Animal biotechnology : Culture media composition and growth conditions -part 1

Animal cell culture (ACC) is invitro maintenance and propagation of animal cells using a suitable nutrient media.  Culturing is a process of growing animal cells artificially.  The most important and essential step in ACC is selecting appropriate growth medium for invitro cultivation. 

The selection of the medium depends on the type of cells to be cultured and also the purpose of the culture.  Purpose of ACC can be growth, differentiation or even production of desired products like pharmaceutical compounds.

Types of ACC media:

1.  Natural media

 In the early years, the natural media obtained from various biological sources were used.

Body fluids:
Plasma, serum, lymph, amniotic fluid, ascitic and pleural fluids, aqueous humour from eyes and insect hemolymph were in common use. These fluids were tested for sterility and toxicity before their utility.

Tissue extracts:
Among the tissue extracts, chick embryo extract was the most commonly employed. The extracts of liver, spleen, bone marrow and leucocytes were also used as culture media.

2.  Synthetic media
The artificial media (containing partly defined components) have been in use for cell culture since 1950.

The minimal criteria needed for choosing a medium for animal cell cultures are listed below:

• The medium should provide all the nutrients to the cells.
• Maintain the physiological pH around 7.0 with adequate buffering.
• The medium must be sterile, and isotonic to the cells.

Physicochemical Properties of Culture Media

The culture media should possess certain physicochemical properties (pH, O₂, CO₂, buffering, osmolarity, viscosity, temperature etc.) to support good growth and proliferation of the cultured cells.

1.  pH:  

Most of the cells can grow at a pH in the range of 7.0-7.4, although there are slight variations depending on the type of cells (i.e. cell lines). The indicator phenol red is most commonly used for visible detection of pH of the media.
 
Its colouration at the different pH is shown below:

• At pH 7.4 — Red                   
• At pH 7.0 — Orange
• At pH 6.5 — Yellow   
• At pH 7.8 — Purple

2.  CO₂, bicarbonate and buffering:

Carbon dioxide in the gas phase dissolves in the medium, establishes equilibrium with HCO3− ions, and lowers the pH. Because dissolved CO2, HCO3−, and pH are all interrelated, it is difficult to determine the major direct effect of CO2.  

The atmospheric CO2 tension will regulate the concentration of dissolved CO2 directly, as a function of temperature. This regulation in turn produces H2CO3, which dissociates according to the reaction

                                        

H2O + CO2 ⇔ H2CO3 ⇔ H+ + HCO3

Addition of sodium bicarbonate (as a component of bicarbonate buffer) neutralizes bicarbonate ions. 
The importance of CO2 cell culture is that, The majority of CO2 exists in the blood in the form of bicarbonate (HCO3-) which acts as a pH buffer to allow for gas, nutrient and metabolites fluctuations without causing wild pH changes. 

In recent years HEPES (hydroxyethyl piperazine 2-sulfonic acid) buffer which is more efficient than bicarbonate buffer is being used in the culture media. However, bicarbonate buffer is preferred because of the low cost, less toxicity and nutritional benefit to the medium. This is in contrast to HEPES which is expensive, besides being toxic to the cells.

A CO2 incubator is used to culture cells to provide it with the optimum temperature, moisture (sterile environment) and to maintain optimum pH. When the media contains carbonate buffer, the CO2 gas from the cylinder is let into the incubator in such a way that the pH remains constant

3. Oxygen:

A great majority of cells in vivo are dependent on the O₂ supply for aerobic respiration. The cultured cells mostly rely on the dissolved O₂ in the medium which may be toxic at high concentration due to the generation of free radicals. 

Therefore, it is absolutely necessary to supply adequate quantities of O₂ so that the cellular requirements are met, avoiding toxic effects. Some workers add free-radical scavengers (glutathione, mercaptoethanol) to nullify the toxicity. Addition of selenium to the medium is also advocated to reduce O₂ toxicity. 

In general, the glycolysis occurring in cultured cells is more anaerobic when compared to in vivo cells. Since the depth of the culture medium influences the rate of O₂ diffusion, it is advisable to keep the depth of the medium in the range 2-5 mm.

4. Temperature:

In general, the optimal temperature for a given cell culture is dependent on the body temperature of the organism, serving as the source of the cells. Accordingly, for cells obtained from humans and warm blooded animals, the optimal temperature is 37°C.

In vitro cells cannot tolerate higher temperature and most of them die if the temperature goes beyond 40°C. It is therefore absolutely necessary to maintain a constant temperature (± 0.5°C) for reproducible results. Besides directly influencing growth of cells, temperature also affects the solubility of CO₂ i.e. higher temperature enhances solubility.

5. Osmolality:

In general, the osmolality for most of the cultured cells (from different organisms) is in the range of 260-320 mOsm/kg. This is comparable to the osmolality of human plasma (290 mOsm/kg). Whenever there is an addition of acids, bases, drugs etc. to the medium, the osmolality gets affected. The instrument osmometer is employed for measuring osmolalities in the laboratory.

6. Balanced Salt Solutions:

The balanced salt solutions (BSS) are primarily composed of inorganic salts. Sometimes, sodium bicarbonate, glucose and HEPES buffer may also be added to BSS. Phenol red serves as a pH indicator.

The important functions of balanced salt solutions are listed

• Supply essential inorganic ions.
• Provide the requisite pH.
• Maintain the desired osmolality.
• Supply energy from glucose.