Owing to the increase in the global demand of meat, cultured meat technology is being developed to circumvent a shortage of meat in the future. However, methods for construction of millimetre-thick bovine muscle tissues with highly aligned myotubes have not yet been established. Here, we propose a culture method for constructing 3D-cultured bovine muscle tissue containing myotubes aligned along its long-axial direction, which contracted in response to electrical stimulation. First, we optimised the composition of biomaterials used in the construction and the electrical stimulation applied to the tissue during culture. Subsequently, we fabricated millimetre-thick bovine muscle tissues containing highly aligned myotubes by accumulating bovine myoblast-laden hydrogel modules. The microbial content of the bovine muscle tissue cultured for 14 days was below the detection limit, indicating that the muscle tissues were sterile, unlike commercial meat. Therefore, the proposed construction method for bovine muscle tissues will be useful for the production of clean cultured steak meat simulating real meat.

Background--Cardiomyopathy is a leading cause of mortality among Duchenne muscular dystrophy patients and lacks effective therapies. Phosphodiesterase type 5 is implicated in dystrophic pathology, and the phosphodiesterase type 5 inhibitor tadalafil has recently been studied in a clinical trial for Duchenne muscular dystrophy.

Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (P i ), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of P i . Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.yosin VI is unique among the known myosins of animal cells in that it traffics toward the minus end of actin filaments (1). This unique directionality, coupled with its ability to act as both a processive transporter (2, 3) and load-dependent anchor (4), allow myosin VI to play a number of cellular roles that cannot be compensated for by any other myosin motor (5-15). To accomplish these cellular functions, myosin VI has a number of unique structural and functional adaptations, many of which have been debated in the literature, and some are still the subject of controversy (8). This is not surprising because its design features represent significant departures from other characterized myosin motors.Mutations in myosin VI can result in deafness in humans (16)(17)(18)(19)(20). There are three published human mutations (16-18) that cause deafness and result in amino acid changes in the myosin VI motor domain: C442Y, H246R, and E216V. In the mouse, there is one characterized missense mutation (20) in the motor (D179Y) as well as a null mutation (19), both of which result in deafness. All of these mutations likely lead to disruption of the normal organization and maintenance of the stereocilia, the mechanosensing organelles of hair cells, present in the cochlear apparatus, as has been documented in the case of the mouse mutations (19).Myosin VI achieves its ability to walk hand-over-hand along a single actin filament (21) by having a motor that has been kinetically tuned to spend the majority of its time strongly bound to actin (22). Thus, the probability that at least one head will be strongly bound to actin at all times is very high. (The ratio of the occupancy of the strongly bound actin states of the actin-myosin ATPase cycle to that of the weak + dissociated + strongly bound states is called the duty ratio.) Although a high duty ratio is sufficient for processive movement, processivity can be further enhanced by a mechanism known as "gating" whereby strain between the heads essentially stalls the lead head on actin until the rear hea...

This paper describes a co-culture method using cell fiber technology. Cell fibers are cell-laden hydrogel microfibers, in which cells are cultured three-dimensionally and allowed to reach more mature state than the conventional two-dimensional cell culture. Cells in the cell fibers are encapsulated by alginate shell. Only cellular secretome is released into the surrounding environment through the shell while the cells were retained by the fiber. With their high handleability and retrievability, we propose to use the cell fibers for co-culture to ensure steady supply of cellular secretome. We cultured mouse C2C12 myoblasts with mouse 3T3 fibroblasts encapsulated in the cell fibers for two days. The number of C2C12 cells increased proportionally to the number of co-cultured 3T3 fibers, suggesting that the secretome of 3T3 fibers promoted survival and proliferation of C2C12 cells. We believe that cell fiber technology is a useful tool for co-culturing cells, and it will contribute to both basic cell biology and tissue engineering with its unique features.Co-culture, in which two or more types of cells are cultured together, is a major method to study interactions between different types of cells in vitro. Cells interact with each other both directly (via physical contact) and indirectly (via secreted molecules; for example, cytokines, growth factors and hormones) and these interactions have an impact on cellular survival, proliferation, differentiation and maturation. To investigate the indirect cellular interactions, two major methods have been established; one using culture inserts and the other using conditioned medium. Culture inserts make upper and lower compartments in culture wells, which enables a concurrent co-culture. Two different types of cells are plated and cultured in the upper and lower compartments. Only cellular secretome, but not the cells themselves, is then transferred between those two compartments through the pores on the bottom of culture inserts, when the pore size is smaller than the cells. On the other hand, in the method that employs conditioned medium, a certain type of cells are cultured and the supernatant containing their secretome (conditioned medium) is collected to subsequently culture the other type of cells. These methods are often employed and have been shown to be effective to study various cellular interactions 1-3 . However, neither of them is highly space-efficient; the number of available cells that provides secretome is limited because of the culture area.Cell fiber is a unique tool for culturing cells three-dimensionally for a long period until they differentiate into a mature tissue 4 . Cell fibers, which are cell-laden hydrogel microfibers formed by using a double-coaxial laminar-flow microfluidic device, consist of two parts; the core containing cells and extra cellular matrix (ECM) proteins such as collagen, and the alginate shell. Various types of cells have been shown to form three-dimensional (3D) tissues in cell fibers; for example, cardiomyocytes, vascular endoth...

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