Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute

The study addresses key challenges in cultured meat (CM): scalable expansion of anchorage-dependent cells and efficient cell-to-meat processing that yields desirable sensory properties. Building on microcarrier-based expansion used in bioreactors, the authors propose using edible chitosan–collagen microcarriers that obviate costly cell-harvesting steps by incorporating the cellularized microcarriers (microtissues) directly into CM products. They also target the crucial role of fat in meat by developing an oleogel-based fat substitute to mimic tenderness, juiciness, and flavor. The central aim is to establish a scalable platform that integrates edible microcarrier-derived microtissues with an oleogel-based protein-stabilized fat substitute and to process these into CM prototypes with meat-like appearance, texture, and improved nutritional attributes.

Microcarriers have long enabled scalable expansion of adherent cells in stirred bioreactors, offering large attachment areas and homogeneous nutrient distribution; materials used include glass, polystyrene, gelatin, and collagen. Conventional microcarriers typically require cell-harvesting steps, incurring costs and yield loss. Prior work from the authors proposed edible microcarriers that can be directly incorporated into CM to enhance appearance, taste, and nutrition. Texture can be tailored by combining edible biopolymers, crosslinkers, and post-expansion processing (e.g., static aggregation to promote ECM deposition or homogenization followed by enzymatic/physical restructuring). Enzymes like transglutaminase (TG), approved as food additives, can modulate protein crosslinking and improve sensory attributes. Fat is essential for meat quality; oleogelation structures liquid oils into solid-like systems using direct (e.g., monoglycerides, waxes) or indirect (biopolymer) approaches. Incorporating protein shells around oleogel particles can improve functionality and integration as solid-fat replacements. The authors draw on these advances to combine edible microtissues with a protein-stabilized oleogel fat substitute for CM.

• Cell source and carriers: Primary bovine mesenchymal stem cells (bMSCs) were isolated from bovine umbilical cord and cultured in α-MEM with FBS, antibiotics, amphotericin B, and bFGF. Edible microcarriers were produced by electrospraying a chitosan (2% w/v)–collagen (0.2–0.3% w/v) solution into a TPP–EGCG crosslinking bath.
• Scalable expansion optimization: Cultures were conducted in a 500 ml Applikon MiniBio bioreactor (dynamic), 1 L spinner flask (dynamic), and 60 mm suspension plates (static). In the bioreactor, gas sparging was avoided due to foaming; air overlay was used. Stirring regimens compared: 45–70 rpm vs 60–80 rpm; the latter ensured proper suspension and growth. Medium exchange strategy: 50% media replaced on days 3, 5, and 7. Temperature 37 °C, pH 7.2, DO 100% (headspace aeration ~1 ml/min). Viability (AlamarBlue, live/dead), metabolite monitoring (glucose, lactate), and imaging were performed over 8 days.
• Phenotype: Post-expansion, stemness markers were assessed by immunofluorescence and flow cytometry: CD29+, CD44+, CD45−.
• Microtissue aggregation: Post-expansion, cellularized microtissues underwent additional static culture to form disc-like aggregates (24-well, 7 days) or unorganized aggregates (60 mm dish 7 days then 24-well 7 days). Viability tracked over 12 days. Mechanical testing (Young’s modulus) and CHNS elemental analysis (lyophilized samples) were conducted.
• Fat substitute (FS) production: Oleogel-in-water emulsion-templated approach using canola oil structured with glycerol monostearate (GMS). Emulsion: 20% oil (w/w) with 20% GMS (w/w of oil) and 4.5% (w/w) chickpea protein in water; homogenized at 16,000 rpm for 3 min, cooled (RT 4 h, 4 °C 4 h, −80 °C overnight), then lyophilized. Final FS composition: 68% canola oil, 15% chickpea protein, 17% GMS. Characterization: CLSM for droplet size, PLM for crystalline structures, TGA for thermal decomposition, DSC for melting behavior, texture analysis, and instrumental color.
• CM prototype assembly: Two formats—(1) Layered CM: three layers of disc-like aggregates with interlayers of FS + TG paste (TG dispersed in PBS, mixed FS:TG in 3:2 w/w), yielding composition 75% w/w cellularized aggregates, 10% w/w FS, 15% w/w TG in PBS; molded (0.8 cm diameter, 0.4 cm thickness). (2) Burger-like CM: homogenized cellularized microtissues mixed with TG and FS; composition 60% w/w cellularized aggregates/microtissues, 32% w/w FS, 8% w/w TG; molded (2 cm diameter).
• Benchmarking and analyses: Beef patties prepared from lean beef (4.3% fat). Moisture content by lyophilization, cooking loss after frying (layered CM: 150 °C 5 min + 250 °C 1 min; burger-like CM and beef patties: 250 °C 6 min). Texture profile analysis (hardness, adhesiveness, cohesiveness, chewiness, springiness). TGA for compositional profiling. Fatty acid profiles via Soxhlet extraction and GC-FID. Color measured in CIELab (L*, a*, b*). Statistical analyses by t-tests/ANOVA with Holm–Šidák post-test.

• Scalable bioreactor culture: Gas sparging caused foaming and poor suspension; air overlay maintained oxygenation without adverse effects. Stirring at 60–80 rpm ensured proper suspension and robust growth versus 45–70 rpm which led to carrier sedimentation/aggregation. Replacing 50% medium on days 3, 5, and 7 supported growth and waste removal.
• Growth performance: Cell expansion in suspension plate, spinner flask, and bioreactor showed comparable fold increases over 8 days; specific growth rates (day⁻¹): 0.22±0.03 (suspension plate), 0.16±0.04 (spinner flask), 0.19±0.05 (bioreactor); no significant differences. Nuclei counts per microtissue were similar across conditions, with greater homogeneity in dynamic cultures.
• Metabolites: In bioreactor culture, glucose decreased from 4.5±0.2 to 2.8±0.4 mmol/L (day 0 to 7); lactate increased from 0.9±0.1 to 2.5±0.3 mmol/L, well below inhibitory levels reported for MSCs (35.4 mmol/L).
• Phenotype: bMSCs retained stem cell immunophenotype post-expansion (CD29+, CD44+, CD45−); flow cytometry indicated high positivity (e.g., CD29 ~95.9%, CD44 ~98.9%).
• Aggregation effects: Disc-like aggregates achieved the highest viable cell numbers by day 7 of static culture and exhibited ~40-fold higher Young’s modulus than cellularized microtissues and acellular microcarriers. Elemental analysis (mean±SD): N%: 4.07±0.23 (microcarriers), 5.43±0.33 (microtissues), 7.44±0.25 (disc-like aggregates); C%: 24.20±0.70, 27.66±1.50, 36.12±0.50; H%: 4.07±0.22, 4.38±0.26, 5.51±0.16; S%: 0.00, 0.33±0.01, 0.51±0.01.
• Fat substitute (FS) characterization: CLSM showed dense, tightly packed oleogel droplets (1–3 μm) with a thick protein layer; PLM revealed GMS spherulite-like structures (~50 μm) conferring solid-like behavior. TGA: FS displayed two weight loss regions (250–350 °C for GMS; ~427 °C for TAG combustion), closely matching beef fat’s major peak (~420 °C). DSC: Beef fat melting Tp ~42 °C; FS showed a sharper peak ~70 °C with ~2× higher melting enthalpy (GMS-driven). Texture: Raw and cooked FS hardness values were similar to beef fat; FS was more yellow (higher b*) than beef fat in both raw and cooked states.
• CM prototypes: Moisture content—layered CM ~75% (similar to beef patties), burger-like CM ~50%. Cooking loss ~30% for both CM prototypes, comparable to beef patties. During cooking, FS melting contributed to juiciness in burger-like CM. TGA of CM prototypes resembled beef patties; derivative curves showed two peaks attributed to FS incorporation. Fatty acid profile of CM matched FS, with significantly lower saturated fat and inclusion of omega-6 and omega-3 fatty acids compared to beef patties.
• Texture profiles (mean±SD): Layered CM hardness decreased upon cooking (6.43±0.90 N raw to 4.56±1.09 N cooked); burger-like CM hardness increased 1.7-fold upon cooking (1.64±0.18 N to 2.72±0.41 N). Beef patties exhibited a large increase in hardness upon cooking (3.76±0.46 N to 37.70±3.98 N). Other TPA parameters varied; layered CM was stiffer raw, while burger-like CM was softer and more tender. Color of CM prototypes was darker and less red than raw beef; further darkening after cooking was attributed to EGCG crosslinking and Maillard reactions.

The integrated platform successfully addresses two major bottlenecks in CM: scalable cell production and incorporation of a fat component that imparts meat-like sensory qualities. The edible microcarrier approach enabled efficient, homogeneous expansion of bMSCs in a scalable bioreactor without subsequent cell-harvesting steps, preserving stemness and achieving confluence on carriers within 8 days—conditions conducive to industrial translation. Static post-culture aggregation enhanced stiffness (~40× increase in Young’s modulus) and increased elemental indicators of proteinaceous content (via ECM deposition), supporting structural integrity for assembly. The protein-stabilized oleogel fat substitute, based on GMS-structured canola oil and chickpea protein, replicated key thermal and hardness properties of beef fat while improving nutritional quality (lower saturated fat, inclusion of omega-6/omega-3). Its integration into CM enhanced juiciness, reduced cooking loss, and provided a marbling-like appearance. The two processing routes—layer-by-layer assembly of aggregates (layered CM) and homogenization (burger-like CM)—demonstrated tunable texture and structure: layered CM offered higher stiffness and protein content, while burger-like CM delivered marbling aesthetics and tenderness. Overall, the findings demonstrate that combining edible microtissues with oleogel-based fat and enzymatic crosslinking can yield cohesive CM prototypes with sensory and compositional attributes approaching conventional meat.

This work establishes a cultured meat platform integrating edible microcarrier-derived microtissues with a protein-stabilized oleogel fat substitute and enzymatic crosslinking to fabricate layered and burger-like CM prototypes. The study validates scalable, homogeneous bMSC expansion on edible chitosan–collagen microcarriers in a bioreactor, preserves stemness, and leverages post-expansion aggregation to enhance stiffness and potential nutritional value. The oleogel fat substitute mimics beef fat’s appearance, thermal behavior, and hardness while offering improved lipid profiles. Together, these components produce CM with comparable moisture, cooking loss, and desirable texture profiles. Future directions include fine-tuning microtissue and FS size and mechanics, exploring alternative crosslinkers and processing (e.g., 3D printing), optimizing fat-to-meat ratios, enhancing water-holding capacity, and fortifying FS with macro- and micronutrients. Tailoring oleogelators and protein contents can further tune melting and texture to meet consumer expectations.

• Additional static culture for aggregate formation increases time and cost and may limit scalability; unorganized aggregates require substantial culture duration.
• Decreased cell viability observed in disc-like aggregates after day 7 suggests mass transport limitations (oxygen/nutrients) and space constraints.
• Visible spherical microcarrier shapes can detract from marbling aesthetics in layered CM and require further process optimization to mask/fuse beads.
• Burger-like CM exhibited lower moisture content and softer texture than beef; further processing may be needed to improve water-holding and firmness.
• Some textural parameters and color (darker tone; less redness; higher yellowness from FS) remain less comparable to beef; EGCG crosslinking and Maillard effects darken products upon cooking.
• Elemental nitrogen overestimates protein content due to chitosan nitrogen, complicating direct nutritional inference; comprehensive nutrition profiling remains to be improved.