Animal Tissue Culture Media And Its Types
Animal Tissue Culture Media And Its Types
Design of animal tissue culture media is more difficult than that of microorganisms and plant cultures. Animal cell culture media are used to support the survival as well as growth by synthesizing certain chemical constituents from inorganic substances. It is classified as natural and artificial or synthesized media. Selection of media is dependent on the type of cells and the main objective of culture.
These media are obtained from natural sources such as plasma clots or coagulants, biological fluid and tissue extracts.
Plasma clots or coagulants are available commercially as liquid plasma in silicone ampoules or lyophilized plasma. Biological fluids are obtained in the form of serum from human blood, placental cord blood, horse blood, calf blood or in the form of biological fluids such as amniotic fluid, ascitic fluid, coconut water, insect haemolymph serum, aqueous humour from eye, culture filtrate etc.
Blood plasma: Blood plasma provides a nutritive substrate and a supporting structure for many types of cultures. It protects the cells and tissues from excessive traumatic damage during subculture and also used for conditioning the surface of the glass for better attachment of cells. Plasma from the chicken is preferred to mammalian plasma because it forms a clear and solid coagulum even when diluted several times. The plasma is obtained by centrifugation of whole blood before coagulation. The tissue is then placed in plasma and coagulation encouraged by the addition of a small amount of tissue extract or thrombin. This is required for solid support to continue growth and activity for the cells in the culture.
Blood serum: Blood serum (fibrinogen free plasma) with or without other nutritive substances is used in animal tissue culture. It is liquid exuded from coagulating blood and is filtered through Millipore filters. The sera used in tissue culture are calf (bovine), fetal bovine, horse and human serum. Calf and fetal bovine serum are most widely used in animal cell culture. Human serum is sometimes used in conjunction with some human cell lines but it is necessary to screen for viruses such as HIV and hepatitis B. Blood serum is a highly complex mixture of plasma proteins, peptides, lipids, carbohydrates, hormones, enzymes and minerals. The chicken serum is prepared by the coagulation of fluid plasma. The plasma is coagulated by adding embryo tissue extract or equivalent amount of thrombin. The tubes are incubated for several hours at 37°C. The coagulated plasma is broken up into fragments and then serum is separated by centrifugation. The mammalian blood is kept at room temperature for an hour for the coagulation. The clot is removed by a glass rod and then centrifuged at 3000 rpm for 30 minutes and the mammalian serum is separated.
Tissue extracts used in animal cell culture include embryo, spleen, liver, bone marrow, Leukocyte etc Chick embryo extract is most commonly used and substituted by the mixture of amino acids. Chick embryo extract is prepared from 10 to 12 days old embryos. The embryos are isolated from the egg and then mixed by using homogenizer with a measured quantity of balanced salt solutions (e.g. 2 ml/embryo). The crude extract is fractionated to give fractions of either high or low molecular weight. The low molecular weight fraction promoted cell proliferation while the high molecular weight fraction promoted pigment and cartilage cell differentiation. It is centrifuged and again diluted 10 to 15 times. It has been dried from the frozen state and stored.
Synthetic or artificial media are prepared by adding organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, O2 and CO2, gas phases. Different types of synthetic media are prepared for a variety of cells and tissues to be cultured e.g. Minimal essential medium (MEM), CMRL 1066, RPMI 1640, Ham’s F 12 Fischers etc. Synthetic media are classified into two types as serum-containing media and serum-free media.
Serum containing media: In animal cell culture media, 5 to 20% serum is added in many serum-free media. The serum is the source of basic nutrients, provides several hormones (cortisone, testosterone, insulin), growth factors (platelet desired growth factors, PDGF, fibroblast growth factor) and proteins like fibronectin, albumin and transferrin. It also provides minerals (Na, K, Fe, Zn etc.), protease inhibitors and acts as a buffer. It binds and neutralizes different toxins. The serum is an important source of all nutrients still it has many disadvantages.
- Serum is the most expensive ingredient in the culture media.
- It increases difficulties and cost of downstream processing.
- It varies from batch to batch because it is not chemically defined. Changing serum batches requires extensive testing to ensure the replacement is similar or close to previous batches.
- Some growth factors may be inadequate and may not be supplemented.
- Supply of serum is less due to the spread of disease among the cattle are drought in the cattle rearing areas.
- The serum is a source of contamination of viruses, mycoplasma, prions etc
Serum-free media: Serum-free media are developed to overcome the limitations of serum. It has the ability to make the medium more selective for a particular cell type. The overgrowth by stromal fibroblasts can be reduced effectively in breast and skin cultures by using MCDB 170 and 153, melanocytes can be cultivated in the absence of fibroblasts and keratinocytes. Serum variability and toxicity is avoided by replacing serum. Downstream processes and bioassays are easy in the absence of serum. Serum-free media also have many disadvantages in routine use.
- Growth is slow in serum-free media and only for a few generations.
- The purity of reagents and more control about pH and temperature of media is required in serum-free media.
- The availability of properly controlled serum-free media is quite limited, hence preparation of this media requires more time in the laboratory.
- Most of the media are specific to one cell type and laboratories face problems in maintaining cell lines of several different origins.
Physicochemical studies are also important for the growth of different cell lines. The optimal temperature for cell culture is dependent on the body temperature of the animal from which the cells are obtained. The temperature recommended for most human and warm-blooded animal cell lines is 37°C Osmolality between 260 m Osm/kg and 320 m Osm/kg are quite acceptable for most cell lines. Most cultured cells have a fairly wide tolerance for osmotic pressure. Buffers are incorporated into the medium to stabilize the pH. Exogenous CO2 may be required to some cell lines to prevent the total loss of dissolved CO2 and bicarbonate from the medium. Cell lines incubated in a CO2 incubator for growth. Each media is prepared by the addition of bicarbonate and CO, tension for achieving the correct pH and Osmolality. Most cell lines grow well at pH 7.4 phenol red (red at pH 7.4) is commonly used as an indicator for detection of pH. Most of the cells require oxygen for respiration in vivo and it is a major constituent of the gas phase. The viscosity of the culture medium is influenced by the serum content. Cell damage is avoided mainly by increasing the viscosity of the medium with carboxymethyl cellulose (CMC) or polyvinyl pyrrolidone. Balanced salt solution (BSS) is composed of inorganic salts and it is used as a diluent for concentrates of amino acids and vitamins to make complete media.