Technical Feasibility Study On Airlift Reactors For Large-scale Porcine Muscle Stem Cell Culture

The socio-technological significance of large-scale animal cell culture technology,where mammalian cells in large quantities are cultivated in reactors to harvest biological products,is becoming increasingly prominent.Immortalized porcine muscle satellite cell is one of the most promising cell types that have the potential for commercial-scale cultured meat production,yet extra-large bioreactor design remains a challenge.In the late 1980s,airlift reactors were once considered to be the most suitable choice for large-scale animal cell culture due to their simple structure,low cost,and uniform shear stress distribution.However,research and development of air-lift reactors in the field of mammalian cell culture quickly halted when it was suspected that the shear stress due to bursting bubbles was more damaging than agitation.By leveraging the recent development in Computational Fluid Dynamics（CFD）technology,this research re-evaluates the technical feasibility of cultivating porcine skeletal muscle stem cells in large-scale air-lift bioreactors for cultured meat production.A 300 m L bench-top bubble column bioreactor was constructed to study the interaction between the gas,liquid and solid（microcarriers）phases to establish a CFD model.The model was then scaled up to 200 m³ and combined with data from cell culture experiments to analyze the large scale air-lift reactors’applicability as a cell culture reactor.The main conclusions are divided into the following four parts:（1）The commercial microcarriers CytodexⅠwas enhanced by coating with Fe³⁺using a technology developed in this study.The Fe³⁺-CytodexⅠmicrocarrier was found to be superior to off-the-shelf microcarrier products and was selected for porcine skeletal muscle satellite cell culture,and a microcarriers-based suspension culture system was established.It was found that in culture flasks bead-to-bead transfer of cells occurred when the liquid phase shear stress was between 0.025-0.18 Pa.In the laminar flow regime,agglomerates of cells and microcarrier were formed.The doubling time of porcine skeletal muscle satellite cells on Fe³⁺-CytodexⅠmicrocarriers was 24 h,which is similar to the doubling time of flat cultured cells.（2）A 300 m L bubble column reactor was constructed for gas,liquid and solid three-phase cold-flow tests and CFD simulations to study the effects of liquid properties（water v.s.culture medium）,gas sparger and other factors on the reactor’s hydrodynamic characteristics and mass transfer performances.When a 50μm sintered sparger was used,the bubbles produced by the cold-flow system were much smaller hence gas holdup much higher than an air-water system,indicating that data obtained from an air-water system should not be used as references for large-scale reactor design.Within the tested range of superficial gas velocity between 0.04-0.17 cm·s^-1,the microcarriers could be fully suspended.The gas holdup was enhanced by the presence of microcarrier with a volumetric fraction between 14%and 20%,without noticeable impact on the size of the bubbles.Antifoam C at 160 ppm could effectively inhibit foam formation.The cell protection agent,Pluronic F68（PF68）significantly enhanced the volumetric mass transfer coefficient k _La,but the microcarrier showed a negative effect on the liquid film mass transfer coefficient k _L,which counteracted the increase in the specific mass transfer area,a,resulting in a k _La similar to that of an air-water system.Under similar operating conditions,a laser-punched gas sparger with 300μm pore size produced bubbles with an average diameter of about 3.2 mm.A Euler-Euler multiphase CFD model shoed good agreement with the quasi-steady-state experimental data,and can be used as a reference for reactor scale-up design.（3）A microscale bubble column reactor was constructed from a 50 m L glass tube.Air with 5%CO₂ was sparged to the micro bubble column by a miniature peristaltic pump,controlled by an Arduino microcontroller.Two different types of spargers were tested to investigate the damages caused by gas bubbles to porcine skeletal muscle satellite cells.Preliminary results showed that the microporous sparger that produced very fine bubbles caused more damage,whilst the cells tolerated the bubbles produced by a straight stainless-steel pipe with 0.7 mm inner diameter,to some extent.Overall,the cells grew poorly in the bubble column reactor compared to surface aerated vessels such as spinner flasks and T flasks,but the exact cause demands further investigation.（4）Using the CFD model validated by the cold-flow experiments,combined with the results from the micro bubble column reactor cell culture tests,a generic tube-in-tube 3-phase air-lift reactor design was modelled.The bubble diameter was set to 3 mm,comparable to those produced by the laser-punched sparger.The aeration rate was 0.11 vvm,typical for animal cell cultures.At a height-to-diameter ratio of about 3:1,this gas flow resulted in a superficial gas velocity of 0.024 m·s^-1 with respect to the entire cross-section of the reactor.Simulation result showed that microcarriers with a volume fraction of 20%could be completely suspended.The average gas holdup of the reactor was 7.8%,and the calculated average mass transfer coefficient was 47 h^-1,high enough for high density cell culture.The average liquid phase shear stress was 10.9 Pa,but with a local maximum up to 70 Pa at the gas-liquid interface,which is too high for cell culture.It is concluded that the generic air-lift reactor design is not recommended for cell culture applications.