Stretchable zein-coated alginate fiber for aligning muscle cells to artificially produce cultivated meat

Cultivated meat aims to provide animal-cell–derived proteins with reduced environmental and animal welfare impacts relative to conventional livestock production. Production requires scaffolds that support skeletal muscle satellite cell attachment, proliferation, differentiation, and structural alignment to mimic native muscle tissue. Many existing 3D scaffolds are made from synthetic or animal-derived biopolymers and alignment techniques often scale poorly beyond a few strands, posing challenges for mass production. Plant-derived proteins have emerged as promising scaffold materials due to non-animal origin, cost-effectiveness, biocompatibility, biodegradability, and structural diversity. Prior work has shown bovine satellite cell growth in soy-protein porous scaffolds and use of wheat gluten and plant-protein–enriched alginate systems. Zein, a corn prolamine with favorable biocompatibility and ductility, is FDA-recognized as safe but suffers from poor rheological/electrical properties and hydrophobicity that complicate scaffold fabrication. This study hypothesizes that combining hydrophobic zein with hydrophilic alginate via a wet-spun zein-coated alginate fiber can yield a fully plant-based scaffold with improved cell affinity, mechanical robustness, and high ductility, enabling efficient alignment of muscle cells through scalable physical stretching. The objectives were to optimize fiber fabrication (zein concentration, diameter), characterize mechanical and structural properties, assess cell adhesion/viability, quantify alignment and myogenesis under controlled strain, and demonstrate assembly of muscle, fat, and vessel fibers into meat-like constructs.

The paper reviews scaffold needs for cultured meat, noting common reliance on synthetic or animal-derived biomaterials and the importance of ECM-mimicking properties and alignment for functional muscle tissues. Plant-derived materials present advantages (non-animal, low cost, biodegradability). Prior studies include textured soy protein porous scaffolds supporting bovine skeletal muscle maturation, wheat gluten-based porous scaffolds, and alginate modified/mixed with pea or soy protein isolates. Alignment strategies such as electrical stimulation or stretching have been demonstrated but often limited to one or few strands, hindering scalability. Zein has been applied in coatings, films, and drug delivery; when combined with other polymers, zein can enhance stiffness and cytoaffinity, though its hydrophobicity and processing challenges limit pure-zein scaffold applications. Alginate is widely used for wet-spun hydrogel fibers due to rapid Ca2+-mediated crosslinking but lacks cell-adhesive motifs, restricting adhesion and proliferation. The rationale is to exploit zein’s biocompatibility and ductility while using alginate’s processability to create a hybrid fiber that overcomes these limitations.

• Fiber fabrication (wet spinning): A 1% (w/v) sodium alginate solution in PBS was loaded in a syringe and injected (0.05 mL/min) into a coagulation bath containing zein dissolved in 70% ethanol with 5% CaCl2, forming alginate fibers that simultaneously acquired a zein coating via hydrophobic interactions. Fibers were collected on a take-up roller and washed with PBS to remove residual ethanol and Ca2+. Zein concentrations of 10%, 20%, and 30% were tested to optimize coating stability; fiber diameters were tuned by needle gauge (20G, 22G, 26G, 30G).
• Mechanical testing: A and ZA fibers were tested 1 h post-fabrication; “soaked” counterparts were immersed in PBS for 48 h. Samples (20 mm length) were tested in tensile mode (crosshead 0.1 mm/s) using a CT3 Texture Analyzer (4500 g load cell). Stress–strain curves and elastic modulus (slope over 5–15% strain) were calculated.
• Structural characterization: Fibers were frozen (−70 °C), lyophilized, Pt-coated, and imaged by SEM (Hitachi SU-8010) to visualize cross-sections and surfaces, confirming internal alginate cores with external zein shells.
• Cell culture and assays (C2C12 model): C2C12 myoblasts were cultured in DMEM + 10% FBS + 1% P/S at 37 °C, 5% CO2. Cells were seeded on ZA fibers (2 × 10^6 cells/mL). For myogenic differentiation, media were switched to DMEM + 2% horse serum + 1% P/S (MDM) for 6 days. Live/dead viability (Calcein-AM/EthD-1) was assessed at days 1, 3, 6 on 2D culture, A fibers, and ZA fibers.
• Stretching device and alignment protocol: A uniaxial stretching device was 3D printed via DLP using bioresin AESO/PEGDA (80:20) with 0.5% (w/v) LAP and 1% (v/v) tartrazine. Devices were PBS-washed and UV-sterilized. Bundles of ZA fibers seeded with C2C12 were cultured 5 days at 0% strain, then subjected to 0, 25, 50, or 75% static strain for 1 additional day. Alignment and morphology were analyzed via immunofluorescence.
• Immunocytochemistry and imaging: Fixation with 4% PFA; permeabilization with 0.25% Triton X-100; blocking (5% BSA). Primary antibodies: α-tubulin (DSHB, 1:100), desmin (ABclonal, 1:200), VE-cadherin (Abcam, 1:200). Secondary Alexa Fluor antibodies (Invitrogen). Nuclei counterstained with DAPI. Imaging on Lionheart FX microscope. ImageJ used to quantify alignment angles (−90° to 90°), nuclear circularity, α-tubulin area, myotube length, and desmin-positive area.
• Gene expression: RT-qPCR on C2C12 (myogenic markers Pax7, MyoD, MyoG, MYH1) and primary adipocytes (PPARγ, CEBPα). RNA isolation via TRIzol; cDNA via HKGscript 5X RT Premix; SYBR Green qPCR on Roche LightCycler 96. Cycling: 95 °C 10 min; 40 cycles of 95 °C 15 s, 60 °C 40 s. Primers provided.
• Primary cell isolation and differentiation: Primary bovine satellite cells (BSCs) isolated from longissimus muscle (3-year-old cow) and bovine preadipocytes from peri-renal fat (4-year-old cow). Disinfection with 70% EtOH, dissection, collagenase I and trypsin digestion, selective adhesion to remove fibroblasts, culture on ECL-coated flasks. BSCs differentiated in MDM for 6 days. Preadipocytes differentiated in ADM (DMEM + 10% FBS + 1% P/S + 10 µg/mL insulin + 300 µM linoleic acid), media refreshed every 2 days. Nile red staining used for lipid droplets.
• Assembly and cooking test: Muscle (BSC myotubes), fat (adipocytes), and vessel (CPAE endothelial) cell-laden ZA fibers were assembled into composite constructs. After 10 days of myoblast culture on ZA fibers, constructs were pan-fried in a preheated cast iron pan; gross morphology and fibrous texture were documented.
• Statistics: Data presented as mean ± s.d.; one-way ANOVA with Tukey’s post hoc or Student’s t test; significance at P < 0.05 (GraphPad Prism 8.0.2).

• Fabrication optimization: Zein coating stability after PBS wash depended on zein concentration: at 10% and 20% zein, 62% and 15% of shell area remained (i.e., substantial loss), whereas at 30% zein, 100% of the zein shell was preserved. Thus, 30% zein was used thereafter.
• Diameter control and swelling: Reducing needle size (20G→30G) decreased fiber diameters. Dry A fibers were <100 µm but swelled ~12.3× after soaking 48 h; dry ZA fibers were >100 µm but showed much lower swelling, yielding smaller soaked diameters than A fibers due to the zein shell acting as a hydrophobic barrier.
• Cell orientation vs. diameter: C2C12 alignment along fibers increased as fiber diameter decreased; best alignment achieved with 30G-made ZA fibers. Optimized fibers had ~124 µm dry diameter and ~220 µm soaked diameter at 30% zein.
• Mechanical properties: Dry A fibers: tensile stress at break 0.68 MPa; elastic modulus 3.04 MPa; strain at break <25%. Dry ZA fibers: 4.61 MPa; 4.63 MPa; strain >60%. Soaked A fibers could not be measured (insufficient elasticity). Soaked ZA fibers: 2.79 MPa tensile stress; 2.54 MPa modulus; strain at break >140%, demonstrating high ductility and stretchability in moist conditions.
• Cell viability/adhesion: ZA fibers supported higher C2C12 adhesion and viability than A fibers over 6 days; alginate alone led to cell clumping due to poor adhesion motifs.
• Stretch-induced alignment: Applying 0, 25, 50, 75% strain to ZA fiber bundles increased cell density and unidirectional alignment with strain. Nuclear circularity decreased with higher strain (indicative of elongation), and α-tubulin-positive area increased.
• Myogenesis enhancement: Under strains of 0, 25, 50, 75%, myotube lengths were 113 ± 19 µm, 155 ± 14 µm, 229 ± 12 µm, and 344 ± 27 µm, respectively; desmin-positive area increased with strain (46%, 55%, 63%, 75%). RT-qPCR at 75% strain showed from day 1 to day 6: Pax7 slightly increased; MyoD ~4.8-fold, MyoG ~15.5-fold, MYH1 ~6.7-fold increases, indicating progression toward myogenic maturation.
• Multitissue assembly and cooking: Primary BSCs formed aligned myotubes on ZA fibers with elevated MyoD/MyoG/MYH1 expression; primary adipocytes differentiated with high PPARγ and CEBPα and lipid droplets (Nile red+); CPAE endothelial cells expressed VE-cadherin on vessel fibers. Assembled muscle/fat/vessel fiber constructs retained fibrous structure after pan-frying, yielding a meat-like texture.
• Scalability aspect: The ductile ZA fiber bundles allowed simultaneous stretching of multiple strands, addressing a common scalability limitation in previous alignment methods.

The study addresses the need for animal-free, scalable scaffolds capable of promoting muscle cell adhesion, alignment, and maturation for cultivated meat. By combining hydrophobic zein with hydrophilic alginate in a wet-spun, zein-coated alginate fiber, the authors achieved improved structural stability, reduced swelling, enhanced mechanical performance, and markedly increased ductility under moist conditions. These properties enabled simple, scalable physical stretching of fiber bundles to align a large number of muscle cells simultaneously, overcoming the throughput limitations of prior alignment techniques. Enhanced adhesion and viability on ZA fibers compared to alginate alone demonstrate that zein confers necessary cytoaffinity. Strain-dependent increases in myotube length, desmin-positive area, and upregulation of myogenic genes confirm that mechanical stretching on ZA fibers accelerates myogenic differentiation and maturation, directly addressing the aim to generate aligned, mature muscle-like tissues. The successful culture and assembly of muscle, fat, and vessel fibers into composite constructs that retained fibrous texture upon pan-frying demonstrate the feasibility of producing structured, meat-like products using exclusively plant-based scaffolds. Collectively, the findings suggest that ZA fibers provide a practical route toward scalable production of aligned muscle tissues and assembled meat components for cultivated meat applications.

This work presents a simple, cost-effective, and fully plant-based scaffold—zein-coated alginate fibers—capable of supporting muscle cell adhesion, growth, alignment, and maturation without toxic chemical modifications. Optimization identified 30% zein as necessary for coating integrity and smaller fiber diameters (via needle gauge) to promote alignment. The fibers exhibit superior mechanical properties and high ductility when hydrated, enabling bundle-level stretching to align many strands simultaneously. Strain application significantly enhanced myotube metrics and myogenic gene expression. The approach further enabled assembly of muscle, fat, and vessel fiber constructs that maintained fibrous texture after cooking, underscoring translational potential for structured cultivated meat. Future research should focus on mitigating zein’s characteristic aroma/flavor, optimizing scalability and cost, and managing batch-to-batch variability of plant-derived materials. Refinement of fiber architecture, dynamic/mechanical conditioning regimens, and integration with serum-free media and edible crosslinking strategies could further advance mass production of high-fidelity cultivated meat.

• Sensory properties: Zein’s distinctive aroma and flavor may affect taste and consumer acceptance; strategies to reduce or mask these sensory attributes are needed.
• Scalability and cost: While bundle stretching improves throughput, large-scale manufacturing, process economics, and regulatory considerations require further development.
• Material variability: Plant-derived material properties can vary by source and batch, potentially impacting reproducibility and performance.
• Scope of biological validation: While C2C12 and primary bovine cells were tested, long-term functionality, contractility, and nutritional equivalence of assembled tissues were not comprehensively evaluated.
• Cooking and texture assessment: Post-cooking evaluation was qualitative; quantitative texture, sensory, and shelf-life analyses are needed.