RESUMO
The kinetics of 18 amino acids, ammonia (NH3) and urea (UREA) in 18 liver cell bioreactor runs were analyzed and simulated by a two-compartment model consisting of a system of 42 differential equations. The model parameters, most of them representing enzymatic activities, were identified and their values discussed with respect to the different liver cell bioreactor performance levels. The nitrogen balance based model was used as a tool to quantify the variability of runs and to describe different kinetic patterns of the amino acid metabolism, in particular with respect to glutamate (GLU) and aspartate (ASP).
Assuntos
Aminoácidos/metabolismo , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Hepatócitos/metabolismo , Fígado Artificial , Modelos Biológicos , Engenharia Tecidual/métodos , Células Cultivadas , Simulação por Computador , Humanos , Cinética , Taxa de Depuração MetabólicaRESUMO
Complex medium additives such as yeast extract or peptone are often used in industrial cell culture processes to prolong cell growth and/or to improve product formation. The quality of those supplements is dependent on the preparation method and can differ from lot to lot. To guarantee consistent production these different lots have to be tested prior to use in fermentation processes. Because a detailed qualitative and quantitative analysis of all components of such a complex mixture is a very difficult task, another assessment method has to be chosen. The best way to evaluate the effect of such supplements is to monitor cell activity during real cultivation conditions with and without the added supplement lot. A bioreactor-based test system has been developed to determine the oxygen requirement of the cells as a response to the addition of a supplement to be tested under standardized conditions. Investigations were performed with a mouse-mouse hybridoma cell line and yeast extracts as an example for complex medium additives. The results showed differences in the impact between different extract lots and between different concentrations of an extract.
Assuntos
Automação , Técnicas de Cultura de Células/métodos , Meios de Cultura , Animais , Reatores Biológicos , Hibridomas , CamundongosRESUMO
A Super-Spinner was Modified by mounting a stainless steel filter(pore size 75 microns) to the impeller shaft to retain cells while fresh nutrient is perfused. Using Macroporous microcarrier Cytopore 1, continuously perfused cultivation of a recombinant CHO cell line, CHO2DS producing prothrombin was performed with the perfusion of a protein-free medium DF6S. The cell retention rate was more than 90% during the 24 days continuously perfused cultivation. The viable cell density of CHO2DS and prothrombin concentration reached 4.62 x 10(6)(cells.m/L) and 11.3(mg/L) respectively after 9 days culture.
Assuntos
Protrombina/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Cricetinae , Engenharia Genética , PerfusãoRESUMO
Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 mum spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mumspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 mum was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s(-1) (141 rpm) - furtherenhancement of spinfilter rotation velocity up to0.56 m s(-1) (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s(-1)(35 rpm) and 1.26 m s(-1) (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.