Hydrodynamics And Mass Transfer In Stirred Bioreactor For Animal Cell Culture

Recently,the production of cell-based meat by culturing animal muscle cells in vitro has received extensive attention.Current reports mainly focus on isolation and differentiation of animal satellite cells,rather than proliferation in bioreactors.The flow field in bioreactors determines the mass transfer,mixing and shear stress,which have an impact on cell metabolism,limiting cell density and production scale,while are still lacking in literature.This study investigated the mass transfer and hydrodynamics of spinner flask and constructed a microcarrier suspension scale-down culture system for porcine skeletal muscle cells,and further explored the feasibility of scale-up production.The main results are as follows.1.The flow field was obtained by cold-flow experiments and CFD simulations,providing fundamental hydrodynamic data.The mass transfer rate was measured by sodium sulfite oxidation method and dynamic oxygenation method.k_La under animal cell culture condition is1.5～4.5 h^-1.Single-phase and two-phase CFD models were used to calculate mixing time and to simulate microcarrier suspension process.The tracer method and a Python-Opencv based machine vision algorithm were used to experimentally measure the mixing time to validate the CFD model.The mixing time under animal cell culture condition is 5～10 s,and 5～30 g·L^-1microcarrier can reach a complete suspension at 30 rpm.The validated CFD model was used to calculate energy dissipation rate（?）and Kolmogorov length scaleη,2×10^-4～1.5×10^-3 W·kg^-1and 160～253μm respectively under animal cell culture condition.2.The temperature,DO and CO₂ concentrations were monitored and controlled by a modified spinner flask,where immortalized porcine muscle cells were cultured in suspension using Fe³⁺-coated CytodexⅠmicrocarriers.The optimal batch culture conditions were 30 rpm intermittent stirring for 24 h and 60 rpm continuous stirring until 96 h.The microcarrier concentration was4 g·L^-1 and cell inoculation density was 1×10⁵ cells·m L^-1 under constant environment of 37℃and 5%CO₂.Cells proliferated 3.64±0.28 folds after 96 h.Cells digested by trypsin maintained normal proliferative capacity and cell specificity after resumption of flat culture.It was also found that the addition of new microcarriers with intermittent agitation during high cell metabolism period facilitated bead-to-bead transfer.The scale-down model combined flow field with cell growth properties to obtain the hydrodynamics conditions for microcarrier-based suspension culture of animal muscle cells,guiding rational bioreactor scale-up design.3.A 20 m³ stirred tank bioreactor was rationally designed and optimized,and Eulerian three-phase CFD model was carried out under different conditions.At 20 rpm stirring speed and 0.12vvm aeration rate,20%microcarriers could be completely suspended.Average gas holdup was2.6%and DO was in the 40%～50%range,k_La was 8.4 h^-1,which could theoretically meet the demand of microcarriers suspension and mass transfer for high-density animal cells culture.The average Kolmogorov length was 88.1μm,and the shear was more severe after the introduction of the gas phase.The model combining cell reaction kinetics with CFD provides the basic data for the actual scale-up process,emphasizing the importance of mitigating mechanical and fluid shear stress in large-scale stirred tank bioreactor.