Slide 1: BILAL 1 ANIMAL CELL/TISSUE CULTURE : BILAL 2 ANIMAL CELL/TISSUE CULTURE PREPARED BY
BILAL AHMAD GHALLOO Definations : 3 BILAL Definations Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells Principles : 4 BILAL Principles Animal cell culture is playing a vital role in the various modern researches.
Recent developments in the field of biotechnology and molecular biology have given various new dimensions to this technology.
Cell culture has provided a new tool for the better understanding of diagnosis and molecular biology in many areas of biological systems including bacterial, viral and parasitic diseases. Slide 5: 5 BILAL The different cell cultures are being used in virology for the last five decades and one of its application has been utilized successfully in various types of vaccine production.
More recent discoveries such as production of various proteins and enzymes through recombinant DNA technology require the use of cell culture to study the exact gene expression, have fully utilized where the available evidences justify it and where their use is consistent with the effort involved and their routine application is recommended Terminologies : 6 BILAL Terminologies Primary culture a cell or tissue culture started from material taken directly from an organism.
Secondary culture a cell or tissue culture started from material taken directly from an organism.
Cell lineA cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space Introduction : 7 BILAL Introduction Many animal cells can, with special care, be induced to grow outside of their organ or tissue of origin.
Isolated cells or tissues can be grown in plastic dishes when they are kept at defined temperatures using an incubator and supplemented with a medium containing cell nutrients and growth factors.
The in vitro cultivation of tissues and cells is collectively known as tissue culture, and is used in many areas of science. Cells that can be grown in culture : 8 BILAL Cells that can be grown in culture The different cell types which can be grown in culture include
Connective tissue elements,
Skeletal tissue (bone and cartilage),
Cardiac and smooth muscle,
Epithelial tissue (liver, lung, breast, skin, bladder and kidney)
Endocrine cells (adrenal, pituitary, pancreatic islet cells)
Tumor cells. Basic equipment and facilities in animal cell culture : 9 BILAL Basic equipment and facilities in animal cell culture Sterile work area A separate room with an air flow Cabinet which supplies filtered air around the work surface. A HEPA (High Efficiency Particle Air Filter). A Laminar flow hood.
The cell culture laboratory will need to be furnished with an incubator or hot room to maintain the cells at 30-40 ºC. The incubation temperature will depend on the type of cells being cultivated. A CO2 controlled incubator. : 10 BILAL A CO2 controlled incubator. Slide 11: 11 BILAL Refrigerators and freezer (-20 ºC)
Both items are very important for storage of liquid media at 4ºC and for enzymes (e.g., trypsin) and some media components (e.g., glutamine and serum) at -20 ºC.
A simple inverted microscope is essential so that cultures can be examined in flasks and dishes. It is vital to be able to recognize morphological changes in cultures since these may be the first indication of deterioration of a culture. Slide 12: 12 BILAL Tissue culture ware
A variety of tissue culture plastic ware is available, the most common being specially treated polystyrene. A: Multi-well plate (6wells) Slide 13: 13 BILAL Washing up and sterilizing facilities
Glassware such as pipettes should be soaked in a suitable detergent, then soaked and washed with distilled water and then sterilized. All other equipment, such as automatic pipette tips and bottles (lids loosely attached) are autoclaved at 121 ºC for 20 min. Slide 14: 14 BILAL Liquid N2 / deep freezer
Invariably for continuous and finite cell lines, samples of cultures will need to be frozen down for storage. They should be frozen in phase of growth with a suitable preservative, usually dimethylsulfoxide (DMSO).
Water still or reverse osmosis apparatus
A double distilled or reverse osmosis water supply is essential for preparation of media, and rinsing glassware. Slide 15: 15 BILAL Filter sterilization
Media that cannot be autoclaved must be sterilized through a 0.22 mm pore size membrane filter. Slide 16: 16 BILAL Facilities for counting cells
It is possible to monitor cell growth by, however, more accurate cell counts are required for most experimental purposes.
The most commonly used device is the Improved Neubauer hemocytometer originally designed for counting blood cells. It consists of a thickened slide with a central chamber of known depth. The counting chamber is prepared and loaded with a suspension of single cells for counting. It is Slide 17: 17 BILAL important to aspirate the cell suspension adequately before loading the chamber in order
to break up clumps of cells which are difficult to count accurately. The counting chamber is examined under a microscope. Culture media : 18 BILAL Culture media A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells. The components of a suitable culture media include:
Glutamine & other amino acids
Antibiotics & antimycotics Supply and preparation of culture media : 19 BILAL Supply and preparation of culture media The choice of culture media used will depend on the type of primary cell, cell line, and the incubation conditions.
Culture media have a limited storage life and the recommendations indicated by the supplier should be followed.
Culture media can be supplied in powdered form which requires dissolving and filter sterilizing.
Bottles of media should be prepared in small batches, for instance two weeks supply at a time. Culturing animal cells : 20 BILAL Culturing animal cells Selecting sources of tissue for culture
Adult or embryonic tissue
Cultures can be derived from adult tissue or from embryonic tissue. Cultures derived from embryonic tissue generally survive and grow better than those taken from adult tissue. Tissues from almost all parts of the embryo are easy to culture, whereas tissues from adult are often difficult or even impossible to culture. Slide 21: 21 BILAL Normal or neoplastic tissue
Normal tissue usually gives rise to cultures with a finite lifespan while cultures from tumors can give continuous cell lines. Selecting types of animal cell culture : 22 BILAL Selecting types of animal cell culture Early attempts at culturing tissues relied upon the explantation of whole tissue or organ which could be maintained in vitro for only very short periods. Nowadays it is more usual to grow specific cell types from tissues, although there are still some situations where it is necessary to grow a whole organ (or a part of it). Slide 23: 23 BILAL Adherent or suspension culture
Cells may grow as an adherent monolayer or in suspension. Adherent cells are said to be anchorage-dependent and attachment to a substratum is a prerequisite for proliferation. They are generally subjected to contact inhibition, which means they grow as an adherent monolayer and stop dividing when they reach such a density that they touch each other. Basic techniques in animal cell culture : 24 BILAL Basic techniques in animal cell culture Aseptic techniques
In culturing animal cells, it is essential that all procedures are carried out using aseptic or sterile techniques. laminar flow facilities or sterile rooms provide a suitable environment, but even then aseptic techniques should be employed.
Basic aseptic technique is to ensure that the work area is clear, swabbed down regularly with 70% ethanol and that all the equipment used has been sterilized. Clean laboratory coats are also essential. . A: Performing all procedures in a laminar flow hood. : 25 BILAL . A: Performing all procedures in a laminar flow hood. . B: Using flames to fix microorganisms on container necks : 26 BILAL . B: Using flames to fix microorganisms on container necks C: Holding a bottlecap with the little finger. : 27 BILAL C: Holding a bottlecap with the little finger. D: Avoid touching tops of open vessels while transferringtheir content. : 28 BILAL D: Avoid touching tops of open vessels while transferringtheir content. Checking and prevention for contamination : 29 BILAL Checking and prevention for contamination Affected cultures should be disposed of into 2.5% hyperchlorite solution.
Media bottles known, or suspected, to be contaminated should be disposed of as well.
Some basic preventive measures may be taken:
1. checking sterilizing procedures (autoclave and oven procedures)
2. checking sterility of laminar flow hoods,
3. Regular checks on cultures;
4. disposal of contaminated cultures rather than attempting to decontaminate them. Preparation of primary cultures : 30 BILAL Preparation of primary cultures It is not always necessary to disaggregate tissue before culturing. Some embryonic tissues can be cultured simply by leaving the whole tissue on the flask surface and individual cells will simply grow out from the whole tissue and proliferate. After a few days the original tissue can be removed and the culture medium replenished to allow the new cells to continue growing. This method works for some tissues but it is not suitable for growing specific cells from a piece of tissue. Maintaining the culture : 31 BILAL Maintaining the culture If a primary culture is not to be used as such, it may be sub-cultured to produce a cell line. Some cells, such as macrophages and neurons do not divide in vitro and can only be used as primary cultures. Sub-culturing from primary to secondary cell culture : 32 BILAL Sub-culturing from primary to secondary cell culture A primary culture contains a very heterogeneous population of cells from the original explant. Some of these cells will die, some will fail to grow, and others will grow quickly and become the dominant cell type present. On sub-culturing the primary cell culture, the dominant types will become even more dominant. Sub-culturing, therefore, enables us to produce cell lines from our original explant Producing cell lines of a particular cell type : 33 BILAL Producing cell lines of a particular cell type We really have only two main options here. Firstly, of course, we must choose the appropriate tissue in the primary explant stage. It is no good attempting to produce a particular epithelial cell line if we use tissues which do not contain the appropriate cell type. Secondly, not all cells will grow equally well in the same medium. So, in principle, by selecting our medium carefully we may provide the condition most suited to our cells of interest. Propagating a cell line : 34 BILAL Propagating a cell line Once a cell line is established, it needs to be propagated in order to produce sufficient cells for characterization and storage, as well as for particular experiments. A cell line is given a name or code which identifies its source (for example HuT, Human T cells. Quantitation of cells in cell culture : 35 BILAL Quantitation of cells in cell culture We can divide the methods available for determining cell growth into two sub-groups. These are:
1- Direct methods
In the direct method, cell numbers are determined directly either by counting using a counting chamber or by using an electronic particle counter. In the indirect methods measurement of some parameters such as DNA content or protein content related to cell number is used as the method of estimating biomass. Direct methods for quantitation of cells in culture : 36 BILAL Direct methods for quantitation of cells in culture Counting chambers
Coulter counters Electronic coulter counter : 37 BILAL Electronic coulter counter Slide 38: 38 BILAL Electronic counters, of which the Coulter counter is the most widely used, provide rapid results, but high cell numbers are required to give an accurate count. Another disadvantage is that they cannot distinguish between dead and viable cells and clumps of cells may register as a single pulse thus leading to inaccurate counts. Indirect methods for determining cells in culture : 39 BILAL Indirect methods for determining cells in culture Other methods of quantitation such as radioisotope labelling and estimation of total DNA or protein are used less frequently. They are useful when cells are grown in microwell plates or as hanging drop cultures. DNA and protein assays are inaccurate, particularly if cells are multinucleated. They do not distinguish viable and non-viable cells. Cell viability determination : 40 BILAL Cell viability determination When cells are freshly isolated from a tissue or confluent monolayers are subcultured, the proportion of living, or viable, cells should be determined before they are used. This is most often determined by assessment of membrane permeability, under the assumption that a cell with a permeable membrane has suffered severe, irreversible damage. LDH leakage : 41 BILAL LDH leakage Leakage of cytosolic enzymes such as lactate dehydrogenase (LDH) provides a test that is similar in sensitivity to dye exclusion and can be more accurately quantitated but takes rather longer to perform. Applications of animal cell culture in pharmaceuticals : 42 BILAL Applications of animal cell culture in pharmaceuticals Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticance agents Slide 43: 43 BILAL Slide 44: 44 BILAL Tissue culture and engineering : 45 BILAL Tissue culture and engineering Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo.Tissue engineering has been defined as "understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use." Examples : 46 BILAL Examples Bioartificial liver device — several research efforts have produced hepatic assist devices utilizing living hepatocytes.
Artificial pancreas — research involves using islet cells to produce and regulate insulin, particularly in cases of diabetes.
Artificial bladders — Anthony Atala (Wake Forest University) has successfully implanted artificially grown bladders into seven out of approximately 20 human test subjects as part of a long-term experiment.
Cartilage — lab-grown tissue was successfully used to repair knee cartilage.
Artificial skin constructed from human skin cells embedded in collagen
Artificial bone marrow Vaccines : 47 BILAL Vaccines Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector, or the use of adjuvants. References : 48 BILAL References Pharmaceutical Microbiology By Purohit / Saluja
Biotechnology and pharmacy by Micheal E.Johnson and Henri R.Manase
Human Cell Culture Protocols Edited by Joanna Picot