Anchorage dependent cell lines

Transformed and tumorigenic cells are different from normal cell lines in that they are not usually anchorage dependent. They exhibit a spherical shape, increased life span and lateral diffusivity of membrane proteins, decreased cell receptors and membrane proteins, and a different cytoskeletal structure. The decrease in the concentration of the cell adhesion molecules in the cell membrane of these cells causes the anchorage independence. Transformed cell lines also do not assemble a normal ECM. It is important to note that some cell lines (e.g., lymphocytes) that are normally anchorage dependent can be induced and then adapted to become anchorage independent. This is of tremendous importance to recombinant protein production as discussed later, because the scale-up of suspension cultures is easier than that of anchorage-dependent cell lines. 

As microcarriers provide a good surface area for attachment per unit volume, various types have been routinely used to grow anchorage-dependent cell lines in bioreactors primarily used for suspension cultures. Airlift reactors can also be operated using microcarriers. Wang et al. reported the use of a fluidized-bed... 

Adherent Cell Culture. Adherent or anchorage-dependent cell lines require attachment to a surface for their survival and replication. These cultures are labor... 

Wamock and Al-Rubeai (29) have reviewed bioreactor systems for the production of biopharmaceuticals via culture of animal cells. They describe the advantages and disadvantages of several types of bioreactors and the huge expansion of commercial-scale culture facilities in the early 2000s. Early bioreactor designs for culture of animal cells focused on anchorage-dependent cell lines (see below), but in many cases these bioreactors may also be adapted for the culture of suspended cells. 

Panjehpour M, Karami-Tehrani F (2007) Adenosine modulates cell growth in the human breast cancer cells via adenosine receptors. Oncol Res 16(12) 575—585 Panjehpour M, Karami-Tehrani F (2004) An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines. Int J Biochem Cell Biol 36(8) 1502—1509... 

Another important aspect involved in the selection of transfected lines is the capacity to grow without physical support, since the scale-up of such processes is much simpler than those designed for growth of anchorage-dependent cells. Thus, cells that grow naturally in suspension are preferred, such as myeloma cells (Sp2/0 and NSO), or others that can be easily adapted to this form of cultivation, such as CHO and BHK (Chu and Robinson, 2001). 

Traditionally, the production of mAbs uses complex culture media containing glucose and amino acids as the main sources of carbon for cell metabolism, as well as vitamins, micronutrients and sometimes animal serum, usually fetal bovine serum. Chapter 5 provides a discussion on composition of culture media and recent trends in the search for formulas that do not require the use of animal serum, or of proteins of animal origin. These serum-free formulations use substitutes such as peptones, epithelial and fibroblast growth factors, hydrolysates, yeast extract, choline, and inositol. For the production of mAbs, various serum-free formulas are available, some of these developed specifically for a given cell line (Chu and Robinson, 2001). The development of those media is easier for non-anchorage-dependent cells, such as those used for mAb production. Thus, approximately 50% of the antibodies for therapeutic use are already produced using serum-free media. In some circumstances, the elimination of serum should be accompanied by the addition of other substances with the same shear stress protective effect of serum proteins,... 

To circumvent mass transfer limitations, shear sensitivity, and equipment footprint problems associated with the scale-up of processes involving anchorage-dependent cells, one may resort to use of cell lines that have been adapted for growth in suspension culture. In such cases, stirred-tank reactors akin to those used in the culture of microorganisms can be employed first in batch and subsequently in fed batch culture of animal cells. Wamock and Al-Rubeai (29) provide useful discussions of the relative merits of stirred tanks and several other reactor configurations and modes of operation that have been used in the manufacture of biopharmaceuticals. During the past half-century, especially in... 

A cell line is perpetuated by the subculture technique. For anchorage-dependent cells attached to a substrate, this entails detachment from the substrate by an enzyme (usually 0.25% trypsin) followed by dilution in fresh medium in a 1 2 to 1 20 ratio, depending upon cell type. Cells growing in free suspension are concentrated (by gentle centrifugation) and redistributed into fresh medium. 

Cell Attachment to MAP. The attachment of two cell types, one anchorage-dependent and the other anchorage-independent, was compared over time on Cell-Tak adhesive-coated dishes with the attachment on other commercially available factors. BHK-21 cells normally attach and grow in monolayers. Figure 1A graphically shows the attachment of BHK-21 cells, which attained 70 to 90% efficiency in 20 min to laminin, fibronectin, and Cell-Tak adhesive and 60% to poly-D-lysine. Maximum attachment occurred at a faster rate on Cell-Tak protein, where 90% efficiency was achieved in 12.5 min. The anchorage-independent, or suspension cell line, U-937, achieved 75-85% attachment efficiency on Cell-Tak adhesive (Figure IB) but less than 30% attachment to any of the other factors. 

Human diploid cell line WI-38 is derived from embryonic lung tissue with fibroblast morphology (Hayflick Moorhead, 1961). The cell line has a broad range of virus susceptibility and is used for the production of human virus vaccines rhinoviruses, measles, mumps, rubella, polio, rabies and yellow fever. The cells are anchorage dependent but will form a multilayered culture when held for long periods at 37°C with periodic pH adjustments. The cells have a hfe span of 50 10 population doublings, with a doubling time of 24h. 

Suspension culture is the preferred method for scale-up because it is easier and cheaper, it requires less space, cell growth can be monitored and environmental parameters can be controlled more easily. However, many cell lines have higher specific productivity when attached to the substrate. In the urge to move to suspension systems, the following advantages of anchorage-dependent systems should not be overlooked ... 

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