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Part A Animal Cell Biotechnology
Part A
Animal Cell Biotechnology



Chapter 1
Chapter 1

Culture Media


The artificial environment created in the laboratory is generally known as media. A media comprises an appropriate source of energy for the cells which they can easily utilize and compounds which regulate the cell cycle. The choice of media is cell type specific and often empirical and there is no “all purpose” medium. It should provide many nutrients, buffering capacity, isotonic, and should be sterile. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient formulations. A wider range of ingredients needed to support survival and proliferation or differentiation. In vitro animal cell cultivation requires a complex combination of nutrients, considering glucose and glutamine as main carbon, energy and nitrogen sources. Mineral salts, amino acids and vitamins are also required; other essential nutrients, like growth factors, hormones, and receptor and transport proteins are required in small quantities as well. The pH was maintained at 7.4 and often it includes pH indicator phenol red (red at 7.4, yellow at 6.5, purple at 7.8). A typical media may or may not comprise of serum. The latter is called a serum-free media. Some of the common sources of serum can be fetal bovine serum, equine serum, calf serum etc. both the types of media have their own set of advantages and disadvantages. The culture media is prepared in such a way that it provides

1. The optimum conditions of factors like pH, osmotic pressure, etc.

2. It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions. Glucose is added as energy source—its concentration varying depending on the requirement. Phenol Red is added as a pH indicator of the medium.

Basic Components in the Culture Media

Most animal cell culture media are generally having following 10 basic components and they are as follows:

1. Energy sources: Glucose, Fructose, Amino acids

2. Nitrogen sources: Amino acids



3. Vitamins: Generally water soluble vitamines B & C

4. Inorganic salts: Na + , K + , Ca 2+ , Mg 2+

5. Fat and Fat soluble components: Fatty acids, cholesterols

6. Nucleic acid precursors

7. Antibiotics

8. Growth factors and hormones

9. pH and bufferig systems

10. Oxygen and CO 2 concentration.

Animal cell culture media vary in their complexity but most contain:

Amino acids

0.1-0.2 mM


ca. 1 µM


NaCl 150 mM


KCl 4-6 mM CaCl 1 mM 5-10 mM


A cell culture medium is composed of a number of ingredients and these ingredients vary from one

culture medium to another. The nutrient media used for culture of animal cells and tissues must be able

to support their survival as well as growth, i.e., must provide nutritional, hormonal and stromal factors

(See Chapter 4). The various types of media used for tissue culture may be grouped into two broad categories:

1. Natural media

2. Artificial media.

The choice of medium depends mainly on the type of cells to be cultured (normal, immortalized or transformed), and the objective of culture (growth, survival, differentiation, production of desired proteins). Nontransformed or normal cells (finite life span) and primary cultures from healthy tissues require defined quantities of proteins, growth factors and hormones even in the best media developed so far. But immortalized cells (spontaneously or by transfection with viral sequences) produce most of these factors, but may still need some of the growth factors present in the serum. In contrast, transformed cells (autonomous growth control and malignant properties) synthesize their own growth factors; in fact, addition of growth factors may even be detrimental in such cases. But even these cultures may require factors like insulin, transferrin, silenite, lipids, etc.

Natural Media

These media consist solely of naturally occurring biological fluids and are of the following three types:

1. Cagula or clots

2. Biological fluids

3. Tissue extracts.

The natural biological fluids are generally used for organ culture. For cell cultures, artificial media with or without serum are used.




The most commonly used clots are plasma clots, which have been in use for a long time. Plasma is now commercially available either in liquid or lyophilized state. It may also be prepared in the laboratory, usually from the blood of male fowl, but blood clotting must be avoided during the preparation.

Biological Fluids

Of the various biological fluids used as culture medium, serum is the most widely used. Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum may be obtained from adult human blood, placental cord blood, horse blood or calf blood (foetal calf serum, newborn calf serum, and calf serum); of these foetal calf serum is the most commonly used. Serum is the liquid exuded from coagulating blood. Different preparations of serum differ in their properties; they have to be tested for sterility and toxicity before use.

Tissue Extracts

Tissue or organ extracts and/or hydrolysates (e.g., bovine pituitary extract (BPE), bovine brain extract, chick embryo extract and bovine embryo extract), and animal-derived lipids and fatty acids, peptones, Excyte, sterols (e.g., cholesterol) and lipoproteins (e.g., high-density and low-density lipoproteins (HDLs and LDLs, respectively) are used in culturing of animal cells. Tissue extracts for example, Embryo extracts—Other biological fluids used as natural media include amniotic fluids, ascetic and pleural fluids, aqueous humour (from eye), serum ultra filtrate, insect haemolymph etc. Chick embryo extract is the most commonly used tissue extract, but bovine embryo extract is also used. Other tissue extracts that have been used are spleen, liver, bone marrow, etc. extracts. Tissue extracts can often be substituted by a mixture of amino acids and certain other organic compounds. Embryo extract is the most commonly used tissue extract. It is a crude homogenate of a l0-day chick embryo clarified by centrifugation. Cohn (1966) fractionated crude extract to give high and low molecular weight fractions. The low molecular weight fraction promoted cell proliferation, while the high molecular weight fraction promoted differentiation of pigment and cartilage cells. These fractions were not fully charac- terized, but recent evidences suggest that the low molecular weight fraction probably contained pep- tide growth factors and high molecular weight fraction contained proteoglycans and other matrix constituents. Smith and Schroedl (1992) showed that the embryo extract could be replaced by hemin in the induction of skeletal muscle differentiation.

Artificial Media

Different artificial media have been devised to serve one of the following purposes:

1. Immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate),

2. Prolonged survival (a balanced salt solution supplemented with serum, or with suitable formu- lation of organic compounds),

3. Indefinite growth

4. Specialized functions.



The various artificial media developed for cell cultures may be grouped into the following four classes:

(i) Serum containing media

(ii) Serum free media

(iii) Chemically defined media

(iv) Protein free media.


Liquid yellowish, clear content left over after fibrin and cells are removed from the blood is known as serum. Calf (bovine), foetal bovine, or horse are used, in some cases human. Fetal bovine serum (FBS) (10-20% v/v) is the most commonly applied supplement in animal cell culture media. Normal growth media often contain 2-10% of serum. These supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors, protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; and protect cells from physical stress and damage. Thus, serum and/or animal extracts (Table 1.1) are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells. The role for all constituents (more than 200) is not clear proteins, peptides, special factors released during platelet aggregation e.g., PDGF, TGF-β, lipids, lipid transport proteins, carbohydrates, micronutrients such as minerals, etc.

Table 1.1: Serum Contents


Concentration Range

Proteins and Polypeptides Albumin Fetuin Fibroncetin Globulins Protease inhibitors (α1-anti-trypsin) Transferrin Amino Acids Lipids Cholesterol Fatty acids Linoleic acids Phospholipids Carbohydrate Glucose Hexosamine Lactic acid Pyruvic acid

40-80 mg/ml

20-50 mg/ml

10-20 mg/ml

1-10 µg/ml

1-15 µg/ml

0.5-2.5 mg/ml

2-4 mg/ml

0.01-1.0 µM

2-10 mg/ml

10 µM

0.1–1.0 µM

0.01-0.1 µM

l0.7-3.0 mg/ml

1.0-2.0 mg/ml

0.6-1.2 mg/ml

0.6-1.2 mg/ml

0.5-2.0 mg/m

2-10 µg/ml





Concentration Range

Polyamines Putrescine, Spermidine Urea Inorganics Ca, Cl, Fe, K, P, Se, Na, In Hormones Hydrocortisone Insulin Triiodothyronine Thyroxine Vitamins Vitamin A Folate

0.1-1.0 µM

170-300 µg/ml

0.14-0.16 M

0.1-200 nM 10-200 nM 1-100 ng/ml 20 nM 100 nM 10 ng-10 µg/ml 10-100 ng/ml 5-2 ng/ml

Functions of Serum in the Culture Medium



provides the basic nutrients for cells; the nutrients are present both in the solution as well as are

bound to the proteins.



provides several hormones, e.g., insulin, which is essential for growth of nearly all cells in

culture, cortisone, testosterone, prostaglandin, etc.



contains several growth factors, e.g., platelet derived growth factor (PDGF), transforming

growth factor β (TGF-β), epidermal growth factor, etc.; these are present in concentrations of µg/l.

Both hormones and growth factors are involved in growth promotion and specialized cell function.


given hormone or growth factor may stimulate growth of one cell type, may have no effect on

another and may even be inhibitory to some others. For example, PDGF induces proliferation in fibroblasts, but induces differentiation of some types of epithelia.

Further, proliferation of a single cell type may be induced by more than one growth factor, e.g., fibroblasts respond to PDGF, epidermal growth factor, fibroblast growth factor and somatomidins.



major role of serum is to supply proteins, e.g., fibronectin, which promote attachment of cells to

the substrate. It also provides spreading factors that help the cells to spread out before they can

begin to divide. Although cells do produce these factors, but trypsinized cells are usually unable to attach to the substrate.



provides several binding proteins, e.g., albumin, transferrin, etc., which carry other molecules

into the cell. For example, albumin carries into cells lipids, vitamins, hormones, etc. Transferrin

usually carries Fe in a nonbasic form, but binding of transferrin to its receptor in cell membrane is believed to be mitogenic.



increases the viscosity of medium and thereby, protects cells from mechanical damages, e.g.,

shear forces during agitation of suspension cultures.


Protease inhibitors present in the serum protect cells, especially trypsinised cells, from proteolysis.


The serum also provides minerals, like Na + , K + , Fe 2+ , Zn 2+ , etc.



also acts as a buffer.



Unfortunately, the use of serum or animal extracts in tissue culture applications has several drawbacks. For example, the chemical composition of these supplements may vary between lots, even from a single manufacturer. The supplements of animal or human origin may also be contaminated with infectious agents (e.g., mycoplasma and viruses) which can seriously undermine the health of the cultured cells when these contaminated supplements are used in cell culture media formulations and may pose a health risk in cell therapy and other clinical applications. A major fear is the presence of prions causing spongiform encephalopathy in humans or animals. Cell surface chemistry, which is a critical portion of the in vitro microenvironment for many cell types, can be adversely modified via adsorption or incorporation of serum or extract proteins. The use of undefined components such as serum or animal extracts also prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells, thus eliminating the ability to study, in a controlled way, the effect of specific growth factors or nutrients on cell growth and differentiation in culture. Moreover, undefined supplements prevent the researcher from studying aberrant growth and differentiation and the disease- related changes in cultured cells. Using cell culture media in the industrial production of biological substances, serum and animal extract supplementation of culture media can also complicate and increase the costs of the purification of the desired substances from the culture media due to nonspecific co- purification of serum or extract proteins.


To overcome these drawbacks of the use of serum or animal extracts, a number of serum-free media have been developed. These media, which often are specifically formulated to support the culture of a single cell type, incorporate defined quantities of purified growth factors, lipoproteins and other pro- teins usually provided by the serum or extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known, these media are generally referred to as “defined culture media” and often as “serum-free media” or “SFM.” A number of SFM formulations are com- mercially available, such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, fibroblasts, neurons, lymphocytes, chondrocytes or hepatocytes which are available from Life Technologies, Inc. (Rockville, Md.). SFM generally provide several distinct advantages to the user. For example, the use of SFM facilitates the investigation of the effects of a specific growth factor or other medium component on cellular physiology, which may be masked when the cells are cultivated in serum- or extract-contain- ing media. In addition, SFM typically contain much lower quantities of protein (indeed, SFM are often termed “low protein media”) than those containing serum or extracts, rendering purification of biologi- cal substances produced by cells cultured in SFM far simpler and more cost-effective. Some extremely simple SFM, which consist essentially of vitamins, amino acids, organic and inorganic salts and buffers have been used for cell culture. Such media (often called “basal media”), however, are usually seriously deficient in the nutritional content required by most animal cells. Accordingly, most SFM incorporate into the basal media additional components to make the media more nutritionally complex, but to maintain the serum-free and low protein content of the media. Examples of such components include serum albumin from bovine (BSA) or human serum albumin (HSA), animal-derived lipids such as human Excyte, sterols, etc., and certain growth factors or hormones derived from natural (animal) or recombinant sources. The use of such animal-derived supplements in cell culture media, however, also has certain drawbacks. For example, there is a risk that the culture medium and/or products purified from it may be immunogenic, particularly if the supplements are derived from an animal different from the source



of the cells to be cultured. Thus, if biological substances to be used as therapeutics are purified from such culture media, certain amounts of these immunogenic proteins or peptides may be co-purified and may induce an immunological reaction, up to and including anaphylaxis, in an animal receiving such therapeutics. To overcome this potential problem, supplements derived from the same species as the cells to be cultured may be used. For example, culture of human cells may be facilitated using HSA as a supple- ment, while media for the culture of bovine cells would instead use BSA. This approach, however, runs the risks of introducing contaminants and adventitious pathogens into the culture medium (such as HIV or Hepatitis B virus from HSA preparations, or Bovine Spongiform Encephalopathy virus from BSA preparations), which can obviously negatively impact the use of such media in the preparation of animal and human therapeutics. In fact, for such safety reasons, the biotechnology industry and gov- ernment agencies are increasingly regulating, discouraging and even forbidding the use of cell culture media containing animal-derived products which may contain such pathogens.

Non-animal Peptide Supplements

To overcome the limitations of the use of animal proteins in SFM, several attempts have been made to construct animal cell culture media that are completely free of animal proteins. For example, some culture media have incorporated extracts of yeast cells into the basal medium to provide sources of nitrogen and other essential nutrients. In another approach, hydrolysates of wheat gluten have been used, with or without addition of yeast extract, to promote in vitro growth of animal cells. Still other media have been developed in which serum is replaced by enzymatic digests of meat, or of proteins such as α-lactalbumin or casein (e.g., peptone), which have been traditionally used in bacterial culture. None of these approaches, however, provided a culture medium optimal for the cultivation of a variety of animal cells. In fact, the approach using wheat peptides is likely to be quite unfavorable for culture of many animal cells and tissues, since wheat peptides are known to be toxic or to induce toxic effects in vitro and in vivo, particularly in the cells and tissues of the gastrointestinal systems of some mam- mals, including humans. Moreover, extracts from certain plants, including wheat, barley, rye and oats have been shown to inhibit protein synthesis in cell-free systems derived from animal cells, suggesting that the use of peptides derived from these plants in cell culture media may actually inhibit, rather than stimulate, the growth of animal cells in vitro. Thus, there remains a need for a serum-free, low-protein culture medium suitable for cultivation of animal cells, which is completely devoid of animal or human proteins. Such a medium formulation will facilitate studies of the effects of growth factors and other stimuli on cellular physiology, will allow easier and more cost-effective purification of biological substances produced by cultured animal cells in the biotechnology industry, and most importantly will eliminate the risk of the introduction of adventitious animal and human pathogens. Chemically Defined Media: These media contain contamination free ultra pure inorganic and organic constituents, and may contain pure protein additives, like insulin, epidermal growth factor, etc. that have been produced in bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, fatty acids and specific amino acids. The CHO cell lines are widely used for being highly stable expression systems for heterologous genes (those from a different organism), and for its rela- tively simple adaptation to adherence-independent growth in serum and protein free media. Protein-Free Media: In contrast, protein free media do not contain any protein; they only contain non-protein constituents necessary for culture of the cells. The formulations MEM, DME, RPMI-1640, etc. are protein free; where required, protein supplementation is provided. Compared with serum-



supplemented media, use of protein-free media promotes superior cell growth and protein expression

and facilitates downstream purification of the expressed product. The protein-free, chemically defined formulation allows the growth of many cell lines but at the same time this becomes lipid dependence, such as NSO myeloma cells without further lipid supplementation does not grow in protein-free, chemically defined medium. Through the use of cyclodextrin-based additives to medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal- derived components if the supplement is made with plant-derived or synthetic lipids.

A chemically-defined protein-free medium called FMX-Turbodoma has been improved for the

production of monoclonal IgA antibodies by hybridoma cells, using a systematic method by Stoll et al. in 1996 for IgA production and the price of the protein-free medium is about 20% of the serum- containing medium. This makes such a protein-free medium very convenient for laboratory and large- scale production. A recombinant CHO cell clone which has been cultivated in serum- and protein-free medium for at least 40 generations and which expresses a recombinant product. When adapting cells initially grown under serum-containing conditions to protein-free medium, it has repeatedly been found that the yield of expressed protein and the productivity of recombinant CHO cells greatly drops after adaptation in protein-free medium as compared to serum-containing conditions. This is the con- sequence of an instability or reduced growth of the recombinant clones due to the changed culturing conditions. Despite the use of a stable original clone, on account of the altered fermentation condi-

tions, repeatedly a large portion of the cells become cells with reduced expression or also non-produc- ers, which overgrow product producers during the production process, whereby the fermenter culture finally largely consists of non-producers or of such cells having a low expression.

A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foun-

dation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been re- cently developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The periph- eral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic make- up and biochemical environmental history of the individual at the time the cells were formed. Exami- nation of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [ 3 H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte prolif- eration. Many researchers are now produced stable recombinant cell clones which contain the encoding sequence for a recombinant blood factor, such as factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C, an activated form of any one of these factors, and which are capable of stable expression of this factor over several generations.


The animal cell cultures are used for a diverse range of research and development. These areas are:

(a) Production of antiviral vaccines, which requires the standardization of cell lines for the multi- plication and assay of viruses.




Cancer research, which requires the study of uncontrolled cell division in cultures.


Cell fusion techniques.


Genetic manipulation, which is easy to carry out in cells or organ cultures.


Production of monoclonal antibodies requires cell lines in culture.

(f ) Production of pharmaceutical drugs using cell lines.


Chromosome analysis of cells derived from womb.


Study of the effects of toxins and pollutants using cell lines.


Use of artificial skin.


Study the function of the nerve cells.


Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.



Define culture media.


Explain main components of animal cell culture medium.


Differentiate between natural media and artificial media.


Write a note on different natural components used in culture media.


What do you mean by serum and mention its advantages in using for culture media?


Describe different functions played by serum components in animal cell culture media.


What is meant by serum free media?


Mention advantages of using serum free media in animal cell culture.


Explain the need of protein-free media in animal cell culture.


Outline the applications of animal cell culture system.