A serum-free culture medium production system by co-culture combining growth factor-secreting cells and L-lactate-assimilating cyanobacteria for sustainable cultured meat production

Abstract

Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an L-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant L-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat.

Keywords: l-lactate-assimilating cyanobacterium; Co-culture; Cultured meat; Mammalian cell cultures; RL34 cells; Serum replacement.

Figure 1

Evaluation of C2C12 cell proliferation following the addition of RL34 cell culture medium: (a–e) or 50:50 v/v% of DMEM-diluted RL34 cell culture medium: (f–j). (a,f) Fold change of C2C12 cell density relative to the initial cell density (a: 4.5 ± 0.70 × 10⁴ cells/well; f: 4.8 ± 0.84 × 10⁴ cells/well in 12-well plates) after culture with 1 mL of DMEM or serum-free RL34 medium for 2 days (a: n = 4; f: n = 3). The darker color of columns indicates higher seeding density of RL34 cells; 0.25 × 10⁶ (light pink), 1.0 × 10⁶ (pink), and 4.0 × 10⁶ (crimson) cells/well in 6-well plate. (b–e), (g–j) Time-dependent changes in (b,g) glucose; (c,h) lactate; (d,i) ammonium; (e,j) LDH content of RL34 culture medium and 50:50 v/v% of DMEM-diluted RL34 cell culture medium (4.0 × 10⁶ cells/well in 6-well plate, (b–e): n = 4, (g–j): n = 3). Data represent the mean ± SD; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 2

Comparing RL34 monoculture vs. RL34 and KC0110 strain transwell co-culture. The gray columns stand for RL34 monoculture (seeding density of 4.0 × 10⁶ cells/well in 6-well plate for 3 days), and the green columns stand for RL34 and KC0110 strain transwell co-culture. The darker the green color of the column stands for the higher seeding density of KC0110; optical density at 750 nm (OD₇₅₀) 1.0 (light green), 2.0 (green), 4.0 (dark green) cultured with 4.0 × 10⁶ RL34 cells/well in 6-well plate for 3 days. (a) Fold change of RL34 cell density relative to the initial cell density (4.5 ± 0.50 × 10⁶ cells/well in 6-well plates) co-cultured with KC0110 for 3 days (n = 4). (b) Change of OD₇₅₀ to the initial cell density (Low: 0.97 ± 0.058; Mid: 2.0 ± 0.017; High: 4.0 ± 0.015) of KC0110 co-cultured with RL34 for 3 days (n = 3). (c–g) Metabolite content of co-culture medium (seeding density of 4.0 × 10⁶ RL34 cells/well with OD₇₅₀ 2.0 KC0110 in 6-well plate for 3 days) compared to monoculture medium (seeding density of 4.0 × 10⁶ RL34 cells/well without KC0110 in the 6-well plate for 3 days); (c) lactate; (d) ammonium; (e) glucose; (f) pyruvate and (g) LDH (n = 6). (h) 20 amino acids composition (n = 5); (i) total amount of 20 amino acids (n = 5). Data represent the mean ± SD; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant.

Figure 3

Evaluation of the proliferation of C2C12 cells in RL34 and KC0110 co-culture medium (sup.). (a) Fold change of C2C12 cell density relative to the initial cell density (4.1 ± 0.62 × 10⁴ cells/well in 12-well plates) following culture in different media (1 mL per well, respectively) for 2 days (n = 3); DMEM (white); RL34 monoculture medium (gray, seeding density of 4.0 × 10⁶ cells/well in 6-well plate for 3 days); RL34 and KC0110 co-culture medium (light green, OD₇₅₀ 1.0; green, OD₇₅₀ 2.0; dark green, OD₇₅₀ 4.0 KC0110 with 4.0 × 10⁶ RL34 cells/well in 6-well plate for 3 days). **p < 0.01; ns not significant. (b) Immunofluorescence staining for phalloidin (red) together with nucleus (blue) and MyoD (green) in C2C12 cells cultured in DMEM, RL34 monoculture medium, or RL34 and KC0110 (OD₇₅₀ 2.0) co-culture medium (scale bar: 200 µm). Data represent the mean ± SD; **p < 0.01; ns not significant.

Figure 4

Schematic diagram of the experiments (a) Evaluation of C2C12 cell proliferation following the addition of RL34 cell culture medium. (b) Evaluation of C2C12 cell proliferation following the addition of 50:50 v/v% DMEM-diluted RL34 cell culture medium. (c) Evaluation of C2C12 cell proliferation in RL34 and Synechococcus sp. KC0110 strain co-culture medium.