Drug-screening platform based on the contractility of tissue-engineered muscle
ABSTRACT A tissue-based approach to in vitro drug screening allows for determination of the cumulative positive and negative effects of a drug at the tissue rather than the cellular or subcellular level. Skeletal muscle myoblasts were tissue-engineered into three-dimensional muscle with parallel myofibers generating directed forces. When grown attached to two flexible micro-posts (mu posts) acting as artificial tendons in a 96-well plate format, the miniature bioartificial muscles (mBAMs) generated tetanic (active) forces upon electrical stimulation measured with a novel image-based motion detection system. mBAM myofiber hypertrophy and active force increased in response to insulin-like growth factor 1. In contrast, mBAM deterioration and weakness was observed with a cholesterol-lowering statin. The results described in this study demonstrate the integration of tissue engineering and biomechanical testing into a single platform for the screening of compounds affecting muscle strength.
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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.Frontiers in Physiology 11/2013; 4:349. DOI:10.3389/fphys.2013.00349 · 3.50 Impact Factor
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ABSTRACT: Tissue engineered skeletal muscle has great utility in experimental studies of physiology, clinical testing and its potential for transplantation to replace damaged tissue. Despite recent work in rodent tissue or cell lines, there is a paucity of literature concerned with the culture of human muscle derived cells (MDCs) in engineered constructs. Here we aimed to tissue engineer for the first time in the literature human skeletal muscle in self-assembling fibrin hydrogels and determine the effect of MDC seeding density and myogenic proportion on the structure and maturation of the constructs. Constructs seeded with 4 x 105 MDCs assembled to a greater extent than those at 1 x 105 or 2 x 105, and immunostaining revealed a higher fusion index and a higher density of myotubes within the constructs, showing greater structural semblance to in vivo tissue. These constructs primarily expressed perinatal and slow type I myosin heavy chain mRNA after 21 days in culture. In subsequent experiments MACS(R) technology was used to separate myogenic and non-myogenic cells from their heterogeneous parent population and these cells were seeded at varying myogenic (desmin +) proportions in fibrin based constructs. Only in the constructs seeded with 75% desmin + cells was there evidence of striations when immunostained for slow myosin heavy chain compared with constructs seeded with 10 or 50% desmin + cells. Overall, this work reveals the importance of cell number and myogenic proportions in tissue engineering human skeletal muscle with structural resemblance to in vivo tissue.Biomaterials 07/2013; DOI:10.1016/j.biomaterials.2013.04.002 · 8.31 Impact Factor
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ABSTRACT: Contractile C(2)C(12) myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C(2)C(12) myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish.Biotechnology and Bioengineering 01/2010; 105(6):1161-7. DOI:10.1002/bit.22636 · 4.16 Impact Factor