The burgeoning field of cultivated meat faces significant challenges in scaling up production to meet increasing demand for animal protein. Current methods struggle to achieve the high cell densities required for cost-effective production. Conventional cell culture techniques using 2D planar surfaces are inefficient, while methods like aggregates and fixed-bed reactors present challenges in control and cost-effectiveness. Microcarriers offer a superior alternative, providing a high surface-area-to-volume ratio for anchorage-dependent cell growth in suspension bioreactors. However, commercially available microcarriers are often made of non-edible synthetic materials, posing challenges for cell harvesting and potentially introducing undesirable components into the final product. Edible or dissolvable carriers offer a solution to this problem, simplifying the process and allowing seamless integration into the final food product. Previous research demonstrated the potential of inactivated mycelium biomass from *Aspergillus oryzae*, a GRAS (Generally Recognized As Safe) fungus, as a support structure for myoblasts. This current study expands upon this proof of concept by exploring a wider range of fungal species and cell types relevant to cultivated meat production, aiming to optimize the process and enhance the overall efficacy of edible mycelium carriers.

Extensive research has explored microcarrier technology for large-scale cell culture in various applications, including regenerative medicine, tissue engineering, and biopharmaceutical manufacturing. The advantages of microcarriers, such as high cell density, ease of scale-up, and cost-effectiveness compared to other methods, are well documented. However, challenges remain in efficient cell harvesting from conventional, non-edible microcarriers. Trypsinization, a common detachment method, has yielded inconsistent results, often requiring additional steps like carrier sieving. The use of edible or dissolvable carriers addresses this issue, eliminating the need for complex harvesting procedures and potentially reducing waste. Studies on edible carriers are still limited, however, with few commercially available options and scarce data on cell proliferation and differentiation. Previous work by the authors demonstrated the viability of *Aspergillus oryzae* mycelium as a support for myoblast growth. This current study builds on that foundation.

The study utilized several filamentous fungal species (*Aspergillus oryzae*, *Aspergillus awamori*, *Aspergillus sojae*, *Aspergillus nishimurae*, *Aspergillus tubingensis*, *Rhizopus oligosporus*, and *Penicillium chrysogenum*) to produce mycelium carriers. These were inactivated and seeded with C2C12 myoblasts and bovine satellite cells (bSCs). AlamarBlue assays measured cell metabolic activity, serving as a proxy for cell viability and proliferation. Different seeding densities were tested to optimize cell growth. Immunostaining with Hoechst and fluorescent microscopy visualized cell attachment and aggregate formation on the carriers. Quantitative PCR (qPCR) analyzed the expression of myogenic differentiation markers (PAX7, MYOD1, MYOG, MYH2 for C2C12; MYOD1, MYOG, MYH1/2, MYH3 for bSCs) to assess differentiation potential on mycelium carriers compared to Cytodex 3 (a non-edible commercial microcarrier) after induction with differentiation media. Statistical analyses, including ANOVA and t-tests, determined significant differences between groups.

The results revealed a significant impact of fungal species on C2C12 cell attachment and metabolic activity. *Aspergillus oryzae* strains (particularly *A. oryzae* 1) consistently supported superior cell proliferation compared to other fungi, which exhibited poor or negligible cell attachment. Optimal seeding density for C2C12 cells on mycelium carriers was found to be 5 × 10⁵ and 5 × 10⁶ cells/mL, resulting in significant proliferation. High-density seeding resulted in smaller cell aggregates on mycelium carriers compared to Cytodex or cells grown in suspension, suggesting that the mycelium carriers may provide better control of aggregate size. qPCR analysis showed that C2C12 cells on mycelium carriers expressed key myogenic differentiation markers (MYOD1, MYOG, MYH2), indicating successful differentiation. Bovine satellite cells (bSCs) also exhibited strong attachment and proliferation on mycelium carriers, comparable to or exceeding that on Cytodex at certain seeding densities. While differentiation marker expression trends for bSCs were less consistent, the presence of MYOD1 and MYH3 suggested differentiation potential.

This study successfully expanded upon previous findings, demonstrating the versatility of mycelium carriers for supporting both proliferation and differentiation of anchorage-dependent animal cells relevant to cultivated meat production. The significant difference in cell attachment and growth observed among various fungal species highlights the importance of selecting appropriate fungal strains. The observed formation of smaller cell aggregates on mycelium carriers, compared to Cytodex, suggests potential advantages in terms of scalability and mass transfer in bioreactor systems. The expression of myogenic differentiation markers further validates the potential of this technology for creating functional muscle tissue. While the study used a 2D cell culture system, findings suggest the potential of a simplified, one-step process for large scale production. Future work should focus on validating these results at higher scales under dynamic, agitated conditions.

This research demonstrates the feasibility of using edible mycelium carriers derived from *Aspergillus oryzae* for cultivating anchorage-dependent animal cells relevant to cultivated meat production. The ability of these carriers to support both cell proliferation and differentiation while eliminating the need for complex harvesting procedures represents a significant advancement in this field. Future research should focus on scaling up the process to bioreactor levels, optimizing fermentation and culture conditions, and assessing the impact of different fungal strains on the final product's flavor, texture, and nutritional characteristics. Exploring genetic modification of the fungi to further enhance cell growth and product attributes also presents exciting possibilities.

The study was primarily conducted in a 2D static microwell plate system, which may not fully reflect the conditions of a large-scale bioreactor. Future studies are needed to validate the findings under dynamic, agitated conditions. Quantitative assessment of the available surface area for cell attachment on the fungal pellets was limited, impacting a fully comprehensive analysis. While differentiation gene expression was observed, more in-depth analysis of the resulting muscle tissue quality (e.g., contractility, microstructure) is necessary to fully assess the suitability of mycelium carriers for creating high-quality cultivated meat.