An important aspect of any biotechnological processes is the culture of animal cells in artificial media. These animal cells in culture are used in recombinant DNA technology, genetic manipulations and in a variety of industrial processes. Now-a -days it has become possible to use the cell and tissue culture in the areas of research which have a potential for economic value and commercialization. The animal cell cultures are being extensively used in production of vaccines, monoclonal antibodies, pharmaceutical drugs, cancer research, genetic manipulations etc.
Animal cells e. . egg cells are used for multiplication of superior livestock using a variety of techniques like cloning of superior embryonic cells, transformation of cultured cells leading to the production of transgenic animals. The animal cells are also used in vitro fertilization and transfer of embryos to surrogate mothers. Hence the establishment and maintenance of a proper animal culture is the first step towards using them as tools for biotechnology. HISTORY OF ANIMAL CELL CULTURE | It was Jolly, who (1903) showed for the first time that the cells can survive and divide in vitro.
Ross Harrison, (1907) was able to show the development of nerve fibres from frog embryo tissue, cultured in a blood clot. Later, Alexis Carriel (1912) used tissue and embryo extracts as cultural media to keep the fragments of chick embryo heart alive. In the late 1940s, Enders, Weller and Robbins grew poliomyelitis virus in culture which paved way for testing many chemicals and antibiotics that affect multiplication of virus in living host cells. The significance of animal cell culture was increased when viruses were used to produce vaccines on animal cell cultures in late 1940s.
For about 50 years, mainly tissue explants rather than cells were used for culture techniques, although later after 1950s, mainly dispersed cells in culture were utilized. In 1966, Alec Issacs discovered Interferon by infecting cells in tissue culture with viruses. He took filtrates from virus infected cells and grew fresh cells in the filtered medium. When the virus was reintroduced in the medium, the cells did not get infected. He proposed that cells infected with the virus secreted a molecule which coated onto uninfected cells and interfered with the viral entry. This molecule was alled “Interferon”. Chinese Hamster Ovary (CHO) cell lines were developed during 1980s. Recombinant erythropoietin was produced on CHO cell lines by AMGEN (U. S. A. ).
It is used to prevent anaemia in patients with kidney failure who require dialysis. After this discovery, the Food and Drug Administration (U. S. A) granted the approval for manufacturing erythropoietin on CHO cell lines. In 1982, Thilly and co-workers used the conventional conditions of medium, serum, and O2 with suitable beads as carriers and grew certain mammalian cell lines to densities as high as 5×106 cells/ml.
A lot of progress has been also made in the area of stem cell technology which will have their use in the possible replacement of damaged and dead cells. In 1996, Wilmut and co-workers successfully produced a transgenic sheep named Dolly through nuclear transfer technique. Thereafter, many such animals (like sheep, goat, pigs, fishes, birds etc. ) were produced. Recently in 2002, Clonaid, a human genome society of France claimed to produce a cloned human baby named EVE. For animals, if the explant maintains its structure and function in culture it is called as an ‘organotypic culture’.
If the cells in culture reassociate to create a three dimensional structure irrespective of the tissue from which it was derived, it is described as a ‘histotypic culture’. WHAT IS ANIMAL CELL CULTURE ? Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment. In practice, the term “cell culture” now refers to the culturing of cells derived from multi-cellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes, including viruses, bacteriaand protists.
The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Salient Features of Animal cell culture : a) Animal cells can grow in simple glass or plastic containers in nutritive media but they grow only to limited generations. b) Animal cells exhibit contact inhibition. In culture the cancer cells apparently differ from the normal cells. Due to uncontrolled growth and more rounded shape, they loose contact inhibition and pile over each other. c) There is a difference in the in vitro and in vivo growth pattern of cells.
For example (i) there is an absence of cell-cell interaction and cell matrix interaction, (ii) there is a lack of three-dimensional architectural appearance, and (iii) changed hormonal and nutritional environment. They way of adherence to glass or plastic container in which they grow, cell proliferation and shape of cell results in alterations. d) The maintenance of growth of cells under laboratory conditions in suitable culture medium is known as PRIMARY CELL CULTURE. e) Cells are dissociated form tissues by mechanical means and by enzymatic digestion using proteolytic enzymes. )
Cells can grow as adherent cells (anchorage dependent) or as suspension cultures (anchorage independent). g) The primary culture is subcultured in fresh media to establish SECONDARY CULTURES. h) The various types of cell lines are categorized into two types as Finite cell line and Continuous cell line. Finite cell lines are those cell lines which have a limited life span and grow through a limited number of cell generations. The cells normally divide 20 to 100 times (i. e. is 20-100 population doublings) before extinction.
Cell lines transformed under in vitro conditions give rise to continuous cell lines. The continuous cell lines are transformed, immortal and tumorigenic. i) The physical environment includes the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses. j) Nutrient media is the mixture of inorganic salts and other nutrients capable of sustaining cell survival in vitro k) Serum is essential for animal cell culture and contains growth factors which promote cell proliferation.
It is obtained as exuded liquid from blood undergoing coagulation and filtered using Millipore filters. l) Cryo preservation is storing of cells at very low temperature (-1800C to -196 0C) using liquid nitrogen. DMSO is a cryopreservative molecule which prevents damage to cells. m) In order to maintain the aseptic conditions in a cell culture, a LAF hood is used. Based on the nature of cells and organism the tissue culture hoods are grouped into three types: Class I, Class II, and Class III. n) CO2 incubators are used and designed to mimic the environmental conditions of the living cells. ) An inverted microscope is used for visualizing cell cultures in situ p) For most animal cell cultures low speed centrifuges are needed. q) Neuronal cells constitute the nervous system. In culture the neuronal cells cannot divide and grow. r) The cells that form connective tissue (skin) is called fibroblast.
The fibroblast can divide and grow in culture to some generations after which they die. All normal animal cells are mortal. s) Organ culture- The culture of native tissue that retains most of the in vivo histological features is regarded as organ culture. ) Histotypic culture- The culturing of the cells for their reaggregation to form a tissue-like structure represents histotypic culture. u) Organotypic culture- This culture technique involves the recombination of different cell types to form a more defined tissue or an organ. There are certain terms that are associated with the cell lines. These are as follows: (i) Split ratio- The divisor of the dilution ratio of a cell culture at subculture. (ii) Passage number- It is the number of times that the culture has been cultured, (iii) Generation number- It refers to the number of doublings that a cell population has undergone.
In fact these parameters help us to distinguish the cancer cells in culture from the normal cells because the cancer cells in culture, change shape (more rounded), loose contact inhibition, pile on each other due to overgrowth and uncontrolled growth. REQUIREMENTS FOR ANIMAL CELL CULTURE Among the essential requirements for animal cell culture are special incubators to maintain the levels of oxygen, carbon dioxide, temperature, humidity as present in the animal’s body. The synthetic media with vitamins, amino acids and fetal calf serum.
Following parameters are essential for successful animal cell culture: ) Temperature- In most of the mammalian cell cultures, the temperature is maintained at 370C in the incubators as the body temperature of Homo sapiens is 370C. b) Culture media- The culture media is prepared in such a way that it provides- 1) The optimum conditions of factors like pH, osmotic pressure, etc. 2) It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions.
Glucose is added as energy source-it’s concentration varying depending on the requirement. Phenol Red is added as a pH indicator of the medium. There are two types of media used for culture of animal cells and tissues- the natural media and the synthesized media. 3) Natural Media – The natural media are the natural sources of nutrient sufficient for growth and proliferation of animal cells and tissues. The Natural Media used to promote cell growth fall in three categories. i) Coagulant, such as plasma clots. It is now commercially available in the form of liquid plasma kept in silicon ampoules or lyophilized plasma.
Plasma can also be prepared in the laboratory taking out blood from male fowl and adding heparin to prevent blood coagulation. ii) Biological fluids such as serum. Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum is obtained from human adult blood, placental, cord blood, horse blood, calf blood.
The other forms of biological fluids used are coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes etc. iii) Tissue extracts for example Embryo extracts- Extracts from tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow etc are also used for culture of animal cells. Synthetic media Syntheic media are prepared artificially by adding several organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors etc. Different types of synthetic media can be prepared for a variety of cells and tissues to be cultured.
Synthetic media are of two types- Serum containing media (media containing serum) and serum- free media (media with out serum). Examples of some media are: minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12 etc. Advantages of serum in culture medium are: i) serum binds and neutralizes toxins, (ii) serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins, (iii) it contains the protease inhibitors, (iv) it increases the buffering capacity, (v) it provides trace elements. Disadvantages of serum in culture medium are: i) it is not chemically defined and therefore it’s composition varies a lot, (ii) it is sometimes source of contamination by viruses, mycoplasma, prions etc, (iii) it increases the difficulties and cost of down stream processing, (iv) it is the most expensive component of the culture medium. 4) pH- Most media maintain the pH between 7 and 7. 4. A pH below 6. 8 inhibits cell growth. The optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular enzymes and binding of hormones and growth factors to cell surface receptors in the cell cultures.
The regulation of pH is done using a variety of buffering systems. Most media use a bicarbonate-CO2 system as its major component. 5) Osmolality- A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in the growth media determine the osmolality of the medium. All commercial media are formulated in such a way that their final osmolality is around 300 mOsm. HOW CULTURE CELLS ARE OBTAINED ? PROTOCOL A. Culture of Primary Chick Embryo Fibroblasts (CEF) Materials 10- to 12-day-old embryonated eggs Forceps and scissors Sterile petri dishes
Sterile 125-ml Erlenmeyer flask with magnetic stir bar Sterile 25-cm2 flasks containing MEM plus 10% fetal calf serum Sterile 0. 5% trypsin in Saline A Sterile 15-ml centrifuge tubes containing 0. 5 ml of serum Hemacytometers 1-ml and 10-ml pipettes Sterile Saline A Procedure 1. Disinfect the surface of the egg over the air sac. With scissors or the blunt-end of a forceps, break the shell over the air sac. Sterilize forceps by dipping in alcohol and flaming. Cool forceps, then peel away the shell over the air sac, sterilize forceps again, and pull back the shell membrane and chorioallantoic membrane to expose the embryo. . Resterilize the forceps, grasp the embryo loosely around the neck, and remove the entire embryo from the egg to a sterile petri dish.
3. Using two forceps or a scissors plus a forceps, decapitate and eviscerate the embryo. Mince the embryo carcass into very small fragments with a scissors. 4. Add about 10 ml of sterile Saline A to tissue fragments in the petri dish, swirl gently for 1 to 2 minutes to resuspend and wash fragments, and carefully pour entire contents into a 125-ml Erlenmeyer flask. Tilt flask, allow fragments to settle, and gently decant saline. Discard saline. 5.
Add 10 ml of sterile warm trypsin solution to fragments in flask, cover, and stir slowlywith magnetic bar for 5 to 10 minutes. Tilt flask, allow fragments to settle, and pour the trypsin-cell suspension into a 15-ml centrifuge tube containing 0. 5 ml of serum. The serum contains a trypsin inhibitor that will prevent further damage to cell membranes by the enzyme. 6. Add 10 ml of sterile warm trypsin to fragments and repeat step 5. At the end of this second treatment, the size of tissue fragments will be greatly reduced and a large number of single cells should be suspended in trypsin. Note: it is preferable to treat the tissue with multiple short applications of trypsin; however, if time is a limitation, for example in a lab class, this method will work). 7. Visually balance volume in centrifuge tubes (transfer liquid if necessary) and centrifuge at 1,500 rpm for 10 minutes. Carefully decant and discard supernatant, resuspend pooled cell pellets in a total of 5 ml of MEM.
(Resuspend the pellet in one tube, then transfer the suspension to the second tube and resuspend that pellet. ) Mix well for counting in a hemacytometer. Be sure to keep your cell suspension sterile. . In most hemacytometers, each heavily etched square (surrounded by double or triple lines and containing either 16 or 25 smaller squares) is 1 mm on each side and 0. 1 mm deep. Therefore, the area is 1 mm2 and the volume is 0. 1 mm3 (Fig. 1). After centrifugation, the cells should be packed in a tight pellet at the bottom of each tube. 9. Place the cover slip on top of the hemacytometer, bridged on the two glass arms beside the etched pattern. Add one drop of evenly-suspended cell suspension to the groove in the hemacytometer stage and allow it to fill the chamber under the cover slip.
Examine with the 10X objective. If there are too many cells to count (>200), make a 1:10 dilution of the cell suspension in MEM (0. 1 ml of cell suspension plus 0. 9 ml of MEM) for counting. The following example shows how to convert your cell count to the concentration of cells in your original suspension. Assume you made a 1:10 dilution, then counted a total of 168 cells in one 16-square grid: 168 x 104 x 10 = 1. 68 x 107 cells/cm3 What does each term mean? 168 is the number of cells in the grid x 104 converts to cells per ml (104 is the number of 0. mm3 in 1 cm3 (1 ml)) x 10 accounts for the dilution 1. 68 x 107 cells/ml(cm3) is the number of cells in your original suspension
Note: if a really accurate count is needed it is customary to count more than one 1-mm2 grid, then take the average of the number of grids counted. 10. Calculate what volume of the original cell suspension you will need to add to the growth medium in the flask to give between 2 x 105 and 8 x 105 cells/ml (~5 x 105 cells/ml). (Hint: if you have 5 ml of growth medium in the flask, you need a total of 5 ml x 5 x 105 cells/ml = 2. x 106 cells. ) Add the appropriate volume of original cell suspension (not the 1:10 dilution, if you made one) to the medium in your flask. 11. Be sure to examine cell cultures both macroscopically and microscopically each day. Actively growing cells produce acidic metabolic by-products and their medium becomes yellow, and thus the pH of the medium may need to be adjusted by the addition of a drop or two of 7. 5% NaHCO3. If floating (dead) cells and debris are present or the color of the medium indicates a basic pH, the medium should be changed. FIG. 1.
Hemacytometer (improved Neubauer counting chamber). B. Transfer of Cell Cultures After cultured cells have formed a confluent monolayer on the surface of their culture vessel, they may be removed from the surface, diluted, and seeded into new vessels. If the initial culture was primary, the new cultures are called secondary and are likely to consist of fewer cell types. Removal of cells from glass or plastic surfaces may be by either physical methods__scraping with a sterile rubber policeman__or chemical methods__proteolytic enzymes or chelating agents__or a combination of the two.
After removal, cells are pipetted up and down against the bottom of the flask to break up clumps, diluted, and counted. Primary cultures can usually be diluted 1:2 or 1:3 for secondary culturing, and after one becomes familiar with the growth characteristics of a certain cell type, counting can usually be dispensed with. The same procedure can be used to transfer both primary cells and a continuous cell line, removing the cells from flasks with a mixture of trypsin and EDTA in physiological saline (STE stands for saline, trypsin, EDTA). Procedure 1.
Examine the cells growing in a 25-cm2 flask with an inverted microscope to see if they have formed a confluent cell monolayer. If there are sufficient cells, pour off the medium. 2. Wash the monolayer with 2 ml of Saline A. Rinse well without shaking (shaking produces bubbles) and pour off. Repeat. 3. Add 1. 0 ml of STE to the flask and incubate at 37oC for 2 to 10 minutes with STE covering the cells. Observe periodically to determine when cells are loosened from plastic. (Note: STE will contain a pH indicator and should have a pH of 7. 0 to 8. 0. Below pH 7. 0 trypsin is inactive. A pH above 8. 0 is damaging to cells. ) 4.
When cells are seen to detach from the surface upon shaking or jarring against the heel of your hand (you can check in the microscope), add 4 ml of fresh growth medium with 10% serum and suspend cells by pipetting up and down a few times. Count cells in a hemacytometer, calculate the volume of additional medium needed to bring the cell concentration to ~5x105cells/ml, and add this volume to the cell suspension. 5. Seed an appropriate volume for the size of flask, or 1 ml of cell suspension into each well of a 24-well cluster dish, or 5 ml of cell suspension into a new 25-cm2 plastic flask. C. Preservation of Cultured Cells by Freezing
Viability of viruses and bacteria is preserved during freezing, but original attempts to preserve animal cells by freezing resulted in cell death. This was first thought to be due to laceration of cell plasma membranes by ice crystals, but more recent evidence suggests the cause may be osmotic changes during freezing which give rise to irreversible changes in lipoprotein complexes in intracellular membranes. In any event, the answer to animal cell preservation has proved to be the addition of glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO) to the medium and slow freezing, ideally at a cooling rate of one Celsius degree per minute.
Cells must be stored at -70oC or lower (ideally in liquid nitrogen at -196oC), and when they are recovered, thawing must be rapid. With careful technique, 50 to 80% of the cells of a healthy culture will survive freezing. Procedure 1. Remove the confluent cell monolayer from the culture flask by the method described in the cell transfer procedure. Suspend cells in added MEM, transfer to tubes containing fetal bovine serum, and centrifuge at 1,500 rpm for 5 to 10 minutes.
After centrifugation, resuspend cells in 0. 5 ml of cold medium containing 10% serum and 10% DMSO and place in a small cryotube. . Immediately place tubes in an ice bath. They will then be transferred to an insulated container and cooled to –20oC at a rate of -1oC per minute (if you do not own such a container, cells can be stored on ice for 1/2 hour then moved to a -70oC freezer in a box with lots of Kimwipes around the tubes), after a few days these cells can be moved from -70oC to liquid nitrogen for permanent storage. Cells stored at -70oC will not remain viable as long as cells stored in liquid nitrogen. If cells are to be stored in liquid nitrogen, they must be placed in sealed ampules or cryotubes. 3.
To recover cells, remove tubes from -70oC or liquid nitrogen and place directly in 37oC water bath. Note: liquid nitrogen can cause burns, therefore, care should be taken when handling liquid nitrogen and tubes should be held in your fingers for as brief a period of time as is possible. When thawing is barely complete, add contents of the tube to a 25-cm2 flask containing 5 ml of medium with 10% serum. The culture medium should be changed approximately 4 hours later (after cells have attached) to minimize the time of exposure to DMSO at 37oC. EQUIPMENTS REQUIRED FOR ANIMAL CELL CULTURE
Laminar Flow Cabinets LAF hoods are the aseptic working table for inoculation of animal cells. The basic purpose of using a LAF hood is to provide protection from contamination from any organism like fungi or bacterial cells under aseptic conditions, and to protect the operator from potential infection risk of infection from the cultured cells. The working area of LAF hood is first made sterile by using 70% ethanol. When the LAF is kept in “ON” position, the sterile air flows inside the cabinet which maintains the sterile conditions required for the transfer of cultured cells.
Depending on the nature of the cells and organisms being handled, tissue culture hoods can be grouped as follows : a) Class I hoods are found with in specially designed sterile work areas and give good protection to the operator and, to a lesser degree, the cell culture. There is an open front from which the air is drawn over the cell culture and goes out through the top of the hood. b) Class II hoods offer protection to both operator and the cell culture and is the most common type found in a tissue culture laboratory.
The cell culture is protected in a stream of sterile air and the operator is protected from contamination by the inflow of air into the base of the work area. The inflow of stream of sterile air into the base of work area protects the culture and operator from contamination. c) Class III hoods contains a full physical barrier which screens the worker, and is mainly used for working with highly pathogenic organisms. In this, a physical barrier separates the operator from the inoculation work. The open front is replaced with glass or Perspex with a pair of heavy duty gloves attached to it.
All the work is assessed from this glass. The Incubators The CO2 incubators provide the suitable environmental conditions to the growing animal cells. Generally CO2 incubators are used in animal cell cultures. This is a) to maintain the sterility of the chamber for which filtered High Efficiency Particulate Air (HEPA) is used. b) to maintain constant temperature the incubators is made airtight using a silicon gasket on the inner door. c) to keep an atmosphere with a fixed level of CO2 and high relative humidity which prevents the dessication of the medium and maintains the osmolality? Inverted Microscope
This type of microscope is used for visualizing cell cultures in situ. The cells in culture vessel remain at the bottom of the vessel and the medium floats above the growing cells. It is impossible to observe these cells under the ordinary microscope, therefore, the inverted microscope is used for such purposes. The inverted microscope has the optical system at the bottom and the light source at the top, this arrangement helps to observe the cultured cells in the plates. Centrifuges Only low speed centrifuges are used generally at 20oC to avoid disruption of the separated bands of cells.
The motor releases the heat which leads to the increase in temperature. Therefore, use of low temperature for centrifugation is recommended so that cells are not exposed to high temperature. Besides these conditions, the culture rooms should have light (diffused light and darkness each for a period of 12 hours) and temperature maintained at 25+/- 20C, with relative humidity at 98% and uniform air ventilation. The cultures should be monitored at regular intervals under aseptic conditions. Sterilized Glassware, culture media and other equipments
The glassware are thoroughly washed and all the equipment sterilized by heat, steam, or Millipore filter paper. The glassware like glass coverslips, instruments, Pasteur pipettes, test tubes etc are sterilized by dry heat. Apparatus containing glass and silicon tubing, disposable tips for micropipettes, screw caps, Millipore filters etc are sterilized by autoclaving. Isolation of animal material (Tissue) The culture animal material is washed in balanced salt solution to avoid contamination. The tissue to be cultured should be properly sterilized with 70% ethanol and removed surgically under aseptic conditions.
Disaggregation of tissue – To obtain the cell suspension for primary cell culture, the tissue is disintegrated either mechanically or by using enzymes. (i) Physical or mechanical disaggregation- After removing the tissue under aseptic conditions, it is pressed through a sieve of 100 micrometer. It is then kept in a sterile Petri dish containing buffered medium with balanced salt solution. The cells are then alternately passed through the sieve of decreasing pore size (50 micrometer and 20 micrometer mesh).
The debris which remains on the sieve is discarded and the medium containing cells is collected and cells are counted by using haemocytometer. This method is cheap and quick but it damages a lot of cells. (ii) Enzymatic disaggregation- In this method, enzymes are used for dislodging the cells of tissues. The two important enzymes used in tissue disaggregation are-collagenase and trypsin. -a) Collagenase- The intracellular matrix contains collagen therefore collagenase is used for disaggregation of embryonic, normal as well as malignant tissues.
The tissues are kept in medium containing antibiotics and then dissected into pieces in basal salt solution. After washing the chopped tissue with distilled water, it is transferred to complete medium containing collagenase. After a few days (around 5 days), the mixture is pipetted so that the medium gets dispersed. The whole treatment is left for sometimes during which the epithelial cells settle on bottom of test tubes. The enzyme collagenase is removed by centrifugation. Suspension consists of cells which are then plated out on the medium. (b) Trypsin- Use of trypsin for disaggregation is called trypsinization.
On the basis of role of temperature on trypsin, the activity of trypsin is of two types- Cold trypsinization and warm trypsinization. Cold trypsinization- The sample tissue to be disaggregated is chopped into 2-3 small pieces and kept in sterile glass vial. The tissues are subsequently washed with sterile water and dissected and then kept in BSS. The whole content is then placed on ice and soaked in cold trypsin for 4-6 hours to allow the penetration of enzymes in tissue. After this the trypsin is removed and the tissue is incubated at 36. 50C for 20-30 minutes.
About 10 ml of medium containing serum is added to the vials containing the cells and the cells are dispersed by repeated pipetting. The cells are counted by haemocytometer and are plated and incubated for 48-72 hours for cell growth. Warm trypsinization- The initial steps are the same as in cold trypsinization however, in this case the tissue pieces are treated with warm trypsin (36. 50C). The tissues are stirred for 4 hours and then pieces are allowed to settle down. The disassociated cells are collected at every 30 minutes. The process is repeated by adding fresh trypsin back to pieces and incubating the contents.
The trypsin is removed by centrifugation after 3-4 hours during which the complete disaggregation of tissues takes place. The glass vials containing dispersed cells are then placed on ice. The cells are counted using haemocytometer and cell density is maintained at an appropriate number. The cells are then plated on medium and incubated for 48-72 hours for cell growth. (iii) Treatment with chelating agents- The tissues like epithelium (which needs Ca2+ and Mg2+ ions for it’s integrity are treated with chelating agents such as citrate and ethylene-diamine-tetra-acetic acid (EDTA).
Chelating agents are mainly used for production of cell suspensions from established cultures of epithelial type. The animal cell cultures are used for a diverse range of research and development. These areas are: a) production of antiviral vaccines, which requires the standardization of cell lines for the multiplication and assay of viruses. b) Cancer research, which requires the study of uncontrolled cell division in cultures. c) Cell fusion techniques. d) Genetic manipulation, which is easy to carry out in cells or organ cultures. e) Production of monoclonal antibodies requires cell lines in culture. ) Production of pharmaceutical drugs using cell lines. g) Chromosome analysis of cells derived from womb.
h) Study of the effects of toxins and pollutants using cell lines. i) Use of artificial skin. j) Study the function of the nerve cells. Somatic Cell Fusion One of the applications of animal cell culture is the production of hybrid cells by the fusion of different cell types. These hybrid cells are used for a the following purposes: (i) study of the control of gene expression and differentiation, (ii) study of the problem of ‘ malignancy’, iii) viral application, (iv) gene mapping, (v) production of hybridomas for antibody production. In 1960s, in France for the first time, the hybrid cells were successfully produced from mixed cultures of two different cell lines of mouse. Cells growing in culture are induced by some of the viruses such as ‘Sendai virus’ to fuse and form hybrids. This virus induces two different cells first to form heterokaryons. During mitosis, chromosomes of heterokaryon move towards the two poles and later on fuse to form hybrids.
It is important to remove the surface carbohydrates to bring about cell fusion. Some other chemicals like polyethylene glycol also induce somatic cell fusion. Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. FIG SHOWING THE PRODUCTION OF T-PA Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.
Blood Factor VIII Haemophilia A is a blood disorder which is a sex-linked genetic disease in humans. The patients suffering from Haemophilia A lack factor VIII, which plays an important role in the clotting of blood. This factor VIII is secreted by a gene present on X-chromosome but this gene undergoes mutations in people suffering from Haemophilia. Current therapy for this disease is the transfusion of blood factor VIII into patients. Using rDNA technology, Factor VIII has been produced from mammalian cell culture e. g. Hamster kidney cell. Erythropoietin (EPO)
The EPO is a glycoprotein consisting of 165 amino acids and is formed in the foetal liver and kidneys of the adults. It causes proliferation and differentiation of progenitor cells into the erythrocytes (erythroblasts) in the bone marrow. Erythropoietin is hormone-like in nature and is released by the kidney under hypoxic or anoxic conditions caused by anaemia. Amgen Inc. holds US patent for preparation of, eErythropoietin, by recombinant method using Chinese Hamster Ovary cell lines. Erythropoietin (EPO) is a hormone-like substance released by the kidney under hypoxic or anoxic conditions caused by anaemia. -HUEPO- recombinant human erythro- protein has been effectively used to treat anemia associated with AIDS, renal failure etc.
The production of Monoclonal Antibodies using hybridoma technology Antibodies are proteins synthesized in blood against antigens and are collected from the blood serum. The antibodies, which are heterogenous and non specific in action are called polyclonal antibodies. If a specific lymphocyte, after isolation and culture in vitro becomes capable of producing a single type of antibody bearing specificity against specific antigen, it is known as monoclonal antibody.
The monoclonal antibodies are used in the diagnosis of diseases because of the presence of desired immunity. However, these antibody secreting cells cannot be maintained in culture. It was observed that the myeloma cells (bone marrow tumour cells due to cancer) grow indefinitely and also produce immunoglobulins which are infact monoclonal antibodies. In 1974, George Kohler and Milstein isolated clones of cells from the fusion of two parental cell lines – lymphocytes from spleen of mice immunized with red blood cells from sheep and myeloma cells.
These cells were maintained in vitro and produced antibodies. The hybrid cells maintained the character of lymphocytes to secrete the antibodies, and of myeloma cells to multiply in culture. These hybrid cell lines are called “Hybridoma” and are capable of producing unlimited supply of antibodies. Hybridoma are obtained by using an antibody producing lymphocytes cell and a single myeloma cell. Monoclonal antibodies bind very specifically to an epitope (specific domains) on an antigen and by using them it is possible to detect the presence of specific antigens. The
Monoclonal antibodies are used for the treatment of patients with malignant leukaemia cells, B cell lymphomas and allograft rejection after transplantation. CD3 is an antigen present on the surface of mature T- cells lymphocytes. If T- cell population is depleted or controlled, the transplanted organ will not be rejected. An antibody that acts against CD3 surface antigen of T-cells is called OKT3 i. e. anti-CD3 Moab. OKT3 is a monoclonal antibody which has been licensed for clinical use for the treatment of acute renal allograft rejection. OKT3 removes antigen bearing cells from circulation thereby helps in accepting the graft.
FIG SHOWING THE STEPS INVOLVED IN THE PRODUCTION OF MONOCLONAL ANTIBODIES When Monoclonal antibodies are used as enzymes using the technique of enzyme engineering, then they are called abzymes. Using animal cell cultures, it is also possible to produce Polyclonal Antibodies. Polyclonal antisera are derived from many cells therefore contains heterogeneous antibodies that are specific for several epitopes or an antigen. SCALE-UP OF ANIMAL CELL CULTURE Modifying a laboratory procedure, so that it can be used on an industrial scale is called scaling up.
Laboratory procedures are normally scaled up via intermediate models of increasing size. The larger the plant, the greater the running costs, as skilled people are required to monitor and maintain the machinery. The first pre-requisite for any large scale cell culture system and its scaling up is the establishment of a cell bank. Master cell banks (MCB) are first established and they are used to develop Master Working Cell Banks (MWCB). The MWCB should be sufficient to feed the production system at a particular scale for the predicted life of the product.
The cell stability is an important criteria so MWCB needs to be repeatedly subcultured and each generation should be checked for changes. A close attention should be paid to the volume of cultured cells as the volume should be large enough to produce a product in amounts which is economically viable. The volume is maintained by a) increasing the culture volume, (b) by increasing the concentration of cells in a reactor by continuous perfusion of fresh medium, so that the cells keep on increasing in number without the dilution of the medium. A fully automated bioreactor maintains the hysicochemical and biological factors to optimum level and maintains the cells in suspension medium.The most suitable bioreactor used is a compact-loop bioreactor consisting of marine impellers. The animal cells unlike bacterial cells, grow very slowly. The main carbon and energy sources are glucose and glutamine. Lactate and ammonia are their metabolic products that affect growth and productivity of cells. So, the on-line monitoring of glucose, glutamate, and ammonia is carried out by on line flow injection analysis (FIA) using gas chromatography (GC), high performance liquid chromatography (HPLC) etc.
In batch cultures, mainly Roller Bottles with Micro Carrier Beads (for adherent cells) and spinner flasks (for suspension cultures) are used in Scale-up of animal cell culture process. Roller Bottles The Roller bottles provide total curved surface area of the micro carrier beads for growth. The continuous rotation of the bottles in the CO2 incubators helps to provide medium to the entire cell monolayer in culture. The roller bottles are well attached inside a specialized CO2 incubators.
The attachments rotate the bottles along the long axis which helps to expose the entire cell monolayer to the medium during the one full rotation. This system has the advantage over the static monolayer culture: (a) it provides increase in the surface area, (b) provides constant gentle agitation of the medium, (c) provides increased ratio of surface area of medium to its volume, which allows gas exchange at an increased rate through the thin film of the medium over the cells. Typically, a surface area of 750-1500 cm2 with 200-500 ml medium will yield 1-2x108cells.
DIAGRAM SHOWING THE ROLLER BOTTLE CELL CULTURE Micro Carrier Beads Micro carrier beads are small spherical particles with diameter 90-300 micrometers, made up of dextran or glass. Micro Carrier beads, increase the number of adherent cells per flask. These dextran or glass-based beads come in a range of densities and sizes. The cells grow at a very high density which rapidly exhausts the medium and therefore the medium has to be replaced for the optimum cell growth. At the recommended concentration when the microcarriers are suspended they provide 0. 4 m2 area for every 100 ml of culture flask.
Spinner cultures The spinner flask, was originally developed to provide the gentle stirring of microcarriers but are now used for scaling up the production of suspension cells. The flat surface glass flask is fitted with a Teflon paddle that continuously turns and agitates the medium. This stirring of the medium improves gas exchange in the cells in culture. The spinner flask used at commercial scale consists of one or more side arms for taking out samples and decantation as well.