Metabolomic Characterisation to Inform Serum-Free Proliferation Medium Development for Cultivated Meat

Abstract

Cultivated meat has the potential to address ethical, sustainability, and food security concerns associated with conventional livestock production, but significant technical and economic barriers remain. Achieving this potential requires the development of cost-effective and efficient serum-free cell culture medium for muscle stem cell proliferation. This thesis employed metabolomic analysis to characterise proliferative myoblast metabolism and media component stability, which informed targeted supplementation of defined components to an existing serum-free formulation, resulting in enhanced bovine myoblast growth rates.
Most existing intracellular metabolite extraction workflows require mechanical detachment of adherent cells, which is impractical in hollow fibre bioreactors (HFBs). A novel protocol was developed for combined polar metabolite extraction and cell nuclei enumeration, enabling accurate data normalisation to cell number without cell detachment or duplicate cultures for cell counts. This protocol matched the metabolite coverage of established methods while improving reproducibility and recovery in well plate cultures, offering a practical, scalable, and cost-effective alternative to existing workflows. However, unintended cell detachment during extraction in HFB cultures limited its applicability and should be addressed in future work.
Metabolomic analysis of immortalised bovine satellite cell (iBSC) proliferation was used to identify metabolic deficiencies in serum-free Beefy-9 medium. Robust iBSC proliferation in Beefy-9 was achieved only when the recommended truncated vitronectin coating was replaced with 0.5 μg/cm2 full-length laminin-521. Untargeted LC-MS/MS metabolomics revealed four major pathway groups that were broadly downregulated relative to serum-containing controls: lipid and fatty acid metabolism, one-carbon metabolism, uridine and cytidine metabolism, and energy and redox metabolism. Additional analyses of proliferation in well plates and HFBs showed poor adhesion and elevated oxidative stress in the latter.
The stability of new media formulations must be assessed to ensure component compatibility and accurate measurement of nutrient consumption rates. In Beefy-9, a previously unreported rapid degradation of ascorbic acid-2-phosphate (AA2P) was observed, induced by recombinant human serum albumin (rHSA). This rice-expressed rHSA also caused apoptosis in iBSC cultures independent of AA2P. Thus, use of this rHSA is cautioned and future work should examine whether similar cytotoxicity occurs in other mammalian cells. Substitution with bovine serum albumin (BSA), termed B9/BSA, restored cell viability but reduced growth rates.
Targeted screening to address metabolic deficiencies in Beefy-9 yielded a cost-effective improvement to B9/BSA: supplementation with 0.5 mM each of carnitine and betaine achieved the fastest iBSC growth rate for a chemically defined serum-free medium to date. This supplementation reduced the iBSC doubling time from 48.0 to 34.8 hours.
Together, this work identified a previously undescribed rHSA-induced AA2P degradation and cytotoxicity, and improved bovine myoblast proliferation in Beefy-9, thus demonstrating the value of metabolomics-guided optimisation for developing scalable, cost-effective, serum-free media for cultivated meat.

Date of Award	24 Jun 2026
Original language	English
Awarding Institution	University of Bath
Supervisor	Marianne Ellis (Supervisor), Ying Swan Ho (Supervisor), David Tosh (Supervisor) & Tina Düren (Supervisor)