Animal cell culture

December 12, 2020 Gaurab Karki Biotechnology 0




Animal cell culture
Animal cell culture

Introduction to animal cell culture:

  • Cell culture is the technique where cells are allowed to grow under controlled conditions, usually outside of their natural environment.
  • Likewise, animal cell culture is a technique in which the cells are removed and are allowed to grow in a favorable artificial environment.
  • The removal of tissue can either take place from tissue directly or from disaggregation by enzymatic or mechanical means before culture, or they may be originated from a cell line or cell strain that has been established earlier.
  • Even if the basic mammalian/animal cell culture techniques are similar to those that are applied to bacteria, fungi, and yeast, there are however some characteristic differences.
  • Usually, mammalian cells are found to be more delicate and more susceptible to mechanical damage.
  • They have lesser growth rates and need more complex culture media along with special substrates.

Steps to culture animal cells:

  • Harvest cells
  • Isolation of the cells with the use of appropriate enzymes.
  • In a culture dish with appropriate growth media, the cells are placed.
  • The culture dish is now kept in incubator for the culture of cells.
  • Cells can be sub-cultured in order to fix the problem or to get the pure culture.
  • Now, cells are ready to be manipulated or modified for lab procedures.

Media Composition for animal cell culture:

  • The basic components for the animal cell culture are enlisted as bellows:
  • Sources of Energy: Glucose, Fructose, Amino acids
  • Nitrogen sources: Amino acids
  • Vitamins: Usually, water soluble vitamins B & C.
  • Inorganic salts: Na+, K+, Ca2+, Mg2+
  • Fat and Fat soluble components: Fatty acids, cholesterols
  • Antibiotics
  • Growth factors and hormones.
  • Oxygen and CO2 concentration.
  • Physical environment: The physical environment consists of the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses.
  • CO2 incubators are employed and designed to resemble the environmental conditions of the living cells.
  • For visualizing cell cultures in vitro, an inverted microscope is used.
  • Low speed centrifuges are needed for most animal cell cultures.
  • Cryopreservation is the storage of cells using liquid nitrogen at a very low temperature (-180 °C to -196 °C).
  • DMSO is a cryo-preservative molecule that prevents cells from being harmed.
  • For the culture of animal cells, serum is necessary and contains growth factors that promote cell proliferation.
  • Even if animal cell culture media differ in their complexity, most include:
    • Amino acids:   0.1-0.2 mM
    • Vitamins:  1 microM
    •  Salts (NaCl) :  150 mM
    • KCl : 4-6 mM 
    • CaCl2 :  1 mM
    • Glucose : 5-10 mM

Animal Culture media:

  • In animal tissue culture, 2 types of culture media are used:
    • Natural media
    • Artificial media
  • The type of medium relies basically on the type of cells to be cultured and its objectives.

1. Natural media:

  • These media include the naturally occurring biological fluids and are of the following three types:
    • Clots
    • Biological fluids
    • Tissue extract

i) Clots:

  • Plasma clots are the most commonly used clots and has been employed for a long time.
  • In the present time, plasma is commercially found in liquid state that can be prepared in the laboratory.

ii) Biological Fluids:

  • Several biological fluids can be employed as culture media such as amniotic fluid, pleural and ascetic fluids, hemolymph of insects, aqueous humoral from eye, serum etc.
  • Among them, serum is the mostly preferred.

iii) Tissue Extracts:

  • The most commonly used tissue extract is chick embryo extract, however, bovine embryo extract is also used.
  • In the culture media, the extracts from spleen, liver, bone marrow and leucocytes were also used.
  • The substitution for tissue extract can be a mixture of amino acids and certain other organic compounds.

2. Artificial media:

  • For the following purposes, various artificial media have been employed:
  • Immediate survival (a balanced salt solution with specified pH and adequate osmotic pressure)
  • Prolonged survival (a balanced salt solution in addition with serum, or appropriate formulation of organic compounds.
  • Indefinite growth
  • Specialized functions
  • Artificial media may be classified  into following types:
    • Serum containing media
    • Serum-free media
    • Protein free media
    • Chemically defined media
  • Serum:
    • Serum is the yellowish liquid and is a transparent content that remains left over after the removal of fibrin and cells from the blood.
    • 2-10% of serum is often contained by normal growth media.
    • The most commonly employed supplement in animal cell culture is fetal bovine serum (FBS).
    • It supplies the basic nutrients for cells.
    • It also contains several hormones and various growth factor.
    • In addition to it, it also acts as a buffer.
  • Serum containing media:
    • In animal cell culture media, fetal bovine serum is the most common supplement.
    • In order to supply an optimal culture medium, it is employed as an economical supplement.
    • Serum supplies carriers water-insoluble nutrients, protease inhibitors, hormones and growth factors and binds and neutralizes the toxic moieties.
  • Serum free media:
    • In case of immunological studies, presence of serum in media can result to serious misinterpretations.
    • In general, these media are specifically designed to promote the culture of a single type of cell and incorporate specified amounts of purified growth factors, lipoproteins and other proteins normally supplied by the serum.
    • As the components of these media are known, thus it is referred to as ‘defined culture media’
  • Chemically defined media:
    • These media contain ultra-pure inorganic and organic ingredients free of contaminants and may also contain pure protein additives, such as growth factors.
    • Their constituents are produced by genetic modification in bacteria or yeast with the addition of vitamins, cholesterol, particular amino acids, and fatty acids.
  • Protein free media:
    • Protein-free media is devoid of any protein and only include non-protein constituents.
    • Usage of protein-free media enhances superior cell growth and protein expression in contrast to serum-supplemented media and enables downstream purification of any expressed product.

Development of animal culture media:

  • Early efforts to culture cells, such as chick embryo extract, plasma, serum, and lymph, were conducted in natural media based on tissue extracts and body fluids.
  • Demand for greater quantities of a medium of more reliable quality has led to the emergence of chemically defined media.
  • However, Eagle’s Basal medium and subsequently Eagle’s Minimal Essential Medium (MEM) were broadly accepted, variously supplemented with human, horse or calf serum, protein hydrolysates, and embryo extract.
  • In order to choose a medium, the minimum criteria required usually involve:
    • The medium should supply the cells with all of the nutrients.
    • Keep the physiological pH about 7.0 with ample buffering.
    •  The medium should be sterile, and isotonic to the cells.
    • The balanced salt solution that was initially used to establish a physiological pH and osmolarity needed to sustain cells in vitro was the basis for the cell culture media.
    • Various components (glucose, amino acids, vitamins, growth factors, antibiotics etc have been added to promote cell growth and proliferation, and several media have been created.

Physicochemical properties of culture media

  • In order to promote good growth and proliferation of the cultured cells, the culture medium is required to possess certain physicochemical properties (pH, O2, CO2, buffering, osmolarity, viscosity, temperature etc.).
  • pH:
    • Most cells can develop in the 7.0-7.4 pH range, but there are minor differences depending on the cell type.
    • Phenol red is usually used as an indicator.
    • It becomes orange at pH 7.0, yellow at pH 6.5, lemon yellow below pH 6.5, more pink at pH 7.6, and purple at pH 7.8. It is red at pH 7.4.
  •  CO2, bicarbonate and buffering:
    • The medium’s carbon dioxide is in a dissolved state, the concentration of which depends on the atmospheric CO2 tension and temperature.
    • As seen below, CO2 in the medium occurs as carbonic acid (H2CO3), and as bicarbonate (HCO3-) and H+ ions.                                    

                             CO2 + H2O ↔ H2CO3 ↔ H+ + HCO3– 

  • The CO2, HCO3-and pH concentrations are interrelated, as is obvious from the above equation.
  • The pH would be decreased by increasing atmospheric CO2, rendering the medium acidic.
  • Bicarbonate ions are neutralized by the addition of sodium bicarbonate (as a part of bicarbonate buffer).

                              NaHCO3 ↔ Na+ + HCO3–

  •  The recommended bicarbonate concentration and CO2 tension for the appropriate pH are found in commercially available media.
  • In recent years HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer being used in the culture media as it is considered more efficient than the bicarbonate buffer.
  • However, due to the low cost, less toxicity and nutritional value to the medium, bicarbonate buffer is favored.
  • In contrast to it, HEPES is expensive and along with it, it is toxic to the cells.
  • The existence of pyruvate in the medium results in the excessive endogenous production of CO2 by the cells.
  • This is good since the reliance on the exogenous supply of CO2 and HCO–3 would be lower.
  • In such a scenario, high amino acid concentrations can be used for buffering.
  • In summary, cultures in open vessels need to be incubated in the CO2 atmosphere, the concentration of which is in equilibrium with that of sodium bicarbonate in the medium.
  • Oxygen:
    • A large majority of in vivo cells rely on the availability of O2 for aerobic respiration, which is made possible by hemoglobin’s continuous supply of O2 to the tissues.
    • The cultured cells mainly depend on the dissolved O2 in the medium that may be toxic at high concentration due to the production of free radicals.
    • It is also completely necessary to supply appropriate amounts of O2 in order to comply with the cellular requirements, preventing toxic effects.
    • The introduction into the medium of free radical scavengers such as glutathione,2-mercaptoethanol (β-mercaptoethanol) or dithiothreitol erases the toxicity of O2.
    • To decrease O2 toxicity, the addition of selenium to the medium is often recommended.
    • It is because selenium is a cofactor for the synthesis of glutathione.
    • In general, as compared with in vivo cells, glycolysis occurring in cultured cells is more anaerobic.
    • As the rate of diffusion of O2 is affected by the depth of the culture medium, it is recommended to keep the depth of the medium in the range of 2-5mm.
  • Temperature:
    • In particular, the optimum temperature for a provided cell culture is reliant on the body temperature of the organism, acting as the source of the cells.
    • Therefore the optimum temperature for cells obtained from humans and warm blooded animals is 37 ° C.
    • Higher temperatures cannot be handled by in vitro cells and most of them die if the temperature goes beyond 40 ° C.
    • For reproducible outcomes, it is therefore completely important to maintain a steady temperature (± 0.5°C).
    • If the cells are collected from birds, the optimum temperature for culture is slightly higher (38.5°C).
    • The culture temperature can be about 15-25 ° C for cold blooded animals (poikilotherms) that do not control their body heat.
    • In addition to directly affecting cell growth, temperature also influences the solubility of CO2, i.e. higher temperatures increase solubility.
  • Osmolality:
    • Most cultured cells have a reasonably wide osmotic pressure tolerance.
    • This is comparable to human plasma osmolality (290mOsm/kg).
    • Once an osmolality is appointed for a culture medium, it should be sustained at that level (with an allowance of ± 10 mOsm/kg).
    • Osmolality is typically determined by the depression of the medium’s freezing point or vapor pressure elevation.
    • The instrument osmometer is used in the laboratory for osmolality measurement.
    • Osmolality is influenced if acids, bases, medications, etc are applied to the medium.
    • If we make up the medium ourselves, the measurement of osmolality is a useful quality control step, as it helps to protect against errors in weighing, dilution, and the like.
    • Osmolality is usually measured by depression of the freezing point or elevation of the vapor pressure, of the medium.
  • Viscosity:
    • A culture medium’s viscosity is largely affected by the serum content and will have little impact on cell growth in most cases.
    • When a cell suspension is agitated (e.g. when a suspension culture is stirred), or when cells are dissociated after trypsinization, or in low serum concentrations/absence, viscosity becomes especially important.
    • Increasing the viscosity of the medium with carboxymethylcellulose (CMC) or polyvinylpyrrolidone (PVP) will minimize any cell damage that occurs under these conditions.
  •  Surface Tension and Foaming:
    • The consequences of foaming have not been clearly identified, but the rate of denaturation of protein can increase, as might the risk of contamination if the foam enters the culture vessel’s neck.
    • If a film from a foam or spill gets into the capillary space between the lid and the base of a Petri dish or between a slack cap and the neck of a flask, foaming can also restrict gaseous diffusion.
    • In suspension cultures in stirrer vessels or bioreactors, foaming can occur when 5 percent of CO2 in air is bubbled via serum containing medium.
    • The addition of 0.01 percent to 0.1 percent silicone antifoam (Dow Chemical) or Pluronic F68 (Sigma) helps avoid foaming in this situation by minimizing surface tension and can also protect cells from bubble shear stress.