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The object of this study was to develop an in vitro bioengineered three-dimensional vascularized skeletal muscle tissue, named eX-vivo Muscle Engineered Tissue (X-MET). This new tissue contains cells that exhibit the characteristics of differentiated myotubes, with organized contractile machinery, undifferentiated cells, and vascular cells capable of forming "vessel-like" networks. X-MET showed biomechanical properties comparable with that of adult skeletal muscles; thus it more closely mimics the cellular complexity typical of in vivo muscle tissue than myogenic cells cultured in standard monolayer conditions. Transplanted X-MET was able to mimic the activity of the excided EDL muscle, restoring the functionality of the damaged muscle. Our results suggest that X-MET is an ideal in vitro 3D muscle model that can be employed to repair muscle defects in vivo and to perform in vitro studies, limiting the use of live animals.

Skeletal muscle tissue is characterized by a population of quiescent mononucleated myoblasts, localized between the basal lamina and sarcolemma of myofibers, known as satellite cells. Satellite cells play a pivotal role in muscle homeostasis and are the major source of myogenic precursors in mammalian muscle regeneration.This chapter describes protocols for isolation and culturing satellite cells isolated from mouse skeletal muscles. The classical procedure, which will be discussed extensively in this chapter, involves the enzymatic dissociation of skeletal muscles, while the alternative method involves isolation of satellite cells from isolated myofibers in which the satellite cells remain in their in situ position underneath the myofiber basal lamina.In particular, we discuss the technical aspect of satellite cell isolation, the methods necessary to enrich the satellite cell fraction and the culture conditions that optimize proliferation and myotube formation of mouse satellite cells.

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Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response. The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based system allows for an accurate and noninvasive measurement of biological tissues' spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation.

X-MET (Ex-vivo Muscle Engineered Tissue) is a promising 3-dimensional model of skeletal muscle for in vitro tests and in vivo transplant. X-MET is an in vitro cultured tissue and has several properties in common with adult skeletal muscle, from biological and morphological to functional ones. To monitor the X-MET's growing improvements, we developed an experimental system based on Digital Image Correlation (DIC) to precisely measure the tissue's contractile capability, thus trying to prevent the formation of any anisotropic or inhomogeneous parts. We employed a high speed camera mounted on a stereomicroscope, and synchronized the image acquisition with the electrical stimulation and the force response measurement. The capability of measuring the 2-dimensional surface strain field in any desired Region Of Interest (ROI) allowed to obtain a comprehensive monitoring of the tissue's formation, both at a global and a local level. Preliminary results confirmed the adequacy of the system to measure tissue's strain field in complete accordance with the force measurement. Moreover, an in-depth analysis allowed to precisely pinpoint the sub-zones where discontinuities arise during tissue formation, returning essential information to improve X-MET generation process

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The object of this study was the recognition of Regions Of Interest(ROIs) in a time series of digital images of two specific laboratory experiments. It concerns the identification of objects in a tissue surface by high-resolution and high-speed ad-hoc systems for morphological dynamic image analysis. The protocols and the algorithms implemented are developed to retrieve biomechanical properties of two different in vitro systems; the solid filament X-MET (eX-vivo Muscle Engineered Tissue) to measure its reaction to a different frequency stimulation, and a planar system of co-culture of skeletal and cardiac muscle cells, where myotubes and cardiomyocytes coexist, to discriminate the interaction between different cell's type, of its spontaneous pulse. The results of the stimulated X-MET from solid culture system are frequency dependent points of the macroscopic muscular strength and its contractile response. The results for the co-culture planar board measure the correlation of the pulsed movements of the different parts of the tissue.