Animal cell culture media

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.

Besides meeting the basic nutritional requirement of the cells, the culture medium should also have any necessary growth factors, regulate the pH and osmolality, and provide essential gases (O2 and CO2)

the culture medium consists of amino acids, vitamins, minerals, and carbohydrates.

These allow the cells to build new proteins and other components essential for growth and function as well as providing the energy necessary for metabolism

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

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

Natural Media

These media consist of naturally occurring biological fluids and are of the following three types: (1)coagula or clots,(2)biological fluids and(3)tissue extracts.

The natural biological fluids are generally used for organ culture.

For cell cultures, artificial media with or without serum are used.

Clots 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 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 obtained from coagulating blood. Different preparations of serum differ in their properties; they have to be tested for sterility and toxicity before use.

Tissue extracts

Chick embryo extract is most commonly used

Bovine embryo extract is also used

They can be substituted by a mixture of amino acids and certain other organic compounds

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 formulation of organic compounds),

(3) indefinite growth, and

(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, and

(iv) protein free media.

Serum containing media

Various defined media supplemented with 5-20% serum Serum provides various plasma proteins, peptides, lipids, carbohydrates,

minerals and some enzymes Functions:- Provides basic nutrients for cells; nutrients are present both in the solution

as well as are bound to the proteins Provides hormones- insulin, cortisone, testosterone etc Contains growth factors- PDGF( platelet derived growth factors),

endothelial growth factor etc Supply proteins – fibronectin(promote attachment of cells) Provides spreading factors that help the cells to spread out before they can

begin to divide Provides binding proteins- albumin, transferrin( carry other molecules in to

the cell)

Increases the viscosity of medium and protects cells from mechanical damage

protease inhibitors present in the serum protect cells especially trypsinised cells from proteolysis

Provides several minerals- Na+, K+, Fe2+ etc also act as a buffer DISADVANTAGES Serum may inhibit growth of some cell types – epidermal

keratinocytes May contain some cytotoxic or potentially cytotoxic

constituents ( foetal calf serum contains the enzyme polyamine oxidase which converts polyamines like spermidine and spermine (secreted by fast growing cells)in to cytotoxic polyamino aldehydes)

Large variation in serum quality from one batch to another

Some growth factors may be inadequate for specific cell types & may need supplementation

Interferes with downstream processing when cell cultures are used for production of biochemicals

The supply of serum is always lower than its demand

Serum Free Media -In view of the disadvantages due to serum, extensive investigations have been made to develop serum-free formulations of culture media. These efforts were mainly based on the following three approaches:

(1) analytical approach based on the analysis of serum constituents,

(2) synthetic approach to supplement basal media by various combinations of growth factors, and

(3) limiting factor approach consisting of lowering the serum level in the medium till growth stops and then supplementing the medium with vitamins, amino acids, hormones, etc. till growth resumes

. These approaches have resulted in several elaborate media

formulations in which serum is replaced by a mixture of amino acids, vitamins, several other organic compounds, etc.; hormones, growth factors and other proteins are supplemented when required.

However, addition of 5-20% of serum even in these media is essential for optimum growth.

Advantages of Serum Free Media -

1. Improved reproducibility of results from different laboratories and over time since variation due to batch change of serum is avoided.

2. Easier downstream processing of products from cultured cells.

3. Toxic effects of serum are avoided

4. Bioassays are free from interference due to serum proteins.

5. There is no danger of degradation of sensitive proteins by serum proteases.

6. They permit selective culture of differentiated and producing cell types from the heterogenous cultures.

Disadvantages of Serum Free Media

1. Most serum free media are specific to one cell type. Therefore, different media may be required for different cell lines.

2. Reliable serum free preparations, for most of the media formulations are not available commercially. This necessitates time consuming task of preparing the desired formulations in the laboratory.

3. A greater control of pH, temperature, etc. is necessary as compared to that with serum containing media.

4. Growth rate and the maximum cell density attained are lower than those with serum containing media.

5. Cells tend to become fragile during prolonged agitated cultures unless biopolymers or synthetic polymers are added.

Several defined media have been evolved from the Eagle's minimal essential medium (MEM),

e.g., Dulbecco's enriched modification (DME), Ham's F12, :,pMRL1O66, RPMIl640, McCoy's 5A and Iscove's modified Dulbecco’s' (IMDM); all are commercially

available. Often a 1: 1 mixture of DME and F12 is used as a serum free

formulation.

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.

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, RPM-1640, etc. are protein free; where required, protein supplementation is provided.

Advantages and Disadvantages of Serum Containing MediaAdvantages Disadvantages

1. Provides basic nutrient for cells, nutrients being present in the solution and also bound to proteins.

Inhibits growth of some cell types, e.g. epidermal keratinocytes.

2. Provides several hormones e.g. insulin, testosterone, prostaglandin, etc., which are essential for cell growth

May contain some cytotoxic constituents.

3. Contains several growth factors, e.g. platelet derived growth factor (PDGF).

Serum quality varies from batch to batch which requires costly and time-consuming tests for each batch.

4. Supplies proteins, e.g. fibronectin promoting attachment of cells and spreading.

Some growth factors are inadequate for specific cell types.

5. Provides several binding proteins, e.g. albumin and transferrin, which carry other molecules into the cell.

When cell cultures are used for production of biochemicals, the serum interferes with downstream processing.

6. Increases viscosity of the medium. thereby protecting cells from mechanical damage.

Supply of serum is always lower than its demand.

7. Protease inhibitors in the serum protect cells from proteolysis.  

8. Provides several minerals e.g. Na+, K+, etc.  

9. Acts as a buffer.  

No.Advantages Disadvantages

1. Variation due to batch change of serum is avoided, there by improving reproducibility in time and place.

Different media required for different cell types, since serum-free media are specific to one cell type.

2. Easier downstream processing. Desired formulations which are reliable are not commercially available.

3. Toxic effects of serum avoided. A greater control of pH, temperature, etc. is required when compared to serum-containing media.

4. Bioassays free from interference with serum proteins. Growth rate and cell density attained are lower.

5. No danger of degradation of sensitive protein Serum protease. Cells tend to become fragile.

6. Permits selective culture of differentiated cells and producing cell types from a heterogeneous culture.

 

Advantages and Disadvantages of Serum-Free Media.