Cultivated meat (CM), also known as cultured meat, offers a sustainable alternative to traditional animal agriculture. CM production involves growing animal cells in a controlled environment, requiring biocompatible scaffolds to support cell growth and mimic the structure of natural meat. Current challenges include developing animal-free, edible scaffolds that can be scaled up for large-scale production. Plant-based materials are promising candidates due to their abundance, sustainability, and potential for biodegradability. Previous research has investigated decellularized spinach and grass leaves, but their thinness limits their application for creating thick meat samples. Decellularized broccoli florets have also been studied, but their ability to promote muscle differentiation remains unclear. This research addresses the need for a scalable, structured, and edible scaffold by investigating the use of decellularized asparagus. Asparagus was chosen for its readily available, aligned vascular bundles, which provide structural support and facilitate cell alignment, mimicking the natural structure of muscle tissue. The aligned structure offers advantages in promoting cell growth and myotube formation, which are critical for producing realistic CM products that meet consumer expectations. The overall goal is to create a CM prototype that closely resembles traditional meat in terms of texture, appearance, and mouthfeel, addressing the need for voluminous and structured CM products.

Existing literature highlights the importance of scaffolds in cultivated meat production for achieving scalability and mimicking the structure of traditional meat. Macro-porous scaffolds support cellular proliferation and mechanical support, and techniques like anisotropic architecture and surface patterning can enhance scaffold texture, promoting cell alignment and myotube formation. Ideal scaffolds should be edible or biodegradable, and plant-based materials offer a sustainable solution. Decellularization removes plant cells while preserving the overall structure, creating a scaffold with inherent structural complexity. Previous studies have explored the use of decellularized spinach, grass, and broccoli, but limitations such as thinness and insufficient muscle differentiation promotion remain. This study aims to overcome these limitations by using decellularized asparagus, leveraging its aligned vascular bundles for improved cell alignment and structural integrity.

White asparagus was obtained and sliced longitudinally to create scaffolds. Decellularization was performed using a multi-step process involving sodium dodecyl sulfate (SDS), Triton X-100, and calcium chloride (CaCl2), followed by ethanol treatment and freeze-drying. DNA quantification confirmed the removal of plant DNA. The scaffolds' physical properties were characterized using Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray (EDX) analysis, Young's modulus testing, scanning electron microscopy (SEM), and micro-computed tomography (Micro-CT). C2C12 myoblasts and porcine adipose-derived mesenchymal stem cells (pADMSCs) were cultured on the scaffolds using a static cell seeding method. Cell viability was assessed using live/dead staining and PrestoBlue assays. Muscle differentiation was evaluated through immunofluorescence staining (Myosin heavy chain (MHC), Myogenin (MYOG), Desmin), creatine kinase (CK) activity assays, and quantitative PCR (qPCR) analysis (MYH1, MYOG). pADMSCs were also differentiated into adipocytes. A co-culture of pADMSC-derived muscle and fat cells was established on the scaffold. Texture profile analysis (TPA) was performed on both uncooked and pan-fried CM prototypes and compared to pork loin. Statistical analysis was conducted using appropriate tests (Welch’s test, one-way ANOVA, Brown-Forsythe and Bartlett’s test, Tukey's multiple comparisons tests).

Decellularization significantly reduced DNA content in asparagus scaffolds. The scaffolds exhibited a macro-porous structure with aligned vascular bundles suitable for cell attachment and alignment. Both C2C12 myoblasts and pADMSCs exhibited robust proliferation and differentiation on the scaffolds, as evidenced by increased CK activity, upregulated MYH1 and MYOG gene expression, and the presence of muscle-specific proteins (MHC, MYOG, Desmin). Successful adipocyte differentiation of pADMSCs was also observed. The co-culture of muscle and fat cells resulted in a CM prototype containing both muscle fibers and adipocytes with lipid droplets. TPA showed no significant difference in most textural parameters between uncooked CM prototypes and pork loin. However, pan-fried CM prototypes showed significant differences in hardness and chewiness compared to pork loin. The study also revealed the thermal stability of the DPS, indicating suitability for high-temperature cooking processes.

This study successfully demonstrated the use of decellularized asparagus scaffolds for cultivated meat production. The aligned vascular bundles facilitated cell alignment and promoted muscle differentiation, leading to the creation of a structured CM prototype. Although the DNA content in the decellularized scaffold exceeded the threshold conventionally used in regenerative medicine, this is not a significant concern for food applications as asparagus is consumed whole. The textural properties of the uncooked CM prototype were comparable to pork loin, suggesting successful mimicry of natural meat structure. However, differences in hardness and chewiness emerged after pan-frying, potentially due to the differences in the composition and structure of the two materials. Future research could focus on optimizing the cooking process and exploring other plant-based materials to further enhance the textural properties of CM products. The study makes a significant contribution to the field of cellular agriculture by offering a promising, sustainable, and scalable approach for producing structured CM products.

This research successfully demonstrated the feasibility of using decellularized asparagus as a sustainable, edible scaffold for cultivated meat production. The aligned vascular bundles in the scaffold promoted cell alignment and muscle differentiation, leading to a CM prototype with textural properties comparable to pork loin (uncooked). Future studies should explore optimization of the decellularization process and other plant species for enhanced scalability and improved textural characteristics.

The DNA content in the decellularized asparagus scaffolds, while significantly reduced, still exceeded the threshold generally considered acceptable in regenerative medicine applications. However, the authors argue this is not a major concern for food applications because asparagus is regularly consumed whole. Further research should explore different decellularization methods to achieve lower DNA levels. The study primarily focused on a single plant species; future work should evaluate other edible plant materials. Additionally, the texture profile analysis showed differences between the pan-fried CM prototype and pork loin, suggesting further optimization of processing methods is needed.