Animal Cell Bioreactor Key Technology Research And Structure Optimization

Newtonian mechanics has ruled the world for hundreds of years;biotechnology will become a pivotal force in leading the development of scienceand technology in the21st century. And bioreactor is the key device for human toproduce and develop the biologic target products. China has many problems in thedevelopment of bioreactor, such as less investment in basic theoretical research,cognitive deficiency on cells’ shear sensitivity semi-empirical method design, lowefficiency reactor, foreign low-end products imitation, etc. CHO cells, also knownas Chinese hamster ovary cells, are one of the most widely used cells in the animalcell engineering. For these problems, the dissertation studied the mechanicalcharacters of CHO cells from the basis; simulation optimized bioreactor’s stirrertank structure, temperature maintenance and feeding mode. The bubble distributoris critical as the supply of oxygen in the bioreactor device, however, the generatedbubble diameter is hard to measure and the high-speed shear can damage cells inthe process of rupture. Thus, these problems can be solved well through simulationand experiments. The dissertation put forward a new method--Shear RateParameter Method (SRPM). It uses existing equipment to verify and evaluate thereactor’s newly developed shear environment in the precondition of non-manufacturing mockup. The SRPM is simple and reliable; it not only shortens thedevelopment cycle, but saves cost.In order to provide the fundamental basis for bioreactor design, it appliesatomic force microscope to have imaging and mechanical measurement on CHOcells. The measured modulus of elasticity of physiological state CHO cells is5.278±0.395kPa. With the numerical calculation and cell culture experiments, toanalyze the force environment of CHO cells in the bioreactor. It shows that theCHO cells can endure the reasonable shear force is approximately0.392Pa,meanwhile, the shearing rate of flow field is391.41(1/s), the maximumdisplacement of U-direction for20μm cell under such shear force is12.9μm. Thus,it offers the basis for the problems solving of fluid shear in the bioreactor design.The homogenization of mixed flow fields and the stirring shear force inbioreactor are the two key factors in process of the cell scale culture. Based on the CH cells’ enduring reasonable shear force in the condition of reducing flow filedshear force and satisfying mixed effect, the flow field effect of optimizing structureis the best, when bioreactor adopts the Elephant Ear stirrer with45oblade includedangle by downdraft installation, the tank high-diameter ratio H/T=1, the radium ofthe tank bottom, the installation height of stirrer C/H=1/3and the number of baffleis3pairs. Temperature in the cell culture process requires strict control. It buildsthe bioreactor’s simulation model of temperature field aiming to have comparativesimulation analyzing for the three heating structure. It indicates that when theoverall wall heating, the fluid within the reactor can be faster to reach temperatureequilibrium. The bioreactor feeding position has an obvious influence on materialmixed effect. It can use tracer method to find better feeding position of the reactoras well as to shorten the mixing time.The bubble distributor as the oxygen supply device of the reactor used togenerate bubble. In process of the cell culture, bubble at reactor orifices from risingto bursting is concerned with three parameters. Study supports, within a certainrange the relation between bubble’s average diameter and orifice ventilation speed,surface coefficient of tension and orifice diameter. It analyzes from the quantitativeangle, the produced in the process of bursting with bubble diameters from4mm-10mm varies from0.97-1.91Pa. It is clear that cell damage happens in the processin terms of the maximum shear force the CHO cell enduring is about0.392Pa.However, the smaller the bubble, the higher the gas-liquid surface area ratio and thebetter the dissolved oxygen in the reactor, in order to meet the dissolved oxygenrequirements in the process of cell culture, the bubble diameter should be controlledat about4mm. During the bubble distributor installation, the distance from orificeto bottom of the bioreactor has little effect on gas holdup. And bubble distributorwith larger distribution ring can balance gas holdup better. Moreover, largedistribution ring ventilation structure can effectively prevent the gas overflowingfrom the area of stirrer raise directly up to the fluid level. It decreases gas holdup;thus, try to choose the large bubble distributor as required in practical application.One of the key problems needs to be solved in bioreactor design andmanufacture is the damage caused by shear force to cell in the process of flowfields mixed. It is hard to measure and verify the problem at the early stage of thebioreactor’s research and development It normally uses mockup to experiment Anew verification method is developed, namely, shear rate parameter method. It can solve the problem through experiments and simulation based on the existingequipments. The dissertation presented a number of techniques, designed reactorexperiment system and verified the SRPM by it. When adopt upper ventilation androtate speed is180r/min, the increment of cell density is the maximum, meanwhile,the average shear rate in main flow field is14.5. It fits the appropriate cell growthshear rate range from11.6-17.4(1/s) presented in the SRPM. The stirrer, tankstructure, heating insulation device in the experiment system all use theoptimization results. It can get better culture effect with such structural parameterswhen adopts upper ventilation and rotate speed is180r/min. It also can get betterculture effect when the bubble diameter is4-8mm, the rotate speed is80r/min andventilate down. The experiment system and commercialization bioreactor havesimilar performance.The dissertation studied the key technology of CHO cell bioreactor; the majorfindings including the mechanical characters of CHO cell, bioreactor structure andthe effect of flow field in bioreactor, bubble distributor structure and the process ofits generation to rupture, verification methods for SRPM in bioreactor, etc, whichlaid a foundation for the practicality of CHO cell scale culture bioreactor.