Primary Human Skeletal Muscle Cells and Growth Media

These products are available in the US, Canada, and select European countries.

• Skeletal Muscle Cell Culture System
• Primary Human Skeletal Muscle Cell Culture System Quality Control
• Materials for Primary Skeletal Muscle Cell Culture

Skeletal Muscle Cell Culture System

New skeletal muscle cells originate from quiescent satellite cells, which reside in the muscle fibers between the basal lamina and the sarcolemma. Quiescent satellite cells are activated by stimuli such as muscle damage. After activation, the cells, now called myoblasts, start to proliferate and fuse with damaged muscle fibers or with one another forming new myotubes. In studying muscle damage, primary human skeletal muscle cells (SkMCs) have been proven useful in elucidating mechanisms of muscle regeneration and differentiation, as well as in modeling neuromuscular disorders and disease.

PromoCell^® Primary Human Skeletal Muscle Cells are produced from normal human skeletal muscle tissue from different locations, including M. pectoralis major or M. gluteus (lot specific source information is available on request). These cells can be induced to differentiate into multinucleated syncytia using PromoCell^® Skeletal Muscle Cell Differentiation Medium. Shortly after isolation, all human skeletal muscle cells are cryopreserved at passage 2 (P2) using a proprietary, serum-free freezing medium, Cryo-SFM (C-29910). Each cryovial contains at least 500,000 viable cells after thawing. Human Skeletal Muscle Cells are also available as proliferating cell cultures generated from cryopreserved cells that have been thawed and cultured for three days.

• Cultivation Protocol for Human Skeletal Muscle Cells

Differentiation Protocol for Human Skeletal Muscle Cells (see below)

The Skeletal Muscle Cell Growth Medium is a low-serum medium developed for the expansion of human skeletal muscle cell cultures. The Skeletal Muscle Cell Differentiation Medium is a serum-free medium developed to induce the fusion of skeletal muscle cells to myotubes with typical multinucleated syncytia. These media are optimized for growing primary human cells, but can also be used for murine, rat, canine, and rabbit skeletal muscle cells. The Skeletal Muscle Cell Growth Medium is available in two formats: “ready-to-use” or “kit”. The “ready-to-use” option consists of a 500 mL bottle of Basal Medium and one vial of SupplementMix. The Skeletal Muscle Cell Growth Medium Kit consists of a 500 mL bottle of Basal Medium and the SupplementPack (a set of individual vials with pre-measured supplements), giving the user full control over the media formulation. The SupplementMix or SupplementPack is added to Basal Medium to prepare the complete Skeletal Muscle Cell Medium. Basal Medium (with or without phenol red), SupplementMix, and the SupplementPack are also available for individual purchase.

Skeletal Muscle Cell Media Supplementation Details

Skeletal Muscle Cell Growth Medium contains all the growth factors and supplements necessary for the optimal growth of human skeletal muscle cells, including epidermal growth factor, basic fibroblast growth factor, and insulin (Table 1). Skeletal Muscle Cell Differentiation Medium is a serum-free medium containing insulin. These media do not contain antibiotics or antimycotics and are formulated for use in an incubator with an atmosphere of 5% CO₂

Final Skeletal Muscle Cell Media supplement concentrations (after addition to the medium)

Component	Skeletal Muscle Cell Growth Medium	Skeletal Muscle Cell Differentiation Medium
Fetal Calf Serum	0.05 mL / mL	—
Fetuin (bovine)	50 μg / mL	—
Epidermal Growth Factor (recombinant human)	10 ng / mL	—
Basic Fibroblast Growth Factor (recombinant human)	1 ng / mL	—
Insulin (recombinant human)	10 μg / mL	10 μg / mL
Dexamethasone	0.4 μg / mL	—

Preparing Supplemented Media for Skeletal Muscle Cell Culture

• Thaw the Skeletal Muscle Cell Media SupplementMix or SupplementPack at 15 – 25 °C.
• Aseptically mix the supplement solutions by carefully pipetting up and down.
• Transfer the entire contents of each supplement to the Skeletal Muscle Cell Basal Medium.
• Close the bottle and swirl gently until a homogenous mixture is achieved.

Instructions for Use of Skeletal Muscle Cell Differentiation Medium

• For differentiating human skeletal muscle cells (SkMC), the cells should be cultured in Skeletal Muscle Cell Growth Medium until they reach approximately 60 – 80% confluence.
• Replace the Growth Medium with Skeletal Muscle Cell Differentiation Medium.
• After 2 – 8 days, extensive formation of multinucleated syncytia may be observed.
• For stable differentiation of SkMC, replace with Skeletal Muscle Cell Growth Medium after 5 days of incubation in Skeletal Muscle Differentiation Medium.

Storage and Stability of Skeletal Muscle Cell Products

• Upon arrival, immediately store the Skeletal Muscle Cell Basal Medium at 4 – 8 °C in the dark; do not freeze. Store the Skeletal Muscle Cell SupplementMix or SupplementPack at -20 °C.
• If stored as directed, the products are stable until the stated expiration date.
• Once supplements are added to the Skeletal Muscle Cell Basal Medium, the shelf life of the complete medium is six weeks at 4 – 8 °C. Do not freeze the complete medium.
• For use, prewarm only an aliquot of the complete Skeletal Muscle Cell Media needed for single use, and keep the remaining medium refrigerated at 4 – 8 °C.
• After delivery, cryopreserved cells should be stored in liquid nitrogen or seeded directly.
• Proliferating cells must be processed immediately.

Primary Human Skeletal Muscle Cell Culture System Quality Control

Human Skeletal Muscle Cells

Rigid quality control (QC) tests are performed for each lot of Human Skeletal Muscle Cells.

• Analysis of cell morphology, adherence, and cell viability.
• Immunohistochemical tests for cell type-specific markers.
• Growth performance, as tested via multiple passages up to 15 population doublings (PD) under culture conditions without antibiotics and antimycotics.
• Differentiation potential, as measured by the capacity to differentiate into multinucleated syncytia.
• Testing for the absence of viral (HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2) and microbial (fungi, bacteria, and mycoplasma) contaminants. For safety precautions, please refer to the appropriate safety data sheet (SDS).
• A detailed certificate of analysis (CoA) for each lot is available for download.

Skeletal Muscle Cell (SkMC) Specifications

Product	Plating Density	Passage after Thawing	Marker	Population Doublings
Human Skeletal Muscle Cells (SkMC)	3,500 - 7,000 cells per cm²	P2	Sarcomeric myosin+ Differentiation tested	> 15

Muscle Cell Growth and Differentiation Media Quality Control

All lots of the Skeletal Muscle Cell Growth Medium are subjected to rigorous and comprehensive quality control tests using primary human SkMC. Each lot is checked for growth promoting activity, adherence rate, and typical morphology of the SkMC used for testing. Additionally, the Skeletal Muscle Cell Differentiation Medium is validated for the capacity to fully induce the differentiation of SkMC. Approved in-house lots of Skeletal Muscle Cell Media are used as a reference.

All lots of media have been tested for the absence of microbial contaminants (fungi, bacteria, mycoplasma). For safety precautions, please refer to the appropriate safety data sheet (SDS).

Note: Due to their low serum content or the absence of serum, PromoCell^® media are not suitable for serum-based trypsin neutralization (as when subculturing the cells). We recommend instead using our DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.

Intended Use of Primary Human Skeletal Muscle Cells and Keratinocyte Growth Media

Primary Human Skeletal Muscle Cells and Skeletal Muscle Cell Growth Media are for in vitro use only and not for diagnostic or therapeutic procedures. For safety information, please see appropriate SDS.