This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes.

Dielectric spectroscopy was applied to two industrial high cell density culture processes and used to determine on-line the concentration of CHO cells immobilized on macroporous microcarriers in a stirred bioreactor and in a packed-bed of disk carriers. The cell concentration predicted from the spectroscopic data was in excellent agreement with off-line cell counting data for both processes. Deviations between the two counting methods only occurred in the case of a significant decrease of the cell viability, from 93% to 64%, which induced a change of the average cell size in the culture. Results for the packed-bed process were further confirmed by the application of indirect yield models based on the measurement of glucose, lactate, and the protein of interest. Moreover, dielectric spectroscopy was used as a tool to characterize the packed-bed process. It was possible to determine both the maximum cell concentration that could be reached in the culture system, 2.0 x 10(11) cell per kg of disk carrier, and to quantify the increase of specific protein productivity induced by the production phase, from 5.14 x 10(-8) microg x cell(-1) x h(-1) to 4.24 x 10(-7) microg x cell(-1) x h(-1).

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