Continuous medium perfusion

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]. 

The Bellco dual overhead drive culture system uses a similar stirring system to the Techne and additionally permits continuous medium perfusion... 

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. 

Liquid concentrate medium has emerged recently as an alternative to powdered medium (33,34). For liquid concentrate preparation, medium components are grouped according to solubility criteria. Liquid medium concentrates allow for the preparation of medium in-line, by automated dilution of the concentrates with water of the appropriate quality (35). This would be particularly useful in continuous or perfused processes that require constant preparation of medium. Medium cost and component stability make it a secondary option for batch or fed-batch processes. 

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].

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. 

The most sophisticated technique for stem cell expansion is the Aastrom-Rephcell system (Aastrom Biosciences Inc., Ann Arbor, Ml, USA), which is an automated clinical system for the onsite expansion of stem cells in cancer therapy. It consists of a grooved perfusion chamber for the retention of the hematopoietic cells, with the medium flow perpendicular to the channel grooves resulting in a continuous supply of fresh nutrients while metabolites are simultaneously removed [47,71,72]. This technique has already been used in a number of clinical studies [73,74]. No incompatibihty of the expanded cells was found,but the expansion of the early progenitor cells was rather low [75]. 

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]. 

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. 

Competing hydrogen donors to horseradish peroxidase must be absent from the sample or perfusion medium, otherwise the decrease in scopoletin fluorescence would not represent the actual rate of hydrogen peroxide formation. A drawback of these assays is that continuous measurement of hydrogen peroxide in neutrophil lysates is not feasible because NADPH interferes with the catalytic action of the peroxidase. 

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