Some calls such as NS0, some hybridoma/ myeloma cells, and hepG2 cells are capable of rapid growth using only basal medium without any further supplement. However, such extraordinary capability is the exception rather than the norm of cells in culture. Most cells in culture require supplementation of at least a number of growth factors and carrier proteins. Many stem cells and other normal diploid cell lines need high concentrations of serum to grow. In fact, for isolation of cells from tissue, serum is still commonly used, at least in the early stage of cell cultivation. In discussing the role of various supplements it is instructive to start by examining the role of animal serum.
Serum is the blood fluid left behind after coagulation; it is free of blood cells and most coagulation proteins. Serum is an extremely complex mixture that contains nutrient substances, metabolites, hormones, plasma proteins, substances released from damaged cells (e.g., hemoglobin and growth factors from platelets), antibody molecules against various antigens to which the animals have been exposed, and may even harbor infectious agents such as viruses carried by the animal. Fetal bovine serum (FBS) is the most widely used serum in animal cell culture because it contains higher concentrations of growth stimulatory factors and lower concentrations of growth inhibitory factors. Other commonly used sera are human, bovine calf, newborn bovine, donor bovine, and donor horse. Serum serves many different and important roles in cell culture. In addition to providing nutrients not sufficiently present in basal medium (e.g., cholesterol), serum provides factors for cell- substrate attachment (e.g., vitronectin, fibronectin) for adherent cells, modulates colloid osmolarity, a physiological property of medium with respect to viscosity. Serum contains protease inhibitors, and neutralizes trypsin used in cell detachment as well as other enzymes released by dead cells. Serum contains the carrier proteins transferrin and serum albumin. Carrier proteins function to chaperone components that are in very low concentration or are otherwise poorly soluble, such as fatty acids (carried by albumin), or are unstable such as ferric ion (carried by transferrin). Serum is rich in “bulk” proteins (e.g., serum albumin) that can prevent non-specific adsorption of critical factors to culture vessels. Serum also plays an important role as a scavenger. In the course of cell cultivation, various contaminants may arise from numerous sources. For example, this could result from leaching of minute chemical components from reactor parts, from the filter used in medium preparation, or even from medium Functions of serum in cell culture medium:
• Protease inhibitors (alpha 2 macroglobin) neutralize proteases used in trypsinization or produced by cells. • Provides hormones and growth factors .
• Provides carrier proteins
• for low molecular weight substances (e .g .,transferrin)
• for nutrients which dissolve poorly (e.g., fatty acids, cholesterol, apolipoprotein)
• Binds compounds which are toxic when present in excessive amounts, and releases slowly
• Binds and/or neutralizes toxic substances (e .g . detergents) .
supplements. Some of these chemicals may be detrimental to cell growth or may have other negative effects on cells. In the presence of serum in the medium, those compounds may be sequestered by adsorption to serum proteins before they can act on cells, thus minimizing potential damage. Animal serum, however, has numerous disadvantages in addition to cost and the difficulty of controlling consistent quality. The most serious concern is the possibility of contamination with animal viruses or prions. The presence of serum in culture medium also makes downstream processing more complicated, and makes the task of final product characterization more complex. Serum carries antibodies, some of which may be against viruses that the animal donors had been exposed to. For virus production processes, if serum antibodies cross-react with the product virus, the production will be drastically affected. Disadvantages of serum in cell culture medium
• potentially introduce animal viruses into cell culture and other undesirable contaminants (e.g. adventitious agents, antibiotics, proteases).
• Availability of high quality serum
• high running costs and unnecessary capital outlay. • Normally purchased in large lot sizes and costly
storage .
• Serum lot testing tedious and costly.
• Increase complexity of downstream processing and final product characterization.
Insulin plays a key role in regulating glucose uptake by many cell types. In addition to modulating glucose metabolism, insulin also exhibits mitogenic effects, and stimulates cell growth through an overlapping pathway with IGF-1. IGF-1 has an acute effect on protein and carbohydrate anabolism by increasing cellular uptake of amino acids and glucose, and by stimulating glycogen and protein synthesis. IGF-1 also affects cell proliferation, differentiation, and apoptosis. It is a potent mitogen acting to increase DNA synthesis and to stimulate the expression of cyclin D in a wide variety of cells. Both insulin and IGF-1 bind to the insulin receptor (IR) and IGF receptor (IGF1R) but with different affinities. After insulin binding, IR or IGF1R is phosphorylated, leading to activation of an insulin receptor substrate (IRS). There are multiple isoforms of IRS that are distributed differently in cells of different tissues. The signal is then relayed to downstream signaling pathways. The response of the cell to insulin and IGF is dependent on the abundance level of the different IRS isoforms. Most cells, including CHO cells, express both IR and IGFR. However, NS0 cells express only IGFR. Differential binding to IR and IGF1R, as well as differential activation of various IRS isoforms leads to different responses to insulin and IGF1. Insulin is used in cell culture at concentrations that are nearly a hundred-fold higher than found in blood. At such high concentrations insulin can trigger a mitogenic response. IGF has a much stronger affinity for IGF1R and stimulates cell growth at much lower concentrations than insulin. • Insulin stimulates glucose uptake by adipocyte
and other cells, also has a mitogenic effect at high concentrations
• Insulin is used in culture at 1 - 10 µg /ml range . Blood insulin level is 4µU/ml or 1 .3 µg / ml (1 µU=0.33 µg) ; IGF1 level is is 100 to 200 ng / mL. • Insulin and IGF have overlapping signaling
pathway through IR and 1GF1R
• IGF1 regulates cell growth, IGF2 invovles in development . IGF1 can replace insulin in cell culture at a much lower concentration
Insulin and Insulin-like growth factor (IGF- 1)
Transferrin is the iron carrier glycoprotein in mammals. Ferric ion is highly oxidative, and exists primarily bound to heme and other proteins with iron centers. In circulation, ferric ion is bound to transferrin. Transferrin has a very high binding constant for iron, but dissociates readily at low pH. Transferrin receptor-bound iron is taken up by cells and translocated to lysosomes, where iron is released for incorporation into cellular proteins. For industrial processing, it is desirable that all growth factors supplemented to culture medium be derived from recombinant DNA expression rather than having been isolated from human or animal sources. Although recombinant insulin and other growth factors have long been available, recombinant transferrin became available only in recent years. The specificity of transferrin binding to cross- species transferrin receptor is not universally high. The recombinant transferrin used commercially is primarily of human origin. The concentration required for cells of different species may differ and must be empirically determined. Transferrin can be replaced by iron chelating agents, including citrate. Most chelating agents have a much lower binding constant for iron than does transferrin, and they are used in higher concentrations than are needed for transferrin.
• Typical concentration: 1 – 30 µg / mL (MW 80kd, 10 µg / mL=0.1 µM)
• 80 kDa glycoprotein with homologous N-terminal and C-terminal iron-binding domains . Binds to iron very strongly with a dissociation constant of approximately 1022M-1 .
• Low interspecific potency; for human and rodent cell lines can be replaced by other iron binding protein (i .e . hemoglobin),
• May be replaced by an iron-chelating agent, such as citrate .
Table 10 . Some Iron Chelators as Transferrin Replacements
Ferric citrate 0 .1 - 0 .5 mM
Ferric iminodiacetic acid 0 .001 µM
Ferric ammonium citrate and
Tropolone 2 - 10 µM
Transferrin
Industrial NS0 cells or other myeloma lines are grown without insulin supplementation, and many CHO cells have been adapted to grow without insulin. It is likely that the signal transduction pathways have been altered downstream of IR or IGFR in those adapted cell lines. Insulin was one of the first recombinant proteins to be made available for therapeutic use. Recombinant insulin has long been used in cell culture. IGF1 is also used in cell culture, often in a commercial form. in cell culture, often in the commercial form.
Anchorage dependent cells are employed in the vaccine industry and in emerging stem cell and cell therapy-based technologies. In this section we will address adhesion of cells only to conventional stationary surface cultivation, not microcarriers. Although stainless steel or chemically modified polymer surfaces are still used, the vast majority of cells are grown on conventional plastic or glass surfaces. Many suspension-adapted cells revert to attachment when grown on adhesive (positively
Cell Adhesion Molecules
The most abundant protein in serum is albumin. Albumin is a versatile molecule and is a carrier for many compounds that may have low solubility in aqueous solution. Most notably, albumin is a carrier for fatty acids, which can be toxic when present in free form at high concentrations. Albumin also binds bilirubin, heavy metal ions, and other agents that may harm cells. Albumin is probably the most important protein that mediates scavenger functions of serum in cell culture medium. Recombinant forms of human albumin are also available. However, unlike other recombinant proteins, which are easily characterized for use in a chemically defined medium, the diverse binding capacity of albumin makes its complete chemical properties harder to define. Not all albumin preparations are the same—albumin from different preparations may be bound to different amounts and varieties of fatty acids or other compounds. In addition to transferrin and serum albumin, serum also contains other carrier proteins as shown in the table. Most are rarely used in culture. Serum Albumin
• used at 0 .1 – 5 mg / mL • High interspecific potency
• Fatty acid composition and content depend on method of preparation and species.
• Most defined medium use fatty acid-free albumin coupled to specific fatty acids, particularly oleic acid or linoleic acid .
Table 11 . Transport and Carrier Proteins
Transport
proteins Source Structure Effects
Serum albumin Plasma 1-chain (MW=68000)
Supplies free fatty acids
Detoxifyer contains trace elements Transferrin Plasma 1-chain (MW=77000) Supplies iron detoxifyer High density lipoprotein (HDL) Plasma Particle (multiple protein subunit) Accepts and transports cholesterol and cholesterol esters Low density lipoprotein
(LDL) Plasma Particle (Apo B)
Transports cholesterol and cholesterol esters
Trenscobalamin Plasma Binds vitamin B12
Ceruloplasmin Plasma 1-chain (MW=
135000) Binds copper
Hemoglobin Red cells 4 subunits (MW
~65000) Transports O2
charged) surfaces. Adhesion can be enhanced in the presence of serum or by coating surfaces with adhesion molecules. For the cultivation of industrial cell lines, there is, in general, no need for coating the surface with adhesion molecules. Adhesion molecules are used to promote adhesion of various stem cells, differentiated cells, or some highly transformed cells that do not attach well to tissue culture flask surfaces. Commonly used adhesion molecules, as shown in the table, include biological molecules (fibronectin, laminin), ECM extract (matrigel, which is rich in laminin), and synthetic molecules (poly-L-lysine or RGD peptide (arg-gly-asp). These adhesion molecules may be used to revert suspension cells to an adherent state for cell cloning. Under adherent conditions, cells form colonies on the surface of culture dishes and can be easily isolated using a cloning ring. In some cases, one might want to prevent cell adhesion to a surface. Inclusion of heparin, heparin sulfate, or Pluronic in the medium, along with use of a non-adhesive surface can minimize cell adhesion.
Table 12 . Adhesion Molecules Used for Cell Culture
Adhesion proteins Source Effects
Fibronectin Plasma, cell lines Promotes attachment growth of mesenchymelly derived cells Laminin Extracellular matrix Promotes attachment and growth of ectodermally and endo-dermally derived cells Collagens (I-IV) Skin, extracellular matrix, placenta Promotes attachment and growth either directly or through the binding of other adhesion proteins
Vitronectin Plasma Promotes attachment and growth of a variety of cell types
Fetuin FBS Promotes attachment of cells to glass and plastic
Poly-d-lysine Synthetic Polymer Promotes attachment of many cell types (even in the presence of serum)
• Cells clumping and cells sticking to vessel surfaces can be partially corrected by adding to medium:
• Pluronic F68 (0 .01 – 0 .1%) • Heparin (10–100 ug / mL)
Protein Hydrolysates Hydrolysates or extracts from animal or plant tissues
were commonly used in cell culture processes to reduce the dependence on serum. A beef extract, Ex-Cyte, was an excellent source of phospholipids; however, the use of animal extracts has been largely discontinued, and has been replaced by hydrolysates from soy, rice, and other plants derived by enzymatic or acid hydrolysis. Lot to lot variation among such extracts is huge. In a transcriptome analysis of CHO cell samples grown under different reactors, pH, and other conditions, it was found that the specific lot of hydrolysate overrode other experimental variables in sample clustering. In a data mining experiment encompassing data from more than 100 manufacturing runs, it was found that hydrolysate lot had a strong correlation with productivity. The roles of hydrolysates are not completely understood. Hydrolysates are complex mixtures of amino acids, peptides, derivatized peptides, carbohydrates, and some lipids. They provide some nutrients and minerals and may also act as scavengers through undefined molecular interactions with possible contaminants. Hydrolysates may have some growth-stimulating or anti-apoptotic activities. Given that synthetic peptides have been found to interfere with signaling pathways by binding to signaling intermediates, the possibility cannot be excluded that some hydrolysates have similar effects.
• Soybean hydrolysate and peptone are commonly used
• Peptones derived from acid or enzyme hydrolysates of casein, gelatin, meat, soy, egg and lactalbumin have been used as supplements in cell culture • Contain a mixture of amino acids, small peptides,
inorganic ions, carbohydrates and vitamins • Possible roles of hydrolysate
• Source of amino acids in the form of oligopeptides
• Some oligopeptides may mimic analogues of signaling molecules by having non-specific binding various cell surface receptors • Such effects may be growth-promoting or
Medium design for industrial cell culture processes encompasses two aspects, cell expansion and production. For cell expansion the focus is on optimal growth and sustained viability; for production, the objective is rapid growth to production cell density, sustained viability, and productivity at high cell density. For the production of non-cell products including recombinant proteins and viruses, the viability of the culture at the end of production may not need to be high; thus an extreme composition that favors production at the expense of viability may be used. In the past decade it has become a common practice to use high osmolarity in the final stage of production culture. Glucose concentrations as high as 15 g/L (83 mM) may be used at the initiation of production culture. At such a high level, glucose contributes significantly to overall osmolarity; thus, the concentration of NaCl is often reduced to compensate for the additional glucose. High osmolarity affects the growth rate of most cells, although a high glucose concentration does not appear to affect growth of some CHO and myeloma cells. As discussed in the section on Fed-Batch cultures, most industrial production is initiated at about 70% of reactor volume, and concentrated nutrient mixtures of amino acids, glucose, etc. are added during cultivation. The concentrated nutrient mixtures usually carry extra salts used to dissolve amino