Spent media analysis suggests cultivated meat media will require species and cell type optimization

The study addresses how to meet growing demand for sustainable protein by advancing cultivated meat (CM). Media cost and formulation are the leading technical hurdles for CM scalability. Conventional practice often uses biomedical media like DMEM without regard to cost or CM-specific needs, and few academic advances target CM media fundamentals. The key research question is whether media formulations can be shared across species and cell types or must be tailored. The authors propose spent media analysis (SMA) to quantify nutrient utilization and waste accumulation to guide targeted, cost-effective CM media design. They focus on CM-relevant cells: primary embryonic chicken muscle precursor cells (CMPCs) and chicken muscle fibroblasts (cMFBs) versus the murine C2C12 myoblast line, noting the utility of chicken primary cells and the dearth of data for cultivated chicken. The goal is to reveal species- and lineage-dependent media requirements by measuring glucose, lactate, amino acids, vitamins, trace elements/minerals, and selected growth factors across proliferation and differentiation in small-scale 2D culture.

• CM media is the major cost driver and barrier to scale; conventional use of DMEM and similar media often ignores cost and scalability considerations.
• Emerging academic efforts seek less expensive and animal-component-free media, but prior work has not quantified CM-relevant cells’ specific nutrient requirements.
• Spent media analysis (SMA) is a standard, simple approach in microbial and animal cell bioprocessing to determine component utilization, accumulation of inhibitory byproducts, and guide feed strategies, yet had not been applied to CM media.
• Many media components may be essential even if not measurably consumed, so SMA should be complemented with other optimization approaches.
• Chicken primary cells are established models in muscle physiology; however, literature on cultivated chicken is limited despite poultry’s large agricultural footprint.

• Cell types: Primary embryonic chicken muscle precursor cells (CMPCs) and primary chicken muscle fibroblasts (cMFBs) isolated from 19-day-old Hy-Line chicken embryos; C2C12 murine myoblasts.
• Chicken cell isolation: Embryos euthanized; pectoral muscles dissociated using gentleMACS kit; pre-plating (25 min) to enrich MPCs; culture on Matrigel for CMPCs; fibroblasts selected by adherence on uncoated plastic (cMFBs). Cells expanded to passage 1, frozen at 3.5×10^6 cells/mL in 70% DMEM/20% FBS/10% DMSO.
• Culture conditions: Experimental medium 40% high-glucose DMEM (with pyruvate and L-glutamine), 40% Ham’s F10, 20% FBS, plus 2.5 ng/mL recombinant human FGF2. Growth on Matrigel-coated vessels; 37 °C, 5% CO2.
• Experimental setup: Matrigel-coated 6-well plates seeded at 1×10^5 cells/well; cMFBs and CMPCs at passage 3; C2C12 at passage 17. Thirty wells per cell type to enable triplicate sampling at multiple time points over up to 3 weeks without media changes. At each time point: collect spent media (record volume), filter (0.22 µm), store at −30 °C; image wells; dissociate and count cells (hemocytometer with trypan blue).
• Analyses:
  • Glucose and lactic acid: HPLC (Agilent 1260 Infinity II) with Aminex HPX-87H column; RI detection; 5 mM H2SO4 mobile phase; isocratic 0.6 mL/min; 50 °C; 20 µL injection; 20 min run. Fresh standards for calibration curves.
  • Free amino acids: Hitachi L-8900 amino acid analyzer with ion-exchange chromatography/post-column ninhydrin; samples acidified to 2% sulfosalicylic acid, incubated, frozen, diluted with AE-Cys Li; detection at 570/440 nm; certain amino acids (e.g., tryptophan) not measured.
  • Water-soluble vitamins: HPLC-DAD (Agilent 1200) with Poroshell 120 EC-C18; mobile phases 25 mM KH3PO4 (pH 7.0) and acetonitrile; gradient 20.1 min; 0.5 mL/min; 35 °C; 20 µL; detection at 205/214/232/266/280 nm; fresh standards shielded from light.
  • Minerals/elements: ICP-MS (Agilent 7850) with He KED in ORS cell; wide dynamic range; calibration across 0.1–100 ppb (trace) and 10–10000 ppb (majors); standards in 2% HNO3/0.5% HCl; internal standards (Sc, Ge, Rh, In, Bi, Lu); autosampler SPS 4.
  • Cytokines/growth factors: Multiplex ELISA panel of 30 bovine cytokines (two biological replicates per time point due to sample limitations) for overall trends; specific bFGF quantified by sandwich ELISA (ThermoFisher EB2RB) with absorbance at 450 nm.
• Data processing and statistics: Concentrations (mass/volume) plotted vs. culture day. Specific cellular consumption/production rates calculated via backward numerical derivatives of concentration profiles normalized by cell counts at each time point. Statistical tests: two-way ANOVA with Holm-Šídák multiple comparisons (α=0.05). GraphPad Prism 9 used.

• Carbohydrates and lactate:
  • Glucose utilization patterns differed markedly: CMPCs consumed glucose more slowly and incompletely, with an approximately linear decrease over time; cMFBs and C2C12 consumed glucose faster, approaching near-complete depletion by day 10, coincident with cell counts plateauing. The expected fresh media glucose (from 40% DMEM + 40% F10) was ~2.24 g/L (not including FBS contribution). Lactate accumulation inversely tracked glucose depletion.
  • Growth/differentiation context: C2C12 proliferated exponentially for ~2 days, then more slowly to day 6; cMFBs for ~7 days; CMPCs for ~4 days. All reached similar maximal cell density by ~day 7 (confluence). CMPCs began myotube differentiation by day 3; C2C12 at day 5; cMFBs did not form myotubes.
• Amino acids (AAs): Several AAs showed little change over time, but key depletions were observed for glutamine (highest absolute use), arginine, serine, isoleucine, leucine, and methionine. In C2C12 cultures, serine approached complete depletion by day 7. Proline increased after day 7 only in cMFB cultures, consistent with collagen synthesis/turnover. During the proliferation phase, no significant differences in AA consumption rates were detected among cell types.
• Vitamins and minerals: No appreciable decreases in water-soluble vitamin concentrations over time. ICP-MS showed no significant decreases in measured elemental minerals (e.g., Na, Mg, K, Ca, Fe, Zn); many trace elements were below detection.
• Cytokines/growth factors: Multiplex screening (n=2 per point) generally showed no significant differential depletion between cell types; notable cases included bFGF (FGF2) and IP-10, NCAM-1, decorin with some trends. Follow-up ELISA indicated bFGF decreased over time in cultures. Decorin levels changed over time, reflecting potential myokine activity.
• Specific rates (per cell): Early in culture, cMFBs exhibited higher specific consumption rates of glucose and glutamine and lower lactate production compared to CMPCs and C2C12, suggesting more complete oxidative glucose metabolism. CMPCs appeared more glycolytic (more lactate per glucose). Differences in specific utilization rates across species/lineages were most pronounced during the first 1–2 days and diminished later.
• Overall: Only a subset of media components were appreciably consumed (notably glucose and a defined set of AAs; bFGF decreased), while many conventional media components showed little to no depletion.

The results directly address whether a single medium can serve diverse CM-relevant cells: nutrient utilization profiles differed substantially across species and lineages, especially early in proliferation, indicating that media must be tailored to cell type and process stage. CMPCs’ slower, more complete glucose metabolism (less lactate accumulation) contrasted with faster glucose use in cMFBs and C2C12; specific per-cell consumption rates highlighted lineage-specific metabolic programs (e.g., fibroblasts’ higher oxidative use). Amino acid utilization emphasized key demands for glutamine, arginine, serine, isoleucine, leucine, and methionine, while many vitamins and minerals were not significantly depleted, suggesting potential to omit or reduce some components to lower cost. Differences in differentiation timing (CMPCs vs C2C12) and proline dynamics in cMFBs (collagen-related) underscore that metabolic requirements shift with lineage and maturation state. These findings imply feed strategies in fed-batch/continuous CM bioprocesses should consider early high-demand phases and lineage-specific kinetics. The observations were made in serum-containing media; although FBS variability complicates interpretation, the trends still indicate that cell species/lineage and differentiation state are primary determinants of media needs. Overall, the study supports targeted, cell-type- and process-specific media optimization rather than adopting universal formulations.

No single, cost-effective medium is likely optimal for multiple cultivated meat cell types. Spent media analysis revealed species- and lineage-dependent nutrient utilization, with only select components (glucose, specific essential and non-essential amino acids, and bFGF) appreciably consumed over time. These insights can guide removal or reduction of unused components to reduce costs and inform feed strategies. Future work should: (1) evaluate chemically defined and complex serum-free media for cell-type dependence and metabolic impacts; (2) assess effects of excessive nutrient supply on growth and differentiation; (3) investigate genetic/immortalization and differentiation-state effects on metabolism; (4) extend to scalable suspension/bioreactor systems and optimize media dynamically using streamlined, robust methods.

• Limited scope: only three cell types (two primary chicken lineages and one murine cell line) in 2D, small-scale cultures; findings may not generalize to other species, lineages, or process formats.
• Serum-containing medium: FBS is undefined and variable; batch variability may influence nutrient and cytokine profiles and cell behavior.
• Cytokine screen underpowered (n=2 per time point), limiting statistical sensitivity for growth factor dynamics.
• Media not changed over extended culture; chemical instability/degradation of components may confound interpretation, though comparative trends remain informative.
• Passage number differences (C2C12 at passage 17 vs chicken cells at passage 3) could influence metabolism and differentiation; effects were not isolated in this study.
• Cell density and differentiation/fusion may alter per-cell consumption calculations later in culture.