Continuous medium perfusion cell culture

New cell culture techniques, which may improve the applicability of renal epithelial cultures, are also required. Currenfly there exist two commercially available cell culture perfusion systems, which allow the continuous perfusion of culture media and optimized oxygenation [243]. These systems allow stable longterm culture of quiescent adherent cells [244]. Continuous medium perfusion furthermore may lead to the re-expression of lost functions in continuous cell hues and the maintenance of differentiated properties in primary cells. Recently our laboratory has demonstrated that LLC-PKj cells maintained in a newly developed perfusion system (EpiFlow ) changed from a glycolytic to a more oxidative phenotype [72]. Evidence is also available from experiments in our laboratory that this mode of cultivation helps to prolong the lifetime of primary cultures of proximal tubular cells. Combining perfusion culture with co-culture of a cell type that is an anatomical neighbour in vivo (e.g. epithelial with endothelial, interstitial or immune cells) may improve the state of differentiation of both partner cells and increase the complexity of autocrine and paracrine interaction [73]. 

Scale and Mode of Operation Scale and also the mode of operation (Figure 1.2, batch, fed-batch, or continuous/perfusion cultivation) have an impact on the design and the interface to the peripheral units. A perfusion cell culture facility may have a relatively small bioreactor vessel but a rather large media preparation area and holding tanks for the perfused medium. 

Culture and differentiation of mESCs in a perfused 3D fibrous matrix has also been reported (Li et al., 2003). In this study, perfusion led to a higher growth rate and final cell density in relation to static conditions. A polyethylene terepthalate (PET) matrix was applied for construction of the scaffold, which provided a larger surface area for adhesion, growth, and reduced contact inhibition. A bioprocess for efficient ESC-derived cardiomyocyte production was also developed (Bauwens et al., 2005). This system was capable of monitoring and control oxygen tension and pH in 500-mL vessels with continuous medium perfusion. Oxygen tension was shown to he a culture parameter that can be manipulated to improve cardiomyocyte yield. 

Similar in principle to chemostat configuration except the cells are retained within the fermenter and roller bottles using inline or external cell separation devices. The hollow fiber filter allows continuous separation of cells from tissue culture fluid containing products. In this configuration, the recombinant product is often designed to be excreted into medium allowing it to be collected in perfusate. 

Figure 1. Time course of medium glucose and lactate concentrations ofLLC-PKI ceiis under conventionai static conditions (A) and with continuous medium renewai (B). (A) LLC-PKi cells were grown to confluence and on 24 well plates, medium was removed from wells every two hours. Two medium replenishment cycles were conducted [r], (B) LLC-PK1 cells were cultured to confluence on micropourus filters and transferred to the EpiFlow perfusion device. Medium perfusion was 2 mi/h. Sampies in the outflowing medium werecoiiectedatreguiarintervais. Giucose and lactate were measured using colorimetric assays. Results represent the mean SEM from 3 experiments. For more information see Felder et. al. 2002 [72].

On the laboratory-scale, batch cell culturing is mainly conducted in flasks, dishes or microtiter plates (Fig. 1), however industrial-scale bioreactors with perfusion (continuous culture) are also available for production of, e.g. antibodies and other proteins. In both laboratory- and industrial-scale systems, a large volume of cell medium is available in comparison with the cell volume to provide the cultured cells with sufficient amount of nutrients. 

To attain consistent and stable production of FVIII from CHO cells adapted to culture in a protein-free medium, continuous (chemostat) perfusion is used in the bioreactor culture system. An important advantage of the system is that the culture conditions can be continuously controlled, monitored, and optimized. 

When the cells in the largest bioreactor have reached an optimum density, continuous (chemostat) perfusion culture is begun. Fresh medium is added, and rFVlll-containing conditioned medium is removed at the same rate. This continuous perfusion culture is maintained for several weeks. Monitoring of culture conditions, cell growth, and microbial sterility continues and provides consistency and stability of cell density and expression of rAHF-PFM over time. The conditioned medium is filtered and rFVIII is then purified. 

Recently, our group has developed a structured polymer scaffold which is made of perforated polycarbonate thin film. The scaffold provides a three-dimensional microenvironment to cell culture and is continuously perfused with the nutrient medium [31]. Therefore, polycarbonate chemistry is of particular interest for our applications with a special focus on the mild reaction conditions. Moreover, most of the methods described above are for post modification of the surface of polycarbonate using aggressive reaction conditions. In this chapter we describe a mild and efficient method for the functionalization of polycarbonate using terminal diamines. 

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

Perfusion systems have also been used for successful scale-up of MoAb production. During the culture period, cell growth occurs exponentially until the cell density reaches a maximum. At that point, the medium needs a continuous supplementation of fresh nutrients and elimination of waste. In perfusion systems, fresh nutrients are supplied and wastes are removed continuously so that the medium meets the physiological needs of the cells. At steady state, the cell concentration is determined by space and other limitations. High cell densities have been achieved by immobilizing the cells in porous ceramic matrices or hollow fiber devices. Intermediate cell densities have been achieved by perfusion reactors with a spin filter, or in a fluidized bed reactor in which the cells are embedded in sponge-like... 

Originally cultures were established in Earle s BSS containing 0.5% lactalbumin hydrolysate, 0.1% yeast extract, 0.1% polyvinylpyrrolidone and 2-5% FBS but they can be maintained successfully in Eagle s MEM (EBSS) with added 7.5% bovine serum and 2.5% FBS or Medium 199 supplemented with 5% FBS. Vero cells are adaptable to batch and continuous perfusion culture. 

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