Cell culture medium perfusion

Microdialysis is used to collect solutes present in the extracellular fluid via a microdialysis probe with a semipermeable membrane at its tip (see Fig. 14.8). The probe is perfused by a buffer (perfusate), and solutes from the environment surrounding the probe diffuse though the membrane into the perfused solution. Perfusate with the solutes (dialysate) is then collected for ex situ analysis. Microdialysis has been used in vivo to monitor the local concentrations of solutes in the extracellular fluids in a number of different tissues. The first microdialysis experiments were conducted on the brain and blood plasma (Bito et al., 1966). It has since been used to study metabolism in numerous tissues such as brain, muscles, tendons, subcutaneous adipose tissue, lungs, kidneys, and liver (Flock and Kloft, 2005 de la Pena et al., 2000 Jackson, 2005 Siddiqui and Shuaib, 2001). Microdialysis is widely used for pharmacokinetic research (Davies, 1999 de Lange et al., 2000 Verbeeck, 2000) and has also been used to monitor cell metabolites in cell culture medium (Wu et al., 2001). 

The use of hydrocyclones for separating mammalian cells from the culture medium opens the possibility of using them to perform perfusion in bioreactors. As hydrocyclones have no moving parts, they are ideally suited for operation under aseptic conditions as required by the biotechnology industry. 

Butler M, Imamura T, Thomas WG, Thilly M (1983), High yields from microcarrier cultures by medium perfusion, J. Cell. Sci. 61 351-363. 

The aim of most technologies is to generate a population of cells each of which secretes a desired product into the medium. For this to occur the cells do not need to be actively dividing and, in fact, this is sometimes counterproductive. Cells can remain viable at high cell densities using perfusion systems (Chapter 3) and the product purified from the spent medium (Spier, 1988). Furthermore, the simpler the growth medium (i.e. the fewer protein factors required) then the easier is the downstream processing and this is one reason for the development of serum-free media especially for the culture of hybridoma cells used for monoclonal antibody production ( 5.8). 

Fig. 10. PCA plot of a perfusion culture with CHO cells producing recombinant hFVIII. The data points in the plot mirror inoculum of the bioreactor and a phase of batch growth (c), transfer from growth to production medium (D), production phase of rFVIII (demarcated with dotted lines) and the deviation of a bacterial infection of the cell culture (E) (from Bachinger et al.[37], with permission)...

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].

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]. 

Abstract. Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and tissue like stmcturing of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers. 

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. 

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