Fungus-derived protein particles as cell-adhesive matrices for cell-cultivated food

Cell-adhesive materials are essential in biomedical and pharmaceutical industries, particularly in tissue engineering for tissue regeneration. Traditionally, animal-derived extracellular proteins like collagen and fibronectin have been used, but sustainability concerns and animal welfare issues necessitate finding alternatives, especially for the burgeoning cell-cultivated food industry. Cell-cultivated meat, milk, and other products require anchorage-dependent cells, which need a suitable support material for growth and proliferation. This support material should ideally be non-animal-derived and edible, eliminating the need for separation from the final product, reducing production time and costs. The Arg-Gly-Asp (RGD) motif is a well-known cell-adhesive peptide sequence. While a few RGD-containing plant proteins have been reported, fungal proteins containing this motif are more prevalent. This study aims to investigate the potential of fungal-derived proteins as a sustainable source of cell-adhesive factors for cell-cultivated food production.

The literature review section of the paper highlights the existing reliance on animal-derived extracellular matrix (ECM) proteins like collagen and fibronectin for cell adhesion in tissue engineering and other applications. The authors discuss the limitations of using animal-derived products, focusing on sustainability and ethical concerns. They mention previous reports on RGD-containing plant proteins and the more widespread presence of RGD motifs in fungal proteins, particularly in pathogenic fungi. The literature review serves as a foundation to justify the exploration of fungal-derived proteins as a potential alternative cell-adhesive matrix. The study also emphasizes the lack of reports on utilizing fungal proteins, especially from edible species, for this purpose, underscoring the novelty of this research.

The methodology section begins with a data mining approach to identify RGD-containing proteins in the fungal and plant kingdoms using the GenBank database. The authors defined an RGD percentage for each species and found that a high percentage of both fungal and plant species possess proteins containing this motif. The focus shifted towards fungi, particularly edible species. Crude extracts were obtained from *Flammulina velutipes*, *Lentinus edodes*, and *Pleurotus eryngii*. The formation of protein particles in these extracts was observed, particularly upon heating, and their sizes were characterized using dynamic light scattering (DLS). A model was developed to understand particle aggregation dynamics. Cell adhesion and alignment were assessed by encapsulating mouse myoblast cells (C2C12) within 3D fibers incorporating the fungal protein particles. Cell alignment was analyzed using image processing techniques. Traction force microscopy (TFM) was employed to measure the traction stress exerted by C2C12 cells on substrates coated with *F. velutipes* particles and fibronectin, using polyacrylamide hydrogels to mimic the stiffness of muscle tissue. Finally, SDS-PAGE coupled with mass spectrometry was used to identify the proteins present in the fungal extracts.

The data mining revealed that approximately 5.5% of the unique proteins in the GenBank database contained RGD sequences. A large majority (98%) of fungal and plant species showed an RGD percentage greater than 1%. The authors observed the spontaneous formation of protein particles in crude fungal extracts, and this process was accelerated by heating. Particle sizes ranged from 146 to 1831 nm. In 3D cell culture assays, the fungal protein particles supported cell alignment, with cell orientation patterns similar to those observed with collagen. Traction force microscopy showed that the traction stresses exerted by C2C12 cells on substrates coated with *F. velutipes* particles were comparable to those on fibronectin-coated substrates. Mass spectrometry analysis identified several RGD-containing proteins, including a previously identified 30 kDa C1 protein from *F. velutipes* and beta-glucosidase. The presence of the C1 protein in *F. velutipes* was corroborated by SDS-PAGE analysis, supporting its role in mediating cell adhesion. Inhibition experiments using RGD peptide confirmed the involvement of RGD in mediating cell adhesion to the fungal particles.

The findings address the research question by demonstrating the feasibility of using fungus-derived protein particles as a sustainable alternative to animal-derived cell-adhesive matrices. The high prevalence of RGD-containing proteins in fungi, along with the ability to easily generate cell-adhesive particles, offers a cost-effective and scalable solution. The comparable cell traction stress exerted on fungal particles versus fibronectin validates their effectiveness in mediating cell adhesion. The identification of specific RGD-containing proteins offers avenues for further optimization and characterization. The results are relevant to the fields of cell-cultivated food and tissue engineering, promoting more sustainable practices and reducing reliance on animal-derived materials. The potential for broad application across various cell types and tissue engineering applications is also highlighted.

This study successfully established a method for identifying and utilizing fungal-derived proteins as sustainable cell-adhesive matrices. The method involves data mining to identify suitable fungal species, extraction and processing of proteins to form particles, and validation of cell-adhesive properties using various assays. The fungus-derived particles offer a promising alternative to animal-derived materials for cell-cultivated food and tissue engineering. Future research should focus on optimizing the production process, further characterizing the protein composition of the particles, and exploring their applications in various cell types and tissue engineering constructs.

The study focused on a limited number of fungal species, and further research is needed to explore the diversity of fungal sources. The mass spectrometry analysis may not have identified all proteins present in the particles, and a more comprehensive proteomic analysis might be beneficial. The 3D cell culture model might not fully capture the complexity of interactions in vivo. The study mainly used C2C12 cells, and further investigations using other cell types are warranted to assess the broader applicability of the fungal particles. Finally, large-scale production and regulatory approval for food applications need further investigation.