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Simple silicone chamber system for in vitro three-dimensional skeletal muscle tissue formation.

Snyman C, Goetsch KP, Myburgh KH, Niesler CU - Front Physiol (2013)

Bottom Line: Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts.We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space.Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

View Article: PubMed Central - PubMed

Affiliation: Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, South Africa.

ABSTRACT
Bioengineering skeletal muscle often requires customized equipment and intricate casting techniques. One of the major hurdles when initially trying to establish in vitro tissue engineered muscle constructs is the lack of consistency across published methodology. Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts. Differences in cell type, number and density, variability in matrix and scaffold usage as well as inconsistency in the distance between and type of adhesion posts complicates initial establishment of the technique with confidence. We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space. Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

No MeSH data available.


Related in: MedlinePlus

Successful generation of mouse and human skeletal muscle constructs using the simple silicone chamber system. (A) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 7 in differentiation media) between pins placed 4 mm apart. (B) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 3 in differentiation media) between pins placed 8 mm apart. (C) When seeded in a matrix of collagen 1 and Matrigel (14%), human skeletal muscle (HSKM) cells formed tissue (day 3 in differentiation media) between pins placed 4 mm apart. (D) After 12 days in differentiation media, actin fibers stained with TRITC-phalloidin and were clearly visible in the differentiated mouse C2C12 myotubes. Nuclei were stained with Hoechst (scale bar = 50 μm). (E) After 15 days in differentiation media, elongated myotubes were aligned and contained desmin, an intermediate filament required for myotube contractile function (scale bar = 50 μm). (F) Thin sections of resin-embedded C2C12 myoblasts culture for 15 days in differentiation media showed the formation of multi-nucleated (arrows) myotubes (scale bar = 20 μm).
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Figure 2: Successful generation of mouse and human skeletal muscle constructs using the simple silicone chamber system. (A) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 7 in differentiation media) between pins placed 4 mm apart. (B) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 3 in differentiation media) between pins placed 8 mm apart. (C) When seeded in a matrix of collagen 1 and Matrigel (14%), human skeletal muscle (HSKM) cells formed tissue (day 3 in differentiation media) between pins placed 4 mm apart. (D) After 12 days in differentiation media, actin fibers stained with TRITC-phalloidin and were clearly visible in the differentiated mouse C2C12 myotubes. Nuclei were stained with Hoechst (scale bar = 50 μm). (E) After 15 days in differentiation media, elongated myotubes were aligned and contained desmin, an intermediate filament required for myotube contractile function (scale bar = 50 μm). (F) Thin sections of resin-embedded C2C12 myoblasts culture for 15 days in differentiation media showed the formation of multi-nucleated (arrows) myotubes (scale bar = 20 μm).

Mentions: This adapted technique proved to be advantageous for skeletal muscle formation with constructs from both C2C12 and HSKM cells successfully spanning the pins after a 3–7 day culture period (Figures 1C, 2A–C). After 12–15 days in culture, differentiated C2C12 myotubes showed clear formation of actin fibers (Figure 2D). In addition, aligned myotubes expressed desmin (Figure 2E) and longitudinal sections showed evidence of organization into multinucleated myotubes (Figure 2F), which is an initial requirement for functionality.


Simple silicone chamber system for in vitro three-dimensional skeletal muscle tissue formation.

Snyman C, Goetsch KP, Myburgh KH, Niesler CU - Front Physiol (2013)

Successful generation of mouse and human skeletal muscle constructs using the simple silicone chamber system. (A) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 7 in differentiation media) between pins placed 4 mm apart. (B) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 3 in differentiation media) between pins placed 8 mm apart. (C) When seeded in a matrix of collagen 1 and Matrigel (14%), human skeletal muscle (HSKM) cells formed tissue (day 3 in differentiation media) between pins placed 4 mm apart. (D) After 12 days in differentiation media, actin fibers stained with TRITC-phalloidin and were clearly visible in the differentiated mouse C2C12 myotubes. Nuclei were stained with Hoechst (scale bar = 50 μm). (E) After 15 days in differentiation media, elongated myotubes were aligned and contained desmin, an intermediate filament required for myotube contractile function (scale bar = 50 μm). (F) Thin sections of resin-embedded C2C12 myoblasts culture for 15 days in differentiation media showed the formation of multi-nucleated (arrows) myotubes (scale bar = 20 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842895&req=5

Figure 2: Successful generation of mouse and human skeletal muscle constructs using the simple silicone chamber system. (A) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 7 in differentiation media) between pins placed 4 mm apart. (B) When seeded in a matrix of collagen 1 and Matrigel (14%), mouse C2C12 cells formed tissue (day 3 in differentiation media) between pins placed 8 mm apart. (C) When seeded in a matrix of collagen 1 and Matrigel (14%), human skeletal muscle (HSKM) cells formed tissue (day 3 in differentiation media) between pins placed 4 mm apart. (D) After 12 days in differentiation media, actin fibers stained with TRITC-phalloidin and were clearly visible in the differentiated mouse C2C12 myotubes. Nuclei were stained with Hoechst (scale bar = 50 μm). (E) After 15 days in differentiation media, elongated myotubes were aligned and contained desmin, an intermediate filament required for myotube contractile function (scale bar = 50 μm). (F) Thin sections of resin-embedded C2C12 myoblasts culture for 15 days in differentiation media showed the formation of multi-nucleated (arrows) myotubes (scale bar = 20 μm).
Mentions: This adapted technique proved to be advantageous for skeletal muscle formation with constructs from both C2C12 and HSKM cells successfully spanning the pins after a 3–7 day culture period (Figures 1C, 2A–C). After 12–15 days in culture, differentiated C2C12 myotubes showed clear formation of actin fibers (Figure 2D). In addition, aligned myotubes expressed desmin (Figure 2E) and longitudinal sections showed evidence of organization into multinucleated myotubes (Figure 2F), which is an initial requirement for functionality.

Bottom Line: Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts.We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space.Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

View Article: PubMed Central - PubMed

Affiliation: Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, South Africa.

ABSTRACT
Bioengineering skeletal muscle often requires customized equipment and intricate casting techniques. One of the major hurdles when initially trying to establish in vitro tissue engineered muscle constructs is the lack of consistency across published methodology. Although this diversity allows for specialization according to specific research goals, lack of standardization hampers comparative efforts. Differences in cell type, number and density, variability in matrix and scaffold usage as well as inconsistency in the distance between and type of adhesion posts complicates initial establishment of the technique with confidence. We describe an inexpensive, but readily adaptable silicone chamber system for the generation of skeletal muscle constructs that can readily be standardized and used to elucidate myoblast behavior in a three-dimensional space. Muscle generation, regeneration and adaptation can also be investigated in this model, which is more advanced than differentiated myotubes.

No MeSH data available.


Related in: MedlinePlus