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Chapter... 16, ANIMAL CELL CULTURE, , ¢ LEARNING OBJECTIVES ¢, After completing this chapter, reader should be able to understand:, © Importance of Cell Culture Technology with Applications, , © Techniques for Growth of Cells, © Different Types of Animal Cell Cultures, , , , 46:4, INTRODUCTION =< F, The animal cell culture is a technique in which cells or tissue are obtained from animals,, , grown and maintained in a suitable medium. Animal tissue culture is divided as organ culture, (organotypic) and cell culture (histotypic). In organ culture, whole embryonic organ or small, tissue fragments are cultured in-vitro in such a manner that they retain their some or all of, the histological features. Cell culture is derived from dispersed cells taken from original, tissue, from a primary culture or from cell line or cell strain by enzymatic or mechanical, disaggregation. The culture produced by the cell or tissue taken from an organism is called, as primary culture. The sequence of culture obtained from the first subcultivation of the, primary culture is called ‘cell line’. Primary cultures are heterogeneous and slow growing. The, animal cells can grow only to a limited generations and it require controlled physicochemical, environment (02, CO2, pH, temperature, osmotic pressure etc.) and defined physiological, , , , conditions., , The first attempt to grow animal cells in culture is attributed to Ross Harrison in 1907., He was able to cultivate frog embryonic nerve cells using the hanging drop technique. The, discovery of antibiotics in the late 1940's led to the development of improved cell culture, techniques by reducing microbial contamination. During this period, many human carcinoma, cell lines (HeLa cell line) were isolated and grown in culture. Wilmut and co-workers, successfully produced a transgenic sheep named Dolly through nuclear transfer technique, , 16.2 GROWTH OF ANIMAL CELLS =, a # 2 & % FATES, Design of animal cell culture media is more difficult than that of microorganisms and, plant cultures. Animal cell culture media (Fig. 16.1) are used to support the survival as well 4, 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, type of cells and main objective of culture., , , , , , , , , (16.1)
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pharmaceutical Microbiology (B.Pharm.Sem.m) 16,2 Animal Cell Culture, , , , , , 16.2.1 Natural Media, , This media are obtained from natural sources such as plasma clots or coagulans,, biological fluid and tissue extracts., , Plasma clots or coagulans 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, humor 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 glass for better attachment, of cells. Plasma from the chicken is preferred to mammalian plasma because it form 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 addition of a small amount of tissue extract or thrombin. This is, require for a 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 8. Blood serum is highly compiles, mixture of plasma proteins, peptides, lipids, carbohydrates, hormones, enzymes and minerals.
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Pharmaceutical Microbiology (B.Pharm. Sem. I) _16.3 Animal Cell Culture, , The chicken serum is prepared by the coagulation of fluid plasma. The plasma js, 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 ang, 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 includes embryo, spleen, liver, bone marrow,, leukocyte etc. Chick embryo extract is most commonly used and substituted by 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 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., , 16.2.2 Artificial Media, , 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, essentiaf 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-medium. 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 buffer. It binds and, neutralizes different toxins. Serum is important source of all nutrients still it has many, disadvantages., , ¢ Serum is most expensive ingredient in the culture media., , « Itincreases difficulties and cost of down stream processing., , * It varius 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 spread of disease among the cattle or drought in the, cattle rearing areas., , *¢ Serum is source of contamination of viruses, mycoplasma, prions etc.
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rmaceutical Microbiology (8.Pharm. Sem, m0) 16.4, , fe Animal Cell Culture, , te mie 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, yergrowth by stromal fibroblasts can be reduced effectively in breast and skin cultures by, using MCDB 170 and ane, be lanocytes can be cultivated in the absence of fibroblasts and, keratinocytes. Serum variability and toxicity is avoided by replacing serum. Down stream, processes and Bipases ys are easy in absence of serum. Serum free media also have many, disadvantages in routine use., ¢ Growth is slow in serum free media and only for few generations., , e Purity of reagents and more control about pH and temperature of media is require in, serum free media., , * The availability of properly controlled serum free media is quite limited, hence, preparation of this media require more time in laboratory., , * Most of the media are specific to one cell type and laboratories face problems in, , maintaining cell lines of several different origins., , , , , , , , , Incubator for growth of cell lines, Physicochemical studies are also important for 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 aan m Osm/kg are quite, acceptable for most cell lines. Most cultured cells have a tally wide tolerance for osmotic, Pressure, Buffers are incorporated into the medium to mer Poa - may, be required to some cell lines to prevent the total loss of disso Ne . 2 ae, ftom the medium. Cell lines incubated '” CO; incubator for growth Is s on in: 7 7 . Eac, , Media is iepared by the addition of biocarbonate and CO; tension for achieving the correct, , Fig. 16.2: CO2
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Pharmaceutical Microbiology (8.Pharm.Sem. I) _16.5 _ Animal Cell Culture, , pH and Osmolality. Most cell lines grow well at pH 7.4 phenol red (red at pH 7,4) ;,, commonly used as an indicator for detection of pH. Most of cells require oxygen for, respiration in vivo and it is 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., , 16.3 PROCEDURE FOR CELL CULTURE. —_~, , A piece of tissue from the organism is usually quite complex and it contains connective, tissue cells, variety of blood cells and reticuloendothelial cells. The first cell suspension is, isolated and then inoculated into a new culture vessel along with fresh medium, such culture, is called primary cell culture or primary cell line. Three stages are used for isolation of primary, cell culture such as (i) isolation of the tissue (ii) disaggregation of tissue and (iii) seeding the, culture into the culture vessel. All stages are performed in laminar bench to avoid, contamination (Fig. 16.3)., , , , , , , , Fig. 16.3: Laminar bench used for isolation and disaggregation of tissue, , (i) Isolation of the tissue: The explant from an excised portion of the body of an, animal is used for the culture of animal cells in a suitable nutrient medium. The major explant, tissues are collected (Fig. 16.4) from laboratory animals like mice, rabbit, guinea pig ete., Mouse embryos are a convenient source of cells for undifferentiated fibroblastic cultures. The, explants from humans such as smooth muscle cells, alveolar cells, macrophages, leukocytes, etc are isolated and cultured in simulated media. Organ from which cells are to be isolated, are surface sterilized with 70% alcohol and then removed aseptically. Excised tissues from the, explant source are immediately transferred in sterile nutrient medium or a well balanced salt, , solution (BSS) containing antibiotics. The tissues isolate from explant is either stored in freeze, or used immediately.
