Preliminary Construction And Evaluation Of A Choanoid Fluidized Bed Bioreactor Using Microcapsules In Vitro

BackgroudSevere hepatitis is a critically ill disease, which is still a worldwide challenge tophysicians. Bioartificial liver (BAL) has been increasingly playing the key role intreatment of severe hepatitis, which can can compensate for the functions of an entireliver. So construction of a BAL has been hot spots of the study on artificial liver.It is well known that a bioreactor is the key device in a BAL. Currently, the hollowfiber bioreactor performs the most application, but it is difficult to magnify and the massdiffuses handicapped by its semipermeable membrane. Also the immunogenicity ofheterologous hepatocytes exists. Based on these, the fluidized-bed bioreactor forcultivated microencapsulated hepatocytes is the optimal selection.About 10¹⁰ hepatocytes are required in a typical bioartificial liver-assisted device.Such a large quantity of hepatocytes, it is very difficult to abtain in conventionalmonolayer culture. Moreover, immunological rejection is also to be solved.Encapsulated biomicapsule has solved the problem. Selective permeability permitsnutrients and micromolecules secreted from hepatocytes to traverse membrane freely,but the macromolecules such as immunoglobulin are isolated by the microcapsules.Presently, microencapsulation has been used for the research on BAL extensively.Spinner cultivation of microencapsulated hepatocytes takes the advantages ofmicrocapsule and spinner Cultivation. Microencapsulation is advantageous tometabolism, high-density culture rapid growth, sythesis, and secretion. Currently, thecultivation of encapsulated hepatocytes in spinners has scarcely been demonstrated. Fluidized-bed bioreactor is a proper circulation device for encapsulatedhepatocytes. In fixed bed, the beads were unable to withstand high stresses caused byhigh perfusion velocity. The fluid often inclines to mobile by preferential channels inthe space devoid of beads. In view of the traditional fluidized bed bioreactors, most ofthem are cylindrical configuration, they still existed the ununiformity of the fluidizationand marginal effect, which led to void volum or dead space. Based on these, wedeveloped a choanoid bioreactor configuration allowing the fluidization of AC beads.This new type fluidized-bed bioreactor was designed to improve the radial mass transferand the mix efficiency so to be convenient for scale-up.In this study, we established the scaling-up encapsulated hepatocytes with spinnercultivation; also constructed a new type of choanoid bioreactor and studied itsconfiguration parameters; The systematic evalution of our choanoid bioreactor byculture media and abandoned plasma of plasma exchange was given in our research.Partâ… In vitro large-scale cultivation and evaluation ofmicroencapsulated immortalized human hepatocytes in spinnersObjective: To develop a large-scale and high-production AC microcapsule spinnerculturefor encapsulation of immortalized human hepatocytes. In this study, the efficacyof encapsulated cells with spinner cultivation was evaluated in vitro.Methods: Microencapsulted immortalized human hepatocytes with high producibilityusing a single-stage procedure grew in large-scale spinner-culture system, free cellscultured in spinners served as controls. The mechanical stability, permeability, andimmuno-isolation of AC microcapsules were investigated, and the growth, metabolismand function of encapsulated cells were also evaluated.Results: The mechanical stability of the microcapsules to withstand the shear stressinduced by high agitation rate was well presented; the microcapsules were not onlypermeable to small molecules up to albumin, but prevented the release of immunoglobulins. AC microcapsules were advantageous to improve the growth,metabolism, and functions (albumin synthesis, ammonia elimination and lidocaineclearance) of immortalized human hepatocytes during different periods of spinnercultivation compared with that of free hepatocytes of spinners.Conclusions: We developed a large-scale and high-production AC microcapsulespinner- culture system for the encapsulation of immortalized human hepatocytes. Onestep procedure is a reliable method for microencapsulation; Large-scale spinnercultivatedencapsulated hepatocytes are a promising candidate for use in future BAL.Partâ…¡Development of a novel choanoid fluidized bed bioreactor andresearch on its fluidization parameters.Objective: We developed a novel choanoid fluidized bed bioreactor configurationconsistent with hydromechanics, which allowed the fluidization of AC beads in whichcells can be immobilized. In the study, we also researched on the appropriatefluidization parameters of this bioreactor for BAL.Materials and methods: A novel type choanoid bioreactor was designed with a certainheight and coning, and the configuration parameters were calculated. We also observedthe changes of fluidized bed from the initial fluidization till to the optimal fluidization.We performed the determination of the gas holdup, apparent rate, and height of theoptimal fluidization. The intact rate of the microcapsules was also observed.Results: The designed choanoid bioreactor is 550 milliliters volume with effectiveheight of 25 centimeters. Process of fluidization is mainly categorized as: fixed bed,fluidized bed, circulating fluidized bed, fully circulating fluidized bed. The gas holdupof this fluidized bed increases accompanied by the solid holdup. The gas holdup reached the maxium of 1.4 percent with 300 gram per liter. So when we took 300 gramper liter solid holdup, optimal fluidization required by pump speed was 85 milliliter perminute and thus the height of optimal fluidization was 23 centimeters. Takingcontinuous fluidzation of 8 hours, the ratio of intact capsules was more than 97 percent.Conclusion: Fluidized bed bioreactor is eligible for artificial liver based onmicroencapsulation, the fluidzation parameters directly infuenced the fluidization effect.So exploring these parameters, it would instructive to microcapsule fluidized bedbioreactor applicable for the research on BAL.Partâ…¢Preliminary evaluation of the novel choanoid fluidized bedbioreactor (CFBB) in vitroObjective: To investigate the fluidization effect of CFBB on microencapsulatedhepatocyte metabolism through cultural media circulation. The exchanged plasmaserved as the plasma model which was used for evaluation of the newly constructedbioreactor. To observe the influence of the encapsulated hepatocyte metabolism infludized bed on the severe hepatitis plasma, and also to observe the interaction betweensevere hepatitis plasma and microencapsulated hepatocytes.Materials and methods: The 2nd week cultural hepatocytes (total numbers 5×10⁹) putinto the fluidized bed bioreactor and DMEM circulated through the bioreactor for 12hours, the 200ml same proportional encapsulated hepatocytes (control, total numers 1×10⁹) cultivated under static conditions in spinner flask during 12h. The samples werecollected for measurement of alanine aminotransferase (ALT), lactate dehydrogenase(LDH), and albumin. The exchanged plasma of severe hepatitis patients was used as theplasma model. 5×10⁹ encapsulated hepatocytes (the viability was above 90%) werecirculated in fluidized bed bioreactor through the half-diluted plasma(1000ml) for 6h, and 1×10⁹ encapsulated hepatocytes were cultured under static conditions for 6h ascontrol. The samples were collected for measurement of ALT, total total bilirubin (TBi),direct bilirubin (DBi), and albumin before and after the experiments. Also, viability ofmicroencapsulated hepatocytes was measured and the microstructure was observed.Results: The level of ALT rose in the 12h circulation of extracorporeal medium. Thestatic culture group rose obviously (H4, p<0.01; H6, p<0.05; H12, p<0.01). The releaseof LDH was higher in static group than that in fluidized bed group after 6h (H6, p<0.05;H12, p<0.01). Whereas, although the albumin synthesis increased in both groups,albumin secretion by fluidized bed was higher than that in static group (H6, p<0.01;H12, p<0.05). The viablity of hepatocytes decreased from 93.5±3.2% to 90.6±6.5% influidized bed compared with that 93.9±4.4% to 86.8±3.8% in static group(p>0.05).In both groups, the level of ALT rose in the severe hepatitis plasma within 6h. Thestatic group rose obviously (p<0.05). After circulation, TBi concentration decreasedobviously in fluidized bed group (p<0.05), there were no obvious changes of TBiconcentration after 6h static culture in the plasma. The albumin concentration was notsignificant difference in both groups after 6h. Although the hepatocyte viablity in bothgroups decreased, the static group decreased obviously (p<0.05). Scanning electronmicroscope showed that cells in static group were damaged with vacuole-likearchitecture. The construction of encapsulated cells in fluidized bed was almost nomal.Conclusions: The newly designed fluidized bed bioreactor facilitated encapsulated cellskeep their viability and function in vitro; The BAL based on a fluidized bed bioreactorwith microencapsulated hepatocytes appeared to be effective in treatment of severehepatitis plasma, with protection of hepatocytes and bilirubin elimination. The efficacyof this novel bioreactor seemed to be promising for human clinical care.