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

The increasing global demand for protein necessitates sustainable and responsible production methods. Cultivated meat (CM) offers a potential solution, but the high cost of cell culture media is a significant obstacle. Current CM research often relies on expensive, commercially available media like DMEM without considering cost-effectiveness or scalability. While efforts are underway to develop cheaper and animal-product-free media, a fundamental understanding of the specific nutrient requirements of CM-relevant cell types is lacking. Spent media analysis (SMA), a simple yet effective technique for media optimization, has been successfully used in various industrial applications but hasn't been extensively applied to CM. SMA helps determine which media components are consumed by cells, which are not, and how waste products accumulate. This study aims to address this gap by performing a comprehensive SMA on three CM-relevant cell types: primary embryonic chicken muscle precursor cells (CMPCs), primary chicken muscle fibroblasts (cMFBs), and the murine C2C12 myoblast cell line. The choice of chicken cells is based on their established use in muscle physiology research and their relatively easy handling. This comparative analysis seeks to uncover species- and cell type-dependent variations in media requirements, paving the way for more targeted and efficient media optimization.

Existing literature highlights the challenges in producing cultivated meat at scale, primarily focusing on the high cost and complexity of cell culture media. Several studies have explored alternative, cheaper media formulations, including those free of animal products. However, a comprehensive understanding of the specific nutrient utilization rates of different CM-relevant cell types, which is critical for effective media optimization, has been lacking. While SMA is a well-established technique for media optimization in other industries, its application to cultivated meat is relatively recent. Previous studies on C2C12 cells and other cell lines have shown variation in metabolic profiles, but a direct comparison across species and cell types relevant to cultivated meat production hasn't been systematically investigated.

Primary CMPCs and cMFBs were isolated from 19-day-old Hy-Line chicken embryos using a method based on existing procedures, involving enzymatic and mechanical tissue dissociation and a pre-plating step to enrich the MPC population. C2C12 cells were obtained commercially. All three cell types were cultured in a medium composed of 40% DMEM, 40% Ham's F10, 20% FBS, and 2.5 ng/mL recombinant human bFGF. For the SMA experiment, cells were seeded in Matrigel-coated 6-well plates. Spent media were collected at various time points (0, 1, 2, 4, 7, 10 and 21 days), filtered, and stored for analysis. Cell counts were taken directly after media collection. Glucose and lactic acid were analyzed using HPLC with RI detection. Amino acid concentrations were measured using a Hitachi L-8900 amino acid analyzer. Water-soluble vitamins were analyzed using HPLC with a diode array detector. Mineral concentrations were measured by ICP-MS. A multiplex ELISA was initially used to screen for 30 bovine cytokines, followed by a standard ELISA for bFGF. Data analysis involved calculating specific cellular rates of nutrient utilization using numerical derivatives and statistical analyses (two-way ANOVA with Holm-Šídák post-test).

SMA revealed significant differences in nutrient utilization patterns among the three cell types. CMPCs exhibited a significantly lower rate of glucose utilization compared to cMFBs and C2C12s. C2C12s and cMFBs depleted glucose more rapidly, almost completely by day 10. Lactate accumulation followed an inverse pattern to glucose consumption. While all three cell types proliferated well, C2C12s showed exponential growth for about two days, cMFBs for 7 days, and CMPCs for 4 days. CMPCs began differentiating into myotubes by day 3, C2C12s by day 5, and cMFBs did not differentiate during the 3-week culture period. Amino acid analysis indicated that several amino acids (glutamine, arginine, serine, isoleucine, leucine, and methionine) were partially depleted, with glutamine being consumed the most. Serine was almost completely depleted by day 7 in C2C12 cultures. No significant decreases were observed in water-soluble vitamin or mineral concentrations. The multiplex cytokine screen revealed variations in cytokine depletion rates, although statistical power was limited by the number of replicates. bFGF displayed significant decreases over time. Analysis of specific nutrient utilization rates (ng/cell/day) revealed that the cell lineage and species origin significantly influenced the early-phase utilization rates of key nutrients. This is less significant later in the culture. CMFBs used glucose and glutamine more rapidly and produced less lactate initially compared to other cell types, suggesting differences in glucose metabolism pathways. The CMPCs did not completely deplete the glucose in the media.

This study's findings confirm that CM-relevant cell types have varied nutrient requirements, disproving the assumption of similar needs. The significant differences in glucose consumption and other nutrient utilization patterns highlight the limitations of using a single, generic medium. The observation that many vitamins and minerals were not significantly depleted suggests potential cost savings by excluding unnecessary components from CM media. The influence of cell density and differentiation status on metabolic activity further complicates media formulation. The relatively high passage number of the C2C12 cells compared to primary chicken cells is a limitation that requires further investigation, as it could affect metabolic patterns. The reliance on FBS in the experimental media introduces batch variability and might also affect results. While this study used FBS to better reflect common laboratory conditions, future studies could utilize chemically defined or serum-free media for comparison.

This study demonstrates the cell-type and species-specific nature of nutrient requirements for cultivated meat production. A one-size-fits-all approach to media formulation is inefficient and costly. Future research should focus on optimizing media for specific cell types, exploring serum-free media alternatives, and investigating the impact of nutrient excess on cell growth. The development of robust, streamlined, and accessible media optimization methods is crucial for advancing the cultivated meat industry.

The use of FBS in the culture media introduces inherent variability due to batch differences, which could confound interpretations. The limited number of biological replicates for the multiplex cytokine analysis reduced statistical power. The study used a 2D culture system, potentially affecting the generalizability of results to larger-scale, 3D or suspension cultures. The differences in passage numbers between the immortalized C2C12 cells and primary chicken cells might influence metabolic activity.