Process intensification of cultivated meat production through microcarrier addition strategy optimisation.

The use of microcarriers (MCs) is currently the most promising method for scaling up bovine satellite cell (bSC) cultures for cultivated meat production. Thanks to the inherent ability of the cells to migrate from one MC to another, also known as bead-to-bead transfer, the need for cell detachment is limited to a minimum, leading to a seamless seeding train. With this study, we aim to intensify the bioprocessing of bSCs in serum-free medium, by exploring the parameters influencing bead-to-bead transfer and cell growth and by optimising the seeding conditions and the MC addition strategy. Keeping production scale bioprocessing requirements into consideration, such as maximisation of fold increase within the same system, we have grown bSCs in up to 80 cm2/ml MC concentrations, using seeding cell densities of 1,000 to 4,750 cells/cm2. We also demonstrated optimisation of the MC addition strategy by determining an optimal confluence range (15,000 to 25,000 cells/cm2) for MC additions and by maximising the MC expansion ratio to 10, without impairing growth. Finally, to ensure scalability of these findings, we successfully applied them at a 3 L bioreactor scale.