Excitability and Isometric Contractile Properties of Mammalian Skeletal Muscle Constructs Engineered in Vitro

Abstract

SUMMARYOur purpose was to engineer three-dimensional skeletal muscle tissue constructs from primary cultures of adult rat myogenic precursor cells, and to measure their excitability and isometric contractile properties. The constructs, termed myooids, were muscle-like in appearance, excitability, and contractile function. The myooids were 12 mm long and ranged in diameter from 0.1 to 1 mm. The myooids were engineered with synthetic tendons at each end to permit the measurement of isometric contractile properties. Within each myooid the myotubes and fibroblasts were supported by an extracellular matrix generated by the cells themselves, and did not require a preexisting scaffold to define the size, shape, and general mechanical properties of the resulting structure. Once formed, the myooids contracted spontaneously at approximately 1 Hz, with peak-to-peak force amplitudes ranging from 3 to 30 N. When stimulated electrically the myooids contracted to produce force. The myooids (n ϭ 14) had the following mean values: diameter of 0.49 mm, rheobase of 1.0 V/mm, chronaxie of 0.45 ms, twitch force of 215 N, maximum isometric force of 440 N, resting baseline force of 181 N, and specific force of 2.9 kN/m 2 . The mean specific force was approximately 1% of the specific force generated by control adult rat muscle. Based on the functional data, the myotubes in the myooids appear to remain arrested in an early developmental state due to the absence of signals to promote expression of adult myosin isoforms.

Prospectus of cultured meat—advancing meat alternatives

“…Small muscle-like organs have been demonstrated to grow from co-cultures of myoblasts and fibroblasts. These organs, termed myooids, are able to contract both spontaneously and by electrical stimulation, albeit with only a fraction of the force observed in control muscles probably because of lack of innervation (Dennis and Kosnik 2000;Dennis et al 2001;Kosnik et al 2001). The diameter of myooids is limited at most to 1 mm due to the lack of perfusion which is probably the biggest problem to overcome in designing an in vitro meat production system.…”

“…Such a design using the artificial capillaries for the purpose of tissue-engineering has been proposed (Technology Review 2003;Zandonella 2003). Like the myooids, it is possible to co-culture the myoblasts with other cell types to create a more realistic muscle structure which can be organized in much the same way as real muscles (Dennis and Kosnik 2000;Dennis et al 2001;Kosnik et al 2001).…”

“…It might be possible that mechanical or electrical stimulation can promote growth and structure of the cultured meat as newly formed myotubes in culture start to contract spontaneously (Wolpert et al 1998) or as a matter of fact, myooids also contract spontaneously at approximately 1 Hz. once formed (Dennis and Kosnik 2000). So exercise by electrical stimulation might be a viable solution to overcome atrophy in an in vitro meat production system.…”

Cartilage and facial muscle tissue engineering and regeneration: a mini review

“…Being highly specific and vital to the growth and survival of bone and bone tissue, vascularization of implanted scaffolds is a process that has already undergone extensive research. Vascularization is especially necessary when planning the creation of skeletal muscle from tissue engineering since the myoblasts can neither differentiate nor proliferate when greater than 150 µm from both an oxygen supply and a nutrient source [87]. This is significant considering that the efficient transport of nutrients, waste products, oxygen, and carbon dioxide through being connected to a muscle construct takes place in muscle that is thicker than 300 µm [35].…”

Commentary:In Vitro-Cultured Meat Production

“…But it is not suitable for producing highly structured meats, such as steaks. To produce these, one would need a more ambitious approach, creating structured muscle tissue as self-organizing constructs 5 or proliferating existing muscle tissue in vitro.…”