Animal biotechnology : Culture media composition and growth conditions -part 2

Complex culture media

In the early years, balanced salt solutions were supplemented with various nutrients (amino acids, vitamins, serum etc.) to push proliferation of cells in culture. Eagle was a pioneer in media formulation. He determined the nutrient requirements for mammalian cell cultures. There are quite a dozen media now available for  various varieties of cultures.

Some of them are given below:

• EMEM—Eagle’s minimal essential medium
• DMEM—Dulbecco’s modification of Eagle’s medium
• CMEM—Glasgow’s modification of Eagle’s medium
• RPMI 1630 and RPMI 1640—Media from Rosewell Park Memorial Institute.

The complete media, in general, contains an oversized number of components amino acids, vitamins, salts, glucose, other organic supplements, growth factors and hormones, and antibiotics, besides serum. Some important aspects of the media ingredients are briefly described.

1. Amino acids:

All the essential amino acids (which can't be synthesized by the cells) must to be added to the medium. Additionaly, even the non-essential amino acids (that will be synthesized by the cells) are usually added to avoid any limitation of their cellular synthesis. 

Among the non-essential amino acids, glutamine and/or glutamate are frequently added in good quantities to the media since these amino acids function as a good sources of energy and carbon.

2. Vitamins:

The quality and quantity of vitamins depends on the medium. As an example, Eagle’s MEM contains only water soluble vitamins (e.g. B-complex, choline, inositol). The different vitamins are obtained from the serum added.

The medium M 199 contains all the fat soluble vitamins (A, D, E and K) also. In general, for the media without serum, more vitamins in higher concentrations are required.

3.  Salts:

The salts present within the various media are basically those found in balanced salt solutions (Eagle’s BSS and Hank’s BSS). The salts contribute to cations (Na⁺, K⁺, Mg²⁺, Ca²⁺ etc.) and anions (CI^–, HCO^–₃, SO^2-₄, PO^3-₄), and are mainly accountable for the upkeep of osmolality. There are another important functions of certain ions contributed by the salts.

1. Ca²⁺ ions are required for cell adhesion, in signal transduction, besides their involvement in cell proliferation and differentiation.
2. Na⁺, K⁺ and CI^– ions regulate membrane potential.
3. PO^3-₄, SO^2-₄ and HCO^–₃ ions are involved within the maintenance of intracellular charge; besides serving as precursors for the assembly of certain important compounds e.g. PO^3-₄ is required for ATP synthesis.

4. Glucose:

Majority of culture media contain glucose which is a crucial source of energy. Glucose is degraded in glycolysis to make pyruvate/lactate. These compounds on their further metabolism enter citric acid cycle and oxidized to CO₂.
Glutamine instead of glucose supplies carbon for the operation of TCA cycle. And for this reason, high content of glutamine required by the cultured cells.

5. Hormones and growth factors:

For the media with serum, addition of hormones and growth factors is typically not required. They're frequently added to serum-free media.

Other organic supplements are:

Several additional organic compounds are added to the media to support cultures which include proteins, peptides, lipids, nucleosides and citric acid cycle intermediates. For serum-free media, supplementation with these compounds is extremely useful.

Antibiotics

In the early years, culture media invariably contained antibiotics. The most commonly used antibiotics were ampicillin, penicillin, gentamycin, erythromycin, kanamycin, neomycin and tetracycline. Antibiotics were added to reduce contamination.

In fact, the use of antibiotics is associated with several disadvantages, which are;

1. Possibility of developing antibiotic-resistant cells in culture.
2. May cause anti-metabolic effects and hamper proliferation.
3. Possibility of hiding several infections temporarily.

The present recommendation is that for the routine culture of cells, antibiotics should not be added. However, they may be used for the development of primary cultures.

SERUM:

Serum is a natural biological fluid, and is rich in various components to support cell proliferation. The most commonly used sera are calf serum (CS), fetal bovine serum (FBS), horse serum and human serum. Approximately 5-20% (v/v) of serum is mostly used for supplementing several media. Some of the important features of the serum constituents are briefly described.

Proteins:

The in vitro functions of serum protein are not very clear. Some of them are involved in promoting cell attachment and growth e.g. fetuin, fibronectin. Proteins increase the viscosity of the culture medium, besides contributing to buffering action.

Nutrients and metabolites:
Serum contains several amino acids, glucose, phospholipids, fatty acids, nucleosides and metabolic intermediates (pyruvic acid, lactic acid etc.). These constituents do contribute to some extent for the nutritional requirements of cells.

Growth factors:

Growth factors in the serum that stimulate the proliferation of cells in the culture:

1. Platelet-derived growth factor (PDGF).
2. Fibroblast growth factor (FGF).
3. Epidermal growth factor (EGF).
4. Vascular endothelial growth factor (VEGF).
5. Insulin-like growth factors (IGF-1, IGF-2).

In fact, almost all these growth factors are commercially available for use in tissue culture.

Hormones:
Hydrocortisone promotes cell attachment, while insulin facilitates glucose uptake by cells. Growth hormone IGFs, promotes cell proliferation.

Inhibitors:

Serum may also contain cellular growth inhibiting factors. Majority of them are artefacts e.g. bacterial toxins, antibodies. The natural serum also contains a physiological growth inhibitor namely transforming growth factor β (TGF-β). Most of these growth inhibitory factors may be removed by heat inactivation (at 56°C for 30 minutes).

Selection of Medium and Serum:

As already stated, there are around a dozen media for the cell cultures. The selection of a particular medium is based on the cell line and the purpose of culturing. For instance, for chick embryo fibroblasts and HeLa cells, EMEM is used. The selection of serum is also based on the type of cells being cultured.

The following criteria are taken into consideration while choosing serum: 

• Batch to batch variations.
• Quality control.
• Efficiency to promote growth and preservation of cells.
• Sterility.
• Heat inactivation.

In recent years, there is a tendency to discontinue the use of serum, and switch over to more clearly defined media.

OTHER SUPPLEMENTS

In addition to serum, tissue extracts and digests have traditionally been used as supplements to tissue culture media are;

• Amino Acid Hydrolysates

Many such supplements are derived from microbiological media. Bactopeptone, tryptose, and lactalbumin hydrolysate  are proteolytic digests of beef heart or lactalbumin and contain mainly amino acids and small peptides.
Bactopeptone and tryptose may also contain nucleosides and other heat-stable tissue constituents, such as fatty acids and carbohydrates. Sterility is easily achieved as they are autoclavable

• Embryo Extract

Embryo extract is a crude homogenate of 10-day-old chick embryo that is clarified by centrifugation.  The crude extract was fractionated to give fractions of either high or low molecular weight. The low-molecular-weight fraction promoted cell proliferation, whereas the high-molecular-weight fraction promoted pigment and cartilage cell differentiation.
Embryo extract was originally used as a component of plasma clots to promote cell migration from the explant and has been retained in some organ culture techniques.