The global demand for meat is increasing, leading to concerns about sustainability and future shortages. Cultured meat (CM), produced in vitro, offers a sustainable alternative with potential to address these issues. CM aims to provide meat-like structures with sensory experiences comparable to animal-derived meat, minimizing environmental impact and enhancing animal welfare. Significant challenges remain, particularly in scalable cell expansion and efficient cell-to-meat processing. Scalable cell expansion requires efficient methodologies to generate large cell quantities for further processing. Microcarriers, providing a large surface area for anchorage-dependent cell attachment and proliferation, offer a solution for scalable expansion in bioreactors. While commercially available microcarriers exist, they often involve costly cell harvesting steps and reduce cell yield. This research proposes using edible microcarriers, integrating the cellularized microcarriers (microtissues) directly into the final CM product, eliminating harvesting steps and potentially enhancing the product's appearance, taste, and nutritional value. The research focuses on developing a CM production method using edible microcarrier-derived microtissues and oleogel-based fat substitutes, exploring scalable cell expansion, various processing approaches, and analyzing the resulting CM prototypes.

Previous research has explored various approaches to cultured meat production, focusing on scalable cell expansion methods. The use of microcarriers in bioreactors has been established as a scalable method for expanding anchorage-dependent cells. However, commercially available microcarriers often require costly cell harvesting and may not contribute positively to the final product's sensory properties. The use of edible microcarriers offers a potential solution to these problems. In addition to cell expansion, the creation of a realistic meat analogue requires mimicking the fat component, crucial for meat's texture, juiciness, and taste. Plant-based fat substitutes have been explored, and oleogelation, a process giving solid properties to liquid oil using gelling agents, emerges as a powerful approach for creating fat mimetics with reduced saturated fat and potential health benefits. Existing research on oleogel production involves both direct and indirect methods, with the latter requiring additional processing steps. The incorporation of protein-based shells to oleogel particles can facilitate their use as a solid fat replacement in CM.

The study involved several key methodological steps: **1. Production of Edible Cell Microcarriers:** Edible microcarriers were produced using an electrospray system, employing a solution of chitosan and collagen electrosprayed into a sodium tripolyphosphate (TPP) and epigallocatechin gallate (EGCG) crosslinking solution. Equations were used to calculate the total volume and surface area of the microcarriers. **2. bMSC Isolation and Characterization:** Primary bovine mesenchymal stem cells (bMSCs) were isolated from bovine umbilical cords. Immunofluorescence staining and flow cytometry were used to characterize the isolated cells for the presence of typical bMSC surface markers (CD29, CD44, and CD45). **3. bMSCs Expansion on Microcarriers:** bMSCs were expanded on the edible microcarriers using static (suspension plates) and dynamic (spinner flasks and a 500 ml Applikon MiniBio bioreactor) culture methods. The bioreactor culture parameters (aeration, stirring speed, and medium exchange) were optimized. Cell growth was monitored using AlamarBlue and live/dead cell imaging. Glucose and lactate concentrations were measured to assess nutrient consumption and waste production. **4. Characterization of Cellularized Microtissues:** After expansion, the cellularized microtissues were characterized using light-sheet fluorescence microscopy to visualize cell morphology and quantify cell numbers. Immunostaining and elemental analysis (CHNS Analyzer) were performed to assess cell marker expression and nutritional composition. Mechanical properties (Young's modulus) were measured using a microscale mechanical test system. **5. Microtissue Aggregation:** Cellularized microtissues were aggregated into disc-like and unorganized aggregates through static culture, and their cell viability and mechanical properties were evaluated. **6. Oleogel-based Fat Substitute Production:** An oleogel-based fat substitute (FS) was produced using a combination of direct and indirect methods. Canola oil, glycerol monostearate (GMS), and chickpea protein were used. The FS was characterized using confocal laser scanning microscopy (CLSM), polarized light microscopy (PLM), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), and texture analysis. The FS was compared to commercial beef fat. **7. CM Prototypes Assembling:** Two CM prototypes were assembled: a layered prototype using aggregated microtissues and FS and a burger-like prototype using homogenized microtissues and FS. Transglutaminase (TG) was used for crosslinking. The prototypes were characterized for moisture content, cooking loss, texture (hardness, adhesiveness, cohesiveness, chewiness, springiness), and color. **8. Statistical Analysis:** Statistical analysis (ANOVA, t-test) was performed to compare different groups.

The study successfully optimized parameters for scalable expansion of bMSCs on edible microcarriers in a bioreactor, achieving comparable growth rates to laboratory-scale cultures. The optimized bioreactor culture yielded high cell viability and maintained bMSC stemness. The disc-like aggregates generated exhibited significantly higher stiffness (40-fold increase in Young's modulus) compared to cellularized microtissues and acellular microcarriers. Elemental analysis revealed increased nitrogen, carbon, hydrogen, and sulfur content in aggregates compared to microtissues, likely due to ECM deposition. The developed oleogel-based fat substitute (FS) exhibited similar appearance, color, and hardness to beef fat but with a reduced saturated fatty acid content and increased omega-6 and omega-3 fatty acids. The two CM prototypes displayed different characteristics. The layered CM showed higher moisture content and stiffness but visible spherical shapes of the beads. The burger-like CM prototype, produced via homogenization, demonstrated a marbling appearance and softer texture but lower moisture content. TGA analysis showed a similar thermal decomposition profile for CM prototypes and beef patties. Textural analysis revealed that the layered CM’s hardness, adhesiveness, cohesiveness, and chewiness did not improve after cooking, while the burger-like CM displayed a significant increase in hardness post-cooking. The burger-like CM had a more tender texture due to microtissue homogenization, which after cooking resulted in a 1.7-fold increase in hardness. The color of the raw CM prototypes was darker than that of raw beef, possibly due to EGCG crosslinking and further darkening occurred during cooking because of the Maillard reaction.

The findings demonstrate a feasible and scalable platform for cultured meat production. The use of edible microcarriers simplifies the process, eliminates costly cell harvesting, and potentially enhances product quality. The oleogel-based fat substitute successfully mimics the desired sensory properties of animal fat with improved nutritional profiles. The two CM prototypes showcase the versatility of the platform, allowing for the creation of products with different textures and appearances, catering to diverse consumer preferences. The use of TG crosslinking facilitated the creation of cohesive structures. The lower moisture content in the burger-like CM compared to the layered CM and beef patties may be addressed through further optimization of homogenization techniques or the addition of other components to enhance water-holding capacity. Future research could focus on enhancing the marbling appearance in layered CM and adjusting the moisture content and texture of the burger-like CM. The nutritional profile could be further improved by enhancing the protein content and adding various micronutrients to the oleogel-based FS.

This research presents a novel cultured meat platform utilizing edible microcarrier-derived microtissues and a protein-incorporated oleogel-based fat substitute. The platform's scalability, versatility in creating different product types (layered and burger-like), and potential for nutritional enhancement contribute significantly to advancing cultured meat technology. Further research should focus on optimizing the platform's aspects to enhance texture, improve nutritional profiles and achieve a marbling appearance similar to that of animal-derived meat. exploring different microtissue aggregation strategies, improving water-holding capacity and optimizing the fat-to-meat ratio.

The study's limitations include the manual assembly of the CM prototypes, potentially limiting scalability. The relatively small scale of the bioreactor used for cell expansion may not fully represent industrial-scale production. The nutritional analysis was indirect, relying on elemental analysis and TGA, and did not fully encompass the complete nutritional composition of the final products. Future research needs to focus on automation of the assembling process and a large-scale production to confirm the findings. Further, a comprehensive nutritional analysis is needed to give a complete picture of the nutritional value of CM products.