Skeletal muscle cell growth medium

Manufactured by PromoCell

Sourced in Germany, France

Skeletal Muscle Cell Growth Medium is a cell culture medium optimized for the maintenance and growth of human skeletal muscle cells. It provides the necessary nutrients and growth factors to support the proliferation and differentiation of skeletal muscle cells in vitro.

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46 protocols using skeletal muscle cell growth medium

Myoblast Culture and Resveratrol Effects

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The experiments were divided into two main groups depending on the culture conditions of the myoblasts: (I) normal (N)- or, (II) substrate restricted (R)-conditions, both, either without or with 10 µM or 20 µM of RSV. At first, all cells were grown in skeletal muscle cell growth medium (Promocell, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS), GlutaMAX-1 (Gibco, Life Technologies, Grand Island, NY, USA), and Supplement mix (Fetuin (bovine, 50 ng/mL), human epidermal growth factor (hEGF, 10 pg/mL), human basic fibroblast growth factor (hbFGF, 1 pg/mL), Dexamethasone (0.4 pg/mL), and human recombinant insulin (10 ng/mL), Promocell, Heidelberg, Germany). After the first 24 h, the medium was changed and the cells were cultured for another 48 h. In the R group, the medium was replaced by a substrate-limited medium (DMEM with 0.5 mM glucose, 1.0 mM glutamine, and 1% FBS) with or without 10 µM or 20 µM RSV. Promocell skeletal muscle cell growth medium was used either without or with 10 µM or 20 µM RSV in the normal group. Resveratrol (>99% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All cells were maintained in 5% CO₂ at 37 °C.

Motlagh Scholle L., Schieffers H., Al-Robaiy S., Thaele A., Dehghani F., Lehmann Urban D, & Zierz S. (2020). The Effect of Resveratrol on Mitochondrial Function in Myoblasts of Patients with the Common m.3243A>G Mutation. Biomolecules, 10(8), 1103.

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Isolation and Culture of CD8+ T Cells and Primary Human Myoblasts

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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and PM patients by centrifugation on a Lymphoprep™ (Fresenius Kabi Norge AS) density gradient. CD8⁺ T cells were negatively purified using the CD8⁺ T cell Isolation Kit (Miltenyi Biotec) following the manufacturer's instructions. Cell purity was analyzed by flow cytometry and revealed > 95% of CD8⁺ cells. CD8⁺ T cells were cultured in RPMI supplemented with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin.
Primary human myoblasts were isolated from diagnostic muscle biopsy specimens as previously described [10 (link)]. Proliferating myoblasts were further purified by NCAM (neural cell adhesion molecule, CD56, clone 5.1H11, hybridoma supernatant) magnetic bead separation. The purity of myoblast cultures was assessed by flow cytometry and showed > 95% NCAM positive cells (Suppl. Figure 1). Human myoblasts were cultured in skeletal muscle cell growth medium (Promocell) at 37°C, 5% CO₂.

Ruck T., Bittner S., Afzali A.M., Göbel K., Glumm S., Kraft P., Sommer C., Kleinschnitz C., Preuβe C., Stenzel W., Wiendl H, & Meuth S.G. (2015). The NKG2D – IL-15 signaling pathway contributes to T-cell mediated pathology in inflammatory myopathies. Oncotarget, 6(41), 43230-43243.

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Differentiating Human Skeletal Myoblasts

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Human skeletal myoblasts (hSkM) were obtained from Thermo Fisher Scientific (A12555, Lot# 1837192) and maintained in an undifferentiated state with Skeletal Muscle Cell Growth Medium (ready to use; Promocell, C23060) in 10-cm plates (Primaria Cell Culture Dish, Corning). Medium was changed every 2–3 days, and hSkM were passaged using Trypsin (Trypsin-EDTA, 0.25%, phenol red; Life Technologies) when they reached ~80% confluency. hSkM from passages 1 to 4 were used for experiments. For analysis of hSkM differentiation ability, hSkM were plated on wells of 24-well plates (Corning) that had been coated with Geltrex™ (1:50 diluted in DMEM/F12, 1 hour at 37°C; Life Technologies, A1413202). 30,000 hSkM were plated on day 0 in Skeletal Muscle Cell Growth Medium. Medium was replaced the day after plating and every other day after that. When hSkM reached ~90% confluency (2–3 days after plating), Skeletal Muscle Cell Growth Medium was replaced with Skeletal Muscle Cell Differentiation Medium (ready to use; Promocell, C23061). On days 0, 5 and 15, hSkM were washed with PBS once and fixed for 10 minutes with 4% paraformaldehyde (PFA).

Andersen J., Revah O., Miura Y., Thom N., Amin N.D., Kelley K.W., Singh M., Chen X., Thete M.V., Walczak E.M., Vogel H., Fan H.C, & Paşca S.P. (2020). Generation of Functional Human 3D Cortico-Motor Assembloids. Cell, 183(7), 1913-1929.e26.

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Myoblast Culture from Muscular Dystrophy

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Control and patient primary myoblasts (with the R155H mutation) were obtained from Muscle Tissue Culture Collection (MTCC)/EuroBioBank (München, Germany). Cells were maintained in Skeletal Muscle Cell Growth Medium (PromoCell, Heidelberg, Germany) supplemented with the supplement mix, 10% FBS, gentamicin (Life Technologies, Carlsbad, CA) and GlutaMAX-1 in 5% CO₂ at 37 °C. Cultured patient myoblasts were plated in 35 mm dishes with a ConA-laminin coated cover glasses (MatTek Corp, Ashland, MA).

Nalbandian A., Llewellyn K.J., Gomez A., Walker N., Su H., Dunnigan A., Chwa M., Vesa J., Kenney M.C, & Kimonis V.E. (2015). In vitro studies in VCP-associated multisystem proteinopathy suggest altered mitochondrial bioenergetics. Mitochondrion, 22, 1-8.

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Myoblast Differentiation and Lamin Variants

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Human skeletal myoblasts were proliferated in Skeletal Muscle Cell Growth Medium (PromoCell) according to the manufacturer’s instructions except for fetal bovine serum (Invitrogen) that was adjusted to 20%. Differentiation was induced at high cell density by serum starvation [DMEM (Life Technologies) in the presence of gentamicin (50 μg/ml) and insulin (10 μg/ml)]. For DNA methylation analysis and myogenin qRT-PCR experiments, myogenic cells were infected with LMNA variants [wild-type, EDMD (p.R453W), FPLD (p.R482W)]. Myogenic cells were affected at myoblast stage and harvested the next day (myoblasts) or differentiated into myotubes and harvested 4 days after differentiation (myotubes).

Perovanovic J., Dell’Orso S., Gnochi V.F., Jaiswal J.K., Sartorelli V., Vigouroux C., Mamchaoui K., Mouly V., Bonne G, & Hoffman E.P. (2016). Laminopathies disrupt epigenomic developmental programs and cell fate. Science translational medicine, 8(335), 335ra58.

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Culturing C2C12 and Primary Myoblasts

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C2C12 cells were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM; Biochrom) supplemented with 10% FCS, 2 mml-glutamine, and 100 units/ml penicillin/streptomycin. Human primary myoblasts were kindly provided by Prof. S. Spuler (Charité-ECRC, Berlin), which had been obtained from healthy donors by muscle biopsies with permission of the local ethics commission (EA 1/203/08) as described previously (31 (link)). Cells were maintained in skeletal muscle cell growth medium (PromoCell) supplemented with SupplementMix C-39365 (PromoCell), 20% FCS, 2 mml-glutamine, and gentamycin.

Horbelt D., Boergermann J.H., Chaikuad A., Alfano I., Williams E., Lukonin I., Timmel T., Bullock A.N, & Knaus P. (2014). Small Molecules Dorsomorphin and LDN-193189 Inhibit Myostatin/GDF8 Signaling and Promote Functional Myoblast Differentiation. The Journal of Biological Chemistry, 290(6), 3390-3404.

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Culturing Skeletal Muscle Cells from Megaconial CMD Patient

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All experimental protocols in this study were approved by the Hacettepe University Faculty of Medicine Ethical Review Board (GO 15/421-31), and all experiments were performed in accordance with relevant guidelines and regulations. Primary skeletal muscle cells of a Megaconial CMD patient (sample ID: NH10-1412A) harboring a homozygous mutation of c.722A>G (NM_005198.3) in the CHKB gene were supplied by the MRC Centre for Neuromuscular Disease Biobank London (REC reference number 06/Q0406/33). Primary skeletal muscle cells derived from a muscle biopsy with no diagnostic pathology as indicated by normal histomorphology was used as a control. Cells were expanded in Skeletal Muscle Cell Growth Medium (PromoCell) supplemented with 2 mM L-glutamine (Biowest) and 1% Pen/Strep (Biowest). Upon reaching 70–80% confluence, the medium was changed to a differentiation medium based on the Skeletal Muscle Cell Differentiation Medium (PromoCell) supplemented with 1% Pen/Strep (Biowest).

Aksu-Menges E., Eylem C.C., Nemutlu E., Gizer M., Korkusuz P., Topaloglu H., Talim B, & Balci-Hayta B. (2021). Reduced mitochondrial fission and impaired energy metabolism in human primary skeletal muscle cells of Megaconial Congenital Muscular Dystrophy. Scientific Reports, 11, 18161.

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Establishing Primary Fibroblast and Myoblast Cultures

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Primary fibroblast and myoblast cultures were established from skin and muscle biopsies and propagated in a humidified incubator with 5% CO₂ at 37 °C. Fibroblasts were cultured in Eagle's Minimal Essential Medium supplemented with 2 mM l-glutamine, 1 × non-essential amino acids and 10% foetal calf serum (FCS). Myoblasts were grown in Skeletal Muscle Cell Growth Medium (PromoCell GmbH, Heidelberg Germany) with Supplement Mix (Epidermal Growth Factor, basic Fibroblast Growth Factor, and Insulin), 10% foetal calf serum, 50 μg/ml streptomycin and 50 U/ml penicillin and 2 mM l-glutamine. Lentiviral particles containing wild-type FARS2 were purchased from Genecopoeia (LP-10240-Lv105-0200-S). Cells were infected overnight, following viral infection, transduced cells were selected with 2 μg/ml puromycin.

Almalki A., Alston C.L., Parker A., Simonic I., Mehta S.G., He L., Reza M., Oliveira J.M., Lightowlers R.N., McFarland R., Taylor R.W, & Chrzanowska-Lightowlers Z.M. (2014). Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency. Biochimica et Biophysica Acta, 1842(1), 56-64.

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Cell Culture and AR Induction Protocols

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Human embryonic kidney (HEK) 293T and MCF7 cells were obtained from the American Type Culture Collection (ATCC) and cultured as described on the ATCC cell culture guide. Immortalized human myoblast cells (MRC CNMD Biobank London, L954/1284 M-I) were maintained in skeletal muscle cell growth medium (PromoCell) supplemented with supplement mix (PromoCell), 10% (v/v) fetal bovine serum (FBS; Gibco), 1× GlutaMAX (Gibco), and 1× antibiotic antimycotic (Gibco). iPS cells were cultured and differentiated into motor neuron–like cells in the Fischbeck laboratory, as described previously (72 (link)). All cell lines were cultured at 37°C and 5% CO₂. AR induction was performed by addition of DHT (Sigma-Aldrich) to a final concentration of 10 nM in the respective cell culture medium. An equal volume of ethanol was added for the uninduced controls.

Lim W.F., Forouhan M., Roberts T.C., Dabney J., Ellerington R., Speciale A.A., Manzano R., Lieto M., Sangha G., Banerjee S., Conceição M., Cravo L., Biscans A., Roux L., Pourshafie N., Grunseich C., Duguez S., Khvorova A., Pennuto M., Cortes C.J., La Spada A.R., Fischbeck K.H., Wood M.J, & Rinaldi C. (2021). Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity. Science Advances, 7(34), eabi6896.

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Culturing Murine and Human Myoblasts

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Immortalized murine H2K, A/J, BL6 and dysferlin-deficient H2K cells were kindly provided by Professor Terry Partridge (Children's National Medical Center,Center for Genetic Medicine Research,Washington DC, US) and cultured as previously reported 23 , 24 (link), 25 (link). Briefly, cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FBS), 2% chick embryo extract, 2% L-glutamine, 1% penicillin and streptomycin and 20U/mL mouse recombinant IFN-γ (Invitrogen, US) at 33°C in 10% CO₂. Immortalized human myoblasts were kindly provided by Professor Jennifer Morgan (University College London, Institute of Child Health, London, UK) and were cultured in skeletal muscle cell growth medium (PromoCell, Germany) supplemented with 1.5% GlutaMAX (Gibco, US), 20% FBS and 1% penicillin and streptomycin. Myotubes were obtained from confluent H2K, dysferlin-deficient H2K or human myoblasts seeded in gelatin-coated 12-well plates after 3 days of serum deprivation at 37°C under a 5% CO₂ atmosphere (DMEM with 5% horse serum (HS, Hyclone, US)). Murine C2C12 cells and NIH 3T3 cells were kept in house and grown at 37°C in 5% CO₂ in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin.

Dong X., Gao X., Dai Y., Ran N, & Yin H. (2018). Serum exosomes can restore cellular function in vitro and be used for diagnosis in dysferlinopathy. Theranostics, 8(5), 1243-1255.

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