Culture systems high cell density

With cell recycling, chemostat efficiency is improved. To maintain a high cell density the cells in the outlet stream are recycled back to the fermentation vessel. Figure 5.10 represents a chemostat unit with a cell harvesting system. The separation unit is used for harvesting the cells and recycling then to the culture vessel to increase the cell concentration. 

To be useful to both, clinicians and the pharmaceutical industry, a bioartificial liver will need to maintain a large number of hepatocytes at high cell densities and in a fully differentiated state for prolonged periods of time. Development of such a system has been impeded by three principal problems a) a requirement for large numbers of cells (>25 10 ) b) loss of liver-specific functions in cultured cells (primary and immortalized) c) nutrient and waste product gradients in high density cultures leading to lowered cell viability and impaired function. 

For antibodies produced in vitro, as is the case for many other proteins obtained from animal cell cultures, the main question involves the low level of expression of these products in the culture medium. This necessitates the use of large culture volumes for production, thus involving higher costs, especially for purification. In general, the optimization of these in vitro processes attempts to increase the concentration of the product in the medium. This is often possible by using high cell densities. Typical values for traditional processes are in the range of 20-100 mg/L, whereas for optimized systems, this can rise to 4.6 g/L (Kretzmer, 2002 Wurm, 2004). 

Table 18.1 compares the relationship between cell culture surface area and bioreactor volume in many different culture systems usually used with adherent cells. For microcarriers, this coefficient might reach 60 cm2/ml of medium for culture area prepared with 10 mg of microcarriers per milliliter. For Roux bottles, this coefficient is around 3 cm2/ml. In cultures initiated with 2 mg of microcarriers per milliliter of medium, high cell densities of even 3 X 106 cells/ml are often reached, compared with smaller cell densities from 2 to 3 X 105 cells/ml usually observed in Roux bottle systems. Another great advantage of the use of microcarrier culture systems is the possibility of preparing cell cultures with hundreds or even thousands of liters (Montagnon et al., 1984). 

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). 

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... 

The fixed-bed, porous-glass-sphere culture system was designed for the production of secreted cell products and lytic virus. The system is based on the immobilization of cells (anchorage-dependent or suspension) to high cell densities in porous glass spheres (supplied by Schott Glaswerke). 

---