Fixed-bed reactors have gained growing attention for the cultivation of mammalian cells. They allow for a low
shear stress cultivation of adherent and non-adherent cells due to the immobilization of cells within macroporous carriers.
Their potential has been demonstrated for many cell culture purposes. Some of the recent developments are presented in
this review, including improved antibody production by hybridoma cells, high performance cultivation of a hepatoblastoma
cell line and cultivation of cells for the production of retroviral vectors. Furthermore, criteria for the selection of
process strategies and scale-up concepts are addressed.
The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.
A novel bioreactor system was constructed to induce extracellular matrix (ECM) synthesis by intervertebral disc (ID) cells due to intermittent hydrostatic pressure. The developed system is completely sterilizable and reusable. It is viable for cultivation, immobilization, and stimulation of various other cell types and tissues especially for cartilage. The custom made lid allows long-run cultivation through semi-continuous operation. Manual interferences and therefore the risk of contamination are reduced. Sampling, medium changing and addition of supplements are easily performed from the connected conditioning vessel, which could be placed in an incubator.
For the present investigations nucleus pulposus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs which were subjected to intermittent hydrostatic pressure. Afterwards the construct was biochemically examined. The proven constituents of ECM were found to be released in dependence of the magnitude and profile of the applied pressure.
Abstract:The ability of commercial ceramic asymmetric ultrafiltration membranes with a cut-off of 10 and 20kDa to purify retroviral pseudotype vectors derived from the murine leukemia virus carrying the HIV-1 envelop protein MLV(HIV-1) was studied. To optimize the filtration process a mathematical model of batch wise vector purification was set up. 745 to 1794ml batches of supernatant containing a maximum of 3.2 x 10 5 colony forming units per ml (cfu/ml) was produced in a 200 ml fixed bed reactor. By cross flow filtration the vector concentration was increased 10-fold with an average recovery of 84.5 ± 4.5 % of the initial infective capacity. Furthermore membrane layer formation and temperature dependent decay of transduction competent vector particles (decay) was included in the mathematical model. A maximal end point titer of 4.1 x 10 6 cfu/ml was predicted by the model and confirmed reasonably well by experimental data. Transmembrane flow of batch filtration was predicted by solving a set of related differential equations. Our modeling allows scale-up of the process and prediction of process performance including specific issues such as vector degradation.
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