density of 3,000 hSkM per μl. Fifty ml of this viscous cell suspension were aliquoted into silicone wells (Ibidi, 80369) located inside 6-well tissue culture plates (Corning), and incubated for 30 minutes at 37°C to allow Geltrex™ gelling, at which point 4 mL of Skeletal Muscle Cell Growth Medium was added. The next day, silicone wells containing hSkM were placed into 6-well ultra-low attachment plates, and medium was changed every 2–3 days. After 7–10 days, medium was changed to Skeletal Muscle Cell Differentiation Medium to allow for differentiation of hSkM with medium changes every 2–3 days. For some experiments, including ACTA1::GCaMP6s imaging, smaller 3D hSkM were generated by resuspending 24,000 cells in 10 μL of Geltrex™ and aliquoting them in small silicone wells (Ibidi, 80409). 3D hSkM were used for assembloid generation 10 to 25 days after the switch to differentiation medium. Figures 5F and S6H show pictures of the 3D hSkM set-up.
Geltrex
Manufactured by Corning
Geltrex™ is a soluble basement membrane extract of proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is a complex mixture of extracellular matrix proteins, including laminin, collagen IV, heparan sulfate proteoglycans, and entactin. Geltrex™ provides a substrate for the attachment, migration, and differentiation of cells in vitro.
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Lab products found in correlation
2 protocols using geltrex
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3D Human Skeletal Muscle Culture
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Sphere Formation Assay Protocol
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A sphere formation assay was performed as previously described by Zhang et al (39 (link)) with slight modification. Falcon 8-well chamber slides (product no. 354118; Corning, Inc.) were precoated with 50 µl of LDEV-free growth factor-reduced Geltrex (cat. no. A1413201; Thermo Fisher Scientific, Inc.). Cells were plated at 3,000 cells/well in the aforementioned culture medium supplemented with 1% Geltrex. One day later, the cells were treated with 0 or 100 µM of S0859 and incubated at 37°C for 6 days to form spheres. Images of spheres at a magnification of ×10 were captured using a Keyence BZ-X700 fluorescence microscope (Keyence Corporation). To quantify sphere growth, the number of spheres was counted and Feret diameters were measured using the FIJI version of ImageJ.
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