Cell cultures, process control

As outhned earUer every single step of hematopoiesis is regulated and controlled in vivo by the cell s microenvironment. This not only includes the composition and concentration of growth factors, but also the local oxygen concentration, the pH, the osmolaHty, the supply of nutrients and the cellular and molecular surrounding of the cells (cell-cell contact, adhesion molecules and extracellular matrix). All these parameters affect the fate of the cell and, to estabUsh a cell culture process to cultivate or generate a specific subpopulation, the influence of all these factors has to be considered in the experimental set-up. In the following sections these parameters will be discussed in brief. 

Wagner R (1997), Metabolic control of animal cell culture processes. In Hauser H, Wagner R (Eds), Mammalian Cell Biotechnology in Protein Production, Walter de Gruyter, Berlin, pp. 193-232. 

Pattison RN, Swamy J, Mendenhall B, Hwang C, Frohlich BT (2000), Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor, Biotechnol. Prog. 16 69-74. 

Mammalian cell culture processes must be tightly controlled to attain acceptable cell density and maximize product titer. Slight deviations in pH, temperature, nutrient, or catabolite concentrations can cause irreparable damage to the cells. This section covers the effects of pH, shear stress, catabolite, and carbon dioxide accumulation on cell growth and product formation, and discusses the importance of controlling glucose and glutamine concentrations... 

Quality assurance by means of strict quality control of all aspects of a cell culture process has always been of prime importance, given the sensitivity of cells to sub-optimal medium and environmental factors and the ease with which cells can become contaminated with viruses and other microorganisms. The potential for biological changes in scale-up of cell culture processes demands even greater standardization and testing of the system. 

Konstantinov, K. B., Zhou, W., Golini, R, and Hu, W.-S. (1994) Expert systems in the control of animal cell culture processes Potentials, functions, and perspectives. Cvtotechnol.. 14 233-246... 

The enthusiasm for using Caco-2 cells and other epithelial cell cultures in studies of drug transport processes has been explained by the ease with which new information can be derived from these fairly simple in vitro models [7]. For instance, drug transport studies in Caco-2 cells grown on permeable supports are easy to perform under controlled conditions. This makes it possible to extract information about specific transport processes that would be difficult to obtain in more complex models such as those based on whole tissues from experimental animals. Much of our knowledge about active and passive transport mechanisms in epithelia has therefore been obtained from Caco-2 cells and other epithelial cell cultures [10-15]. This has been possible since Caco-2 cells are unusually well differentiated. In many respects they are therefore functionally similar to the human small intestinal enterocyte, despite the fact that they originate from a human colorectal carcinoma [16, 17]. 

Production Culture medium used, cell culture and fermentation techniques, in-process controls, purification steps, and cleaning of chromatographic columns and matrices... 

Over 300 peptides isolated in our laboratory were studied in one or more tumor or normal cell cultures [39-44]. Part of the results obtained is summarized in Table 2.3. Over 75% of the peptides showed pronounced proliferative or antiproliferative activity in at least one cell type (Fig. 2.3). As a rule, tumor cells are more sensitive to peptide action. Besides the cell type, experimental conditions such as cell density or composition of the culture medium also affected the overall effect. In several cases (13%, Fig. 2.3) even the sign of the effect was peptide concentration dependent. Generally, experiments with cell cultures conform with the view that the main physiological function of cell and tissue peptidomes is control of long term processes and the homeostatic balance (i.e. cell differentiation, proliferation and elimination). The overall effect of peptide pools is achieved by concerted action of total sets of peptides rather than by single components. The molecular mechanisms of peptide action in cells requires concrete study in each individual case and are the subject of current research. 

Three different approaches for the cultivation of isolated hematopoietic cells have been described, the static, the stirred and the immobilized culture. Static cultivation takes place in very simple culture systems like well plates, tissue-culture flasks or gas-permeable culture bags [62, 63]. As the first two systems do not allow cell cultivation on a clinical scale, the latter is actually the most often used technique for stem cell expansion. All these systems have the advantage of being easy to handle, single-use devices, which enable an uncomplicated cell harvest. But all of them do not offer possibilities for process control or continuous feeding. This causes variations in culture conditions during fermentation (e.g., oxygen tension, pH, substrate, metabolite and cytokine concentrations). 

Stirred bioreactors are common in animal cell culture, as they offer a homogenous enviroiunent, representative sampling, better access to process control and an increased oxygen transfer. Several of these techniques (spinner flasks and stirred vessel bioreactors) have been tested successfully for the cultivation of hematopoietic cells [58,64-67]. 

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