Culture Media for Animal Cells!
The selection of an appropriate growth medium for the in vitro cultivation of cells is an important and essential step. The mammalian cells of an organ in the body receive nutrients from blood circulation.
For culturing these cells in vitro, it is expected that they should be provided with the components similar to those present in blood. In general, the choice of the medium mostly depends on the type of the cells to be cultured, and the purpose of the culture (growth, differentiation, and production of desired products). The culture media may be natural or artificial.
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. Some workers still prefer natural media for organ culture.
Artificial 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:
i. The medium should provide all the nutrients to the cells.
ii. Maintain the physiological pH around 7.0 with adequate buffering.
iii. The medium must be sterile, and isotonic to the cells.
The basis for the cell culture media was the balanced salt solution which was originally used to create a physiological pH and osmolarity required to maintain cells in vitro. For promoting growth and proliferation of cells, various constituents (glucose, amino acids, vitamins, growth factors, antibiotics etc.) were added, and several media developed.
Addition of serum to the various media is a common practice. However, some workers in recent years have started using serum-free media. The physicochemical properties of media required for tissue cultures are briefly described. This is followed by a brief account on balanced salt solutions, commonly used culture media and the serum-free media.
Physicochemical Properties of Culture Media:
The culture media is expected to possess certain physicochemical properties (pH, O2, CO2, buffering, osmolarity, viscosity, temperature etc.) to support good growth and proliferation of the cultured cells.
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
CO2, bicarbonate and buffering:
Carbon dioxide in the medium is in a dissolved state, the concentration of which depends on the atmospheric CO2 tension and temperature. CO2 in the medium exists as carbonic acid (H2CO3), and bicarbonate (HCO–3) and H+ ions as shown below.
CO2 + H2O ↔ H2CO3 ↔ H+ + HCO–3
As is evident from the above equation, the concentrations of CO2, HCO–3 and pH are interrelated. By increasing the atmospheric CO2, the pH will be reduced making the medium acidic.
Addition of sodium bicarbonate (as a component of bicarbonate buffer) neutralizes bicarbonate ions.
NaHCO3 ↔ Na+ + HCO–3
In fact, the commercially available media contain a recommended concentration of bicarbonate, and CO2 tension for the required pH. 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 by most workers 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. The presence of pyruvate in the medium results in the increased endogenous production of CO2 by the cells. This is advantageous since the dependence on the exogenous supply of CO2 and HCO–3 will be less. In such a case, the buffering can be achieved by high concentration of amino acids.
Oxygen:
A great majority of cells in vivo are dependent on the O2 supply for aerobic respiration. This is in fact made possible by a continuous supply of O2 to the tissues by hemoglobin. 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. This is because selenium is a cofactor for the synthesis of glutathione.
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.
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.
If the cells are obtained from birds, the optimal temperature is slightly higher (38.5°C) for culturing. For cold blooded animals (poikiltherms) that do not regulate their body heat (e.g. cold-water fish), the culture temperature may be in the range of 15-25°C. Besides directly influencing growth of cells, temperature also affects the solubility of CO2 i.e. higher temperature enhances solubility.
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). Once an osmolality is selected for a culture medium, it should be maintained at that level (with an allowance of ± 10 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.
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 hereunder:
i. Supply essential inorganic ions.
ii. Provide the requisite pH.
iii. Maintain the desired osmolality.
iv. Supply energy from glucose.
In fact, balanced salt solutions form the basis for the preparation of complete media with the requisite additions. Further, BSS is also useful for a short period (up to 4 hours) incubation of cells.
The composition of two most widely used BSS namely Earle’s BSS and Hank’s BSS is given in Table 34.1.
Complete Culture Media:
In the early years, balanced salt solutions were supplemented with various nutrients (amino acids, vitamins, serum etc.) to promote proliferation of cells in culture. Eagle was a pioneer in media formulation. He determined (during 1950-60) the nutrient requirements for mammalian cell cultures. Many developments in media preparation have occurred since then. There are more than a dozen media now available for different types of cultures.
Some of them are stated 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 other important culture media are Ham’s F10, and F12, TC 199 and CMRL 1060. The detailed composition of three commonly used media namely Eagle’s MEM, RPMI 1640 and Ham’s F12 is given in Table 34.2. The complete media, in general, contains a large number of components amino acids, vitamins, salts, glucose, other organic supplements, growth factors and hormones, and antibiotics, besides serum. Depending on the medium, the quality and quantity of the ingredients vary. Some important aspects of the media ingredients are briefly described.
Amino acids:
All the essential amino acids (which cannot be synthesized by the cells) have to be added to the medium. In addition, even the non-essential amino acids (that can be synthesized by the cells) are also 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 serve as good sources of energy and carbon.
Vitamins:
The quality and quantity of vitamins depends on the medium. For instance, Eagle’s MEM contains only water soluble vitamins (e.g. B-complex, choline, inositol). The other 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.
Salts:
The salts present in 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, SO2-4, PO3-4), and are mainly responsible for the maintenance of osmolality. There are some other important functions of certain ions contributed by the salts.
i. Ca2+ ions are required for cell adhesion, in signal transduction, besides their involvement in cell proliferation and differentiation.
ii. Na+, K+ and CI– ions regulate membrane potential.
iii. PO3-4, SO2-4 and HCO–3 ions are involved in the maintenance of intracellular charge; besides serving as precursors for the production of certain important compounds e.g. PO3-4 is required for ATP synthesis.
Glucose:
Majority of culture media contain glucose which serves as an important source of energy. Glucose is degraded in glycolysis to form pyruvate/lactate. These compounds on their further metabolism enter citric acid cycle and get oxidized to CO2. However, experimental evidence indicates that the contribution of glucose for the operation of citric acid cycle is very low in vitro (in culture cells) compared to in vivo situation. Glutamine rather than glucose supplies carbon for the operation of citric acid cycle. And for this reason, the cultured cells require very high content of glutamine.
Hormones and growth factors:
For the media with serum, addition of hormones and growth factors is usually not required. They are frequently added to serum-free media.
Other organic supplements:
Several additional organic compounds are usually added to the media to support cultures. These include certain proteins, peptides, lipids, nucleosides and citric acid cycle intermediates. For serum-free media, supplementation with these compounds is very 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. However, with improved aseptic conditions in the present day tissue culture laboratories, the addition of antibiotics is not required. In fact, the use of antibiotics is associated with several disadvantages.
i. Possibility of developing antibiotic-resistant cells in culture.
ii. May cause anti-metabolic effects and hamper proliferation.
iii. Possibility of hiding several infections temporarily.
iv. May encourage poor aseptic conditions.
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 major constituents found in different types of sera are listed in Table 34.3. The most commonly used sera are calf serum (CS), fetal bovine serum (FBS), horse serum and human serum. While using human serum, it must be screened for viral diseases (hepatitis B, HIV).
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. This may however, be insignificant in complex media with well supplemented nutrients.
Growth factors:
There are certain growth factors in the serum that stimulate the proliferation of cells in the culture:
i. Platelet-derived growth factor (PDGF).
ii. Fibroblast growth factor (FGF).
iii. Epidermal growth factor (EGF).
iv. Vascular endothelial growth factor (VEGF).
v. 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, in association with somatomedins (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 medium DMEM can be used for the cultivation of neurons. A selected list of cells and cell lines along with the media and sera used is given in Table 34.4. In fact, information on the selection of appropriate medium for a particular cell line is available from literature.
The selection of serum is also based on the type of cells being cultured.
The following criteria are taken into consideration while choosing serum:
i. Batch to batch variations.
ii. Quality control.
iii. Efficiency to promote growth and preservation of cells.
iv. Sterility.
v. Heat inactivation.
In recent years, there is a tendency to discontinue the use of serum, and switch over to more clearly defined media.
Supplementation of the Medium with Tissue Extracts:
Besides serum, the culture media can also be supplemented with certain tissue extracts and microbial culture extracts. The examples are—chick embryo extract, proteolytic digests of beef heart, bactopeptone, lactalbumin hydrolysate, tryptose. The chick embryo extract was found to contain both high molecular weight and low molecular weight compounds that support growth and proliferation of cells.