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Cultivated meat (CM) provides a potential solution to meet the rising demand for eco-friendly meat supply systems. Recent efforts focus on producing CM that replicates the architecture and textural toughness of natural skeletal muscle. Significance of the regulated role of cellular microenvironment in myogenesis has been reinforced by the substantial influence of mechanical cues in mediating the muscle tissue organization. However, the formation of structured CM has not been adequately described in context of the mechanical microenvironment. In this review, we provide an updated understanding of the myogenesis process within mechanically dynamic three-dimensional microenvironments, discuss the effects of environmental mechanical factors on muscle tissue regeneration and how cell mechanics respond to the mechanical condition, and further highlight the role of mechanical cues as important references in constructing a sustainable Hydrocolloids-based biomaterials for CM engineering. These findings help to overcome current limitations in improving the textural properties of CM.

Overcoming the challenge of creating thick, tissue-resembling muscle constructs is paramount in the field of cultivated meat production. This study investigates the remarkable potential of random cellulose acetate nanofibers (CAN) as a transformative scaffold for muscle tissue engineering (MTE), specifically in the context of cultivated meat applications. Through a comparative analysis between random and aligned CAN, utilizing C2C12 and H9c2 myoblasts, we unveil the unparalleled capabilities of random CAN in facilitating muscle differentiation, independent of differentiation media, by exploiting the YAP/TAZ-related mechanotransduction pathway. In addition, we have successfully developed a novel process for stacking cell-loaded CAN sheets, enabling the production of a three-dimensional meat product. C2C12 and H9c2 loaded CAN sheets were stacked (up to four layers) to form a ~300–400 μm thick tissue 2 cm in length, organized in a mesh of uniaxial aligned cells. To further demonstrate the effectiveness of this methodology for cultivated meat purposes, we have generated thick and viable constructs using chicken muscle satellite cells (cSCs) and random CAN. This groundbreaking discovery offers a cost-effective and biomimetic solution for cultivating and differentiating muscle cells, forging a crucial link between tissue engineering and the pursuit of sustainable and affordable cultivated meat production.

After muscle loss or injury, skeletal muscle tissue has the ability to regenerate and return its function. However, large volume defects in skeletal muscle tissue pose a challenge to regenerate due to the absence of regenerative elements such as biophysical and biochemical cues, making the development of new treatments necessary. One potential solution is to utilize electroactive polymers that can change size or shape in response to an external electric field. Poly(ethylene glycol) diacrylate (PEGDA) is one such polymer, which holds great potential as a scaffold for muscle tissue regeneration due to its mechanical properties. In addition, the versatile chemistry of this polymer allows for the conjugation of new functional groups to enhance its electroactive properties and biocompatibility. Herein, we have developed an electroactive copolymer of PEGDA and acrylic acid (AA) in combination with collagen methacrylate (CMA) to promote cell adhesion and proliferation. The electroactive properties of the CMA + PEGDA:AA constructs were investigated through actuation studies. Furthermore, the biological properties of the hydrogel were investigated in a 14-day in vitro study to evaluate myosin light chain (MLC) expression and metabolic activity of C2C12 mouse myoblast cells. The addition of CMA improved some aspects of material bioactivity, such as MLC expression in C2C12 mouse myoblast cells. However, the incorporation of CMA in the PEGDA:AA hydrogels reduced the sample movement when placed under an electric field, possibly due to steric hindrance from the CMA. Further research is needed to optimize the use of CMA in combination with PEGDA:AA as a potential scaffold for skeletal muscle tissue engineering. [ABSTRACT FROM AUTHOR]

In preclinical drug testing, human muscle tissue models are critical to understanding the complex physiology, including drug effects in the human body. This study reports that a multilayering approach to cell sheet‐based engineering produces an engineered human muscle tissue with sufficient contractile force suitable for measurement. A thermoresponsive micropatterned substrate regulates the biomimetic alignment of myofiber structures enabling the harvest of the aligned myofibers as a single cell sheet. The functional muscle tissue is produced by layering multiple myofiber sheets on a fibrin‐based gel. This gel environment promotes myofiber maturation, provides the tissue an elastic platform for contraction, and allows the attachment of a measurement device. Since this multilayering approach is effective in enhancing the contractile ability of the muscle tissue, this muscle tissue generates a significantly high contractile force that can be measured quantitatively. The multilayered muscle tissue shows unidirectional contraction from electrical and chemical stimulation. In addition, their physiological responses to representative drugs can be determined quantitatively in real time by changes in contractile force and fatigue resistance. These physiological properties indicate that the engineered muscle tissue can become a promising tissue model for preclinical in vitro studies in muscle physiology and drug discovery. [ABSTRACT FROM AUTHOR]

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212 nm vs. 284 ± 130 nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Cultured meat produced using cell culture technology can potentially alleviate many of the ethical, environmental, and public health concerns associated with conventional livestock meat production. The industrialization of cultured meat for wide-spread adoption requires new methods to efficiently collect high-quality cells and to preserve their cell quality. Cryopreservation is a widely used technique to enable the long-term storage of cells without causing severe damage. In this study, we focused on the feasibility of cryopreservation to maintain cell quality for storage of bovine myogenic cells harvested from bovine meat based on our unique primary culture method. Primary bovine cells were incubated in a culture dish and then cryopreserved at −80°C for 1 week or 1 year. After thawing, the cells were further cultured for several passages to evaluate the abilities of the cells to proliferate or differentiate into myotubes. Furthermore, the cells were repeatedly cryopreserved for 1 week each time to investigate the impact of the repeated freezing and thawing. Consequently, long-term (within 1 year) or repeated (up to 3 times for 1 week each) cryopreservation at −80°C caused no degradation in the abilities of the cells to proliferate or differentiate, which is important for cultured meat production. We also confirmed that the cryopreservation did not require any unique cell freezing media. Moreover, based on our tissue engineering technique, our cryopreserved bovine myogenic cells had the ability to form sarcomere structures and produce muscle contractions even after they were frozen for 1 year. Although the bovine muscle tissues described here require more mature structures and functions in order to closely mimic native muscle tissue, we believe that the functional maturation of myogenic cells is essential to produce a “tissue-engineered meat” that will have native-like nutrients, texture, and taste that consumers will expect in the future. These results reveal the potential of cryopreserving quality-controlled bovine myogenic cells to contribute to a stable supply of high-quality cells for cultured meat production.

Developing in vitro models that recapitulate the in vivo organization of living cells in a 3D microenvironment is one of the current challenges in the field of tissue engineering. In particular for anisotropic tissues where alignment of precursor cells is required for them to create functional structures. Herein, a new method is proposed that allows aligning in the direction of a uniform magnetic field both individual cells (muscle, stromal, and stem cells) or spheroids in a thermoresponsive collagen hydrogel. In an all‐in‐one approach, spheroids are generated at high throughput by magnetic engineering using microfabricated micromagnets and are used as building blocks to create 3D anisotropic tissue structures of different scales. The magnetic cells and spheroids alignment process is optimized in terms of magnetic cell labeling, concentration, and size. Anisotropic structures are induced to form fibers in the direction of the magnetic alignment, with the respective roles of the magnetic field, the mechanical stretching of hydrogel or co‐culture of the aligned cells with non‐magnetic stromal cells, being investigated. Over days, spheroids fuse into 3D tubular structures, oriented in the direction of the magnetic alignment. Moreover, in the case of the muscle cells model, multinucleated cells can be observed within the fibers. [ABSTRACT FROM AUTHOR]

Many mammalian tissues have a specific cellular arrangement that enables their unique function. For example, parallel alignment of myofibers enables uniaxial muscle contraction. To engineer structured tissues ex vivo, it is critical to recapitulate this cellular arrangement. Conventional 3D encapsulation often fails to recapitulate this complexity, motivating the need for advanced patterning approaches. In this work, an acoustofluidic device to continuously pattern mammalian cells within hydrogel fibers is engineered. Contactless acoustofluidic forces are used to control the spacing between parallel lines of cells. To enable continuous extrusion of cell‐laden hydrogel fibers, a low friction Teflon tube is integrated into the device. A photopolymerizable hydrogel allows triggering gelation externally with light once the cells are under the influence of the acoustic field, setting the patterned cells within the hydrogel fiber. Using this device, the muscle progenitor cells (myoblasts) within the hydrogel are patterned in parallel lines to mimic the structure of skeletal muscle. The increased formation of myotubes and spontaneous twitching of the myotubes in patterned samples are observed. This approach combining continuous fabrication with the tunability of acoustofluidics can create complex 3D tissues to engineer skeletal muscles as well as tendons, ligaments, vascular networks, or combinations thereof in the future. [ABSTRACT FROM AUTHOR]

Temporary scaffolds that mimic the extracellular matrix's structure and provide a stable substratum for the natural growth of cells are an innovative trend in the field of tissue engineering. The aim of this study is to obtain and design porous 2D fibroin-based cell matrices by femtosecond laser-induced microstructuring for future applications in muscle tissue engineering. Ultra-fast laser treatment is a non-contact method, which generates controlled porosity—the creation of micro/nanostructures on the surface of the biopolymer that can strongly affect cell behavior, while the control over its surface characteristics has the potential of directing the growth of future muscle tissue in the desired direction. The laser structured 2D thin film matrices from silk were characterized by means of SEM, EDX, AFM, FTIR, Micro-Raman, XRD, and 3D-roughness analyses. A WCA evaluation and initial experiments with murine C2C12 myoblasts cells were also performed. The results show that by varying the laser parameters, a different structuring degree can be achieved through the initial lifting and ejection of the material around the area of laser interaction to generate porous channels with varying widths and depths. The proper optimization of the applied laser parameters can significantly improve the bioactive properties of the investigated 2D model of a muscle cell matrix. [ABSTRACT FROM AUTHOR]

Opportunely arranged microscaled fibers offer an attractive 3D architecture for tissue regeneration as they may enhance and stimulate specific tissue regrowth. Among different scaffolding options, encapsulating cells in degradable hydrogel microfibers appears as particularly attractive strategy. Hydrogel patches, in fact, offer a highly hydrated environment, allow easy incorporation of biologically active molecules, and can easily adapt to implantation site. In addition, microfiber architecture is intrinsically porous and can improve mass transport, vascularization, and cell survival after grafting. Anionic polysaccharides, as pectin or the more popular alginate, represent a particularly promising choice for the fabrication of cell‐laden patches, due to their extremely mild gelation in the presence of divalent ions and widely accepted biocompatibility. In this study, to combine the favorable properties of hydrogel and fibrous architecture, a simple coaxial flow wet‐spinning system was used to prepare cell‐laden, 3D fibrous patches using RGD‐modified pectin. Rapid fabrication of coherent self‐standing patches, with diameter in the range of 100–200 μm and high cell density, was possible by accurate choice of pectin and calcium ions concentrations. Cells were homogeneously dispersed throughout the microfibers and remained highly viable for up to 2 weeks, when the initial stage of myotubes formation was observed. Modified‐pectin microfibers appear as promising scaffold to support muscle tissue regeneration, due to their inherent porosity, the favorable cell–material interaction, and the possibility to guide cell alignment toward a functional tissue. [ABSTRACT FROM AUTHOR]

In the present study, it is aimed to fabricate a novel silk sericin (SS)/wool keratin (WK) hydrogel-based scaffolds using an in situ bubble-forming strategy containing an N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling reaction. During the rapid gelation process, CO 2 bubbles are released by activating the carboxyl groups in sericin with EDC and NHS, entrapped within the gel, creating a porous cross-linked structure. With this approach, five different hydrogels (S2K1, S4K2, S2K4, S6K3, and S3K6) are constructed to investigate the impact of varying sericin and keratin ratios. Analyses reveal that more sericin in the proteinaceous mixture reinforced the hydrogel network. Additionally, the hydrogels' pore size distribution, swelling ratio, wettability, and in vitro biodegradation rate, which are crucial for the applications of biomaterials, are evaluated. Moreover, biocompatibility and proangiogenic properties are analyzed using an in-ovo chorioallantoic membrane assay. The findings suggest that the S4K2 hydrogel exhibited the most promising characteristics, featuring an adequately flexible and highly porous structure. The results obtained by in vitro assessments demonstrate the potential of S4K2 hydrogel in muscle tissue engineering. However, further work is necessary to improve hydrogels with an aligned structure to meet the features that can fully replace muscle tissue for volumetric muscle loss regeneration., (© 2024 The Author(s). Macromolecular Bioscience published by Wiley‐VCH GmbH.)

Objective: To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation., Methods: Firstly, hyaluronic acid was oxidized with NaIO 4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR)., Results: FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P <0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P <0.05)., Conclusion: The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.

Several microfabrication technologies have been used to engineer native-like skeletal muscle tissues. However, the successful development of muscle remains a significant challenge in the tissue engineering field. Muscle tissue engineering aims to combine muscle precursor cells aligned within a highly organized 3D structure and biological factors crucial to support cell differentiation and maturation into functional myotubes and myofibers. In this study, the use of 3D bioprinting is proposed for the fabrication of muscle tissues using gelatin methacryloyl (GelMA) incorporating sustained insulin-like growth factor-1 (IGF-1)-releasing microparticles and myoblast cells. This study hypothesizes that functional and mature myotubes will be obtained more efficiently using a bioink that can release IGF-1 sustainably for in vitro muscle engineering. Synthesized microfluidic-assisted polymeric microparticles demonstrate successful adsorption of IGF-1 and sustained release of IGF-1 at physiological pH for at least 21 days. Incorporating the IGF-1-releasing microparticles in the GelMA bioink assisted in promoting the alignment of myoblasts and differentiation into myotubes. Furthermore, the myotubes show spontaneous contraction in the muscle constructs bioprinted with IGF-1-releasing bioink. The proposed bioprinting strategy aims to improve the development of new therapies applied to the regeneration and maturation of muscle tissues., (© 2023 Wiley-VCH GmbH.)

Muscle tissue engineering has been the focus of extensive research due to its potential for numerous medical applications, including ex vivo actuator development and clinical treatments. In this study, we developed a method for harvesting muscle fiber in a floatable and translocatable manner utilizing thermally expandable hydrogels with a chemically patterned polydopamine (PD) layer generated by microcontact printing (μCP). The μCP of PD on the hydrogel facilitated the formation of stripe patterns with varying widths of printed/nonprinted area (50/50, 100/100, and 200/200 μm). The spatially controlled adhesion of C2C12 myoblasts on the PD patterns produced clearly distinguishable muscle fibers, and translocated muscle fibers exhibited preserved extracellular matrix and junction proteins. Furthermore, the development of anisotropic arrangements and mature myotubes within the fibers suggests the potential for functional control of engineered muscle tissues. Overall, the muscle fiber harvesting method developed herein is suitable for both translocation and floating and is a promising technique for muscle tissue engineering as it mimics the structure-function relationship of natural tissue.

The field of tissue engineering has been long seeking to develop functional muscle tissue that closely resembles natural muscle. This study used a bio-inspired assembly based on the surface tension mechanism to develop a novel method for engineering muscle tissue. This approach enabled uniaxially ordered electrospun fibers to naturally collide into an aligned bundle without the need for manual handling, thereby reducing cell damage during the cell culture procedure. During the assembly procedure, C2C12 myoblasts were cultured in a viscous collagen hydrogel that caused wetting while providing adequate structural stability for the cell-fiber construct. In addition, gene expression analysis of the resulting muscle-like fibril bundle revealed improved myogenic differentiation. These findings highlight the potential of using a collagen hydrogel and the surface tension mechanism to construct biologically relevant muscle tissue, offering a promising strategy that may outperform existing approaches. Overall, this study contributes to the development of advanced tissue engineering methods and brings us a step closer to creating functional muscle tissue for therapeutic and regenerative medicine applications., (© 2023 IOP Publishing Ltd.)

• The constructions of current muscle units built in vitro are over-simplified. • The cell helical micromotors are generated from microfluidics. • Under the magnetic field, cell micromotors are assembled into a cell mass. • The cell mass assembled by cell micromotors mimics the tissue units in vivo. Building units play an important and critical role in muscle tissue engineering, which has aroused increasing attention in recent decades. The current muscle units are suffering from over-simplified constructions and complicated fabrication approaches. Thus, we proposed a kind of helical micromotor with the technique of microfluidics to build tissue constructions for muscle tissue engineering. The desired micromotors were achieved by encapsulating muscle cells together with magnetic iron oxide nanoparticles (Fe 3 O 4 NPs) in helical microfibers with biocompatible materials. The helical structures and encapsulated Fe 3 O 4 NPs imparted micromotors with the capacity of advancing in rotation under magnetic fields; while biocompatible components made it possible for cell adhesion, proliferation, and migration. By adjusting the intensity of magnetic fields, the helical pitch of microfibers, or/and the concentration of Fe 3 O 4 NPs, the movement speed of the achieved micromotors could be changed correspondingly. Benefiting from the controllable movement and sufficient cell cultivation, the generated micromotors were capable of assembling together to form a cell mass construction in a relatively safe and convenient environment. Furtherly, fibroblasts could be cultured on the surface of the assembled cell units to achieve complex muscle tissue structures, which would be more similar to in vivo tissue units. These characteristics indicated that the desired helical micromotors had a great application prospect in tissue regeneration, artificial muscle, cell cultured meat and other fields. [ABSTRACT FROM AUTHOR]

Tissue engineering strongly relies on the use of hydrogels as highly hydrated 3D matrices to support the maturation of laden cells. However, because of the lack of microarchitecture and sufficient porosity, common hydrogel systems do not provide physical cell-instructive guidance cues and efficient transport of nutrients and oxygen to the inner part of the construct. A controlled, organized cellular alignment and resulting alignment of secreted ECM are hallmarks of muscle, tendons, and nerves and play an important role in determining their functional properties. Although several strategies to induce cellular alignment have been investigated in 2D systems, the generation of cell-instructive 3D hydrogels remains a challenge. Here, we report on the development of a simple and scalable method to efficiently generate highly macroporous constructs featuring aligned guidance cues. A precross-linked bulk hydrogel is pressed through a grid with variable opening sizes, thus deconstructing it into an array of aligned, high aspect ratio microgels (microstrands) with tunable diameter that are eventually stabilized by a second photoclick cross-linking step. This method has been investigated and optimized both in silico and in vitro , thereby leading to conditions with excellent viability and organized cellular alignment. Finally, as proof of concept, the method has been shown to direct aligned muscle tissue maturation. These findings demonstrate the 3D physical guidance potential of our system, which can be used for a variety of anisotropic tissues and applications.