Perfusion bioreactors

Recently, there has been success in generating cocultures that more faithfully reproduce in vivo metastatic microenvironments. An ex vivo microscale liver perfusion bioreactor was used to assess metastatic seeding, mimicking the salient features of fluid dynamics and functionality of hepatic parenchyma. Invasion and subsequent growth of breast and prostate carcinoma cells were detected by two-photon microscopy of fluorescently labeled cells. Tumors... 

Furthermore, for such application, they would require no maintenance, which would avoid additional risks of contamination and allow a continuous operation of the perfusion bioreactor for several months. 

As a consequence, the perfusion bioreactor can only be operated up to a cell concentration supported by the perfusion rate In this way, spin-filter retention efficiency determines the maximum attainable cell concentration in a given perfusion process. 

The core of double membrane stirrer perfusion bioreactors is a stirrer on which two microporous hollow fiber membranes are mounted, one of them being hydrophobic and used for bubble-free aeration, the second of them being hydrophilic and used for cell-free medium exchange [15]. This system has been reported to provide viable cell densities of 20 million cells per miUiliter for more than two months [106]. Although Lehmann et al. [15] have described the scale-up of this system to the 20-L and 150-L scale, it has been most commonly employed at the bench-scale. 

Perfusion culture (A) typical operational configuration, using an external cell retention device (B) concentration of viable cells and product in a perfusion bioreactor culture of a myeloid transfectoma producing a humanized monoclonal antibody (Center of Molecular Immunology - CIM, Cuba). 

The design of bioreactors for perfusion operation is more sophisticated, which makes the equipment more expensive. However, the productivity increases obtained by perfusion operation allow the use of much more compact systems than those operated under batch or fed-batch mode. In this way, perfusion bioreactors can be up to 10-fold smaller for a given production scale (Bibila and Robinson, 1995), decreasing the costs not only of the bioreactors themselves, but also of storage tanks and downstream processing equipment. 

If a separator, used as a cell retention device in a perfusion bioreactor, is operating with E < 100%, some of the cells are lost in the perfusate. In such a situation, the apparent specific cell growth rate p p is given by Equation (19). 

Consequently, the maximum perfusion rate possible that can be used in a perfusion bioreactor is a function of both the specific cell growth rate and the cell retention efficiency. 

Hydrocyclones are very simple devices and always operate with a flow ratio Rf > 0. They may be easily designed to give a desired separation efficiency (Castilho and Medronho, 2000), and their performance may also be easily predicted (Coelho and Medronho, 2001). In the last few years, it has been shown either theoretically or experimentally that hydrocyclones may be used in animal cell separations (Luebberstedt et al., 2000 Medronho et al., 2005 Elsayed et al., 2006 Pinto et al., 2007) aimed mainly at mammalian cell retention in perfusion bioreactors (Jockwer et al., 2001 Elsayed et al., 2005). 

Figueredo-Cardero A, Chico E, Castilho LR, Medronho RA (in press), CFD study of the fluid and particle dynamics in a spin-filter perfusion bioreactor, In Hauser H (Ed.), Proceedings of the 20th ESACT Meeting (European Society for Animal Cell Technology), Springer, Dordrecht. 

BHK Perfusion bioreactor 2000 Bayer USA, Australia, Europe, Switzerland... 

CHO Perfusion bioreactor 1999 Genetics Institute/Biovitrum AB Europe, USA... 

In the production process of Kogenate , BHK cells are cultivated in perfusion bioreactors up to 500 L in size. The first step of purification involves anion exchange chromatography. After that, an immunoaffinity chromatographic step and gel filtration are carried out, followed by another immunoaffinity and another anion exchange chromatography step (Boedeker, 1992 Bhattacharyya et al., 2003). 

In perfusion bioreactors, supernatant is removed from the bioreactor at certain times, the cells are separated from the supernatant, the supernatant is harvested, and the cells are returned to the bioreactor. Perfusion bioreactors can be operated in a variety of modes. The simplest mode is to consistently remove a certain amount of broth each day (i.e., one bioreactor volume/day) and replace with fresh media. This mode is relatively easy to control. However, as the cell density increases, the required nutrient level may not be met. Also,... 

Dowd, J.E., K.E. Kwok, and J.M. Piret. 2001. Predictive modeling and loose-loop control for perfusion bioreactors. Biochem Eng J 9 1 -9. 

The DON cells were cultured in DMEM/F-12 on Cytodex 1 microcarriers in a 3-1 perfusion bioreactor. 

Fig. 16.4 Annual output using 10 x perfusion bioreactors (x-axis liquid volume of each single bioreactor) with the following assumptions 50 day s perfusion rate at 0.5 L per day overall yield 46.5% inoculation and capture in decoupled work-centers 90% occupation.

FIGURE 26.3 (See color insert following page 40). Modes of bioreactor operation. (A) Batch bioreactor, (B) fed-batch reactor, and (C) perfusion bioreactor. 

Figure 13.11 Schematic representation of generic perfusion bioreactor.

M.E. Gomes, H.L. Holtorf, R.L. Reis, A.G. Mikos, Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor. Tissue Eng. 12 (2006) 801-809. 

L. Fassina, L. Visai, L. Asti, F. Benazzo, P. Speziale, M.C. Tanzi, G. Magenes, Calcified matrix production by S AOS-2 cells inside a polyurethane porous scaffold, using a perfusion bioreactor. Tissue Eng. 11 (5-6) (2005) 685-700. 

Obradovic, B., Bugarski, D., Petakov, M., Jovcic, G., Stojanovic, N., Bugarski, B., and Vunjak-Novakovic, G., Cell support studies aimed for cartilage tissue engineering in perfused bioreactors, in Progress in Advanced Materials Processes, Mater. Sci. Forum, Vol. 453-454, Uskokovic, D.P., Milonjic, S.K., Rakovic D.I., Eds., Trans Tec Publications Ltd., Switzerland, 2004, pp. 549-555. 

Perfusion bioreactors present a highly complex and sophisticated approach for modeling drug-induced hepatotoxicity by offering opportunities to combine a multicellular and 3D hepatic ceU system with structural and perfusion capabilities that better mimic the physiological conditions of the liver (Zeilinger et al., 2011). Human liver cell cultures in 3D formats placed in hollow fiber bioreactors have been... 

Tostoes, R.M., et al.. Human liver cell spheroids in extended perfusion bioreactor cultme for repeated-dose drug testing. Hepatology, 2012. 55(4) p. 1227-36. 

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