Skeletal muscle explants: ex-vivo models to study cellular behavior in a complex tissue environment

Abstract

Purpose/Aim: Skeletal muscle tissue explants have been cultured and studied for nearly 100 years. These cultures, which retain complex tissue structure in an environment suited to precision manipulation and measurement, have led to seminal discoveries of the extrinsic and intrinsic mechanisms regulating contractility, metabolism and regeneration. This review discusses the two primary models of muscle explant: isolated myofiber and intact muscle.Materials and Methods: Relevant literature was reviewed and synthesized with a focus on the unique challenges and capabilities of each explant model.Results: Impactful past, current and future novel applications are discussed.Conclusions: Experiments using skeletal muscle explants have been integral to our understanding of the fundamentals of muscle physiology. As they are refined and adapted, they are poised to continue to inform the field for years to come.

Keywords: Isolated myofiber; ex-vivo stimulation; in-vitro culture; intact muscle; satellite cell.

Figure 1.

An exploded view of hierarchical structural organization of muscle tissue. Long cylindrical myofibers packed with myofibrils are encapsulated in the basal lamina with satellite cells lying between the muscle fiber membrane and basal lamina. Motor neurons and capillary networks interact with each myofiber to transmit signals and supply nutrients. Layers of interstitial ECM that store soluble growth factors and cytokines and guide cellular patterning surround myofibers (endomysium), bundles of myofibers termed fascicles (perimysium), and the whole muscle (epimysium). Inset, the basal lamina and myofiber provide direct mechanical and biochemical cues to the muscle satellite cell that are integral to maintenance of quiescence and self-renewal.

Figure 2.

Graphical illustration of myofiber explant. Striated multi-nucleated myofibers are freed from surrounding muscle tissue with the basal lamina intact. The myofiber explant culture conditions enable precise control of mechanical stretch, soluble factors, interstitial matrix mimics, and genetic manipulations while also allowing direct high-resolution imaging. Inset, Example light microscopy image of a myofiber 48 hours after isolation with associated attached and extravasating satellite cells (upper panel, top and bottom arrows respectively) and fluorescent microscopy image depicting nuclear staining positive for Pax7, the canonical satellite cell marker (lower panel, arrrow). The cell resides in direct contact with the myofiber sarcolemma, labeled with dystrophin (Dmd), included with permission from [65]).

Figure 3.

Graphical illustration of two common whole muscle explant preparations: hindlimb preparations for mechanical manipulation and measurement and ribcage associated muscles for live-cell imaging and direct neural recordings. A, Isolated hindlimb muscles are prepared in a bath, where soluble factors may be manipulated, with attachments near the myotendinous junction. Mechanical stretch of the muscle can be precisely delivered with length controller, electrical stimulation of the muscle through current delivered via electrodes in the bath, and the resulting forces recorded by a force transducer. Inset, Example image of a mouse 5^th toe EDL muscle prepared for mechanical testing as described. B, Depiction of a diaphragm whole muscle explant that includes the associated nerve. While control of length and potentially force recording may be sacrificed due to the complex architecture, it enables inclusion of neuronal stimulation of the muscle as well as recording of muscle afferent signals through an electrode placed on the severed nerve. The thinness of the diaphragm muscle also makes it well suited for live imaging. Inset, Example images of the triangularis sterni muscle explants and its innervation using genetic labeling of thyl with YFP to mark axons (green) and bungarotoxin used to label the motor endplate (red). Left, shows multisegmental innervation of the muscle and right, shows high resolution imaging of an endplate band, included with permission from [58].