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Animal Tissue Culture Principles and Applications

    In this animal tissue culture principles and applications post we have briefly explained about types of animal cell tissue culture, cell line development methods, examples of common cell lines, applications of animal cell tissue culture, advantages and disadvantages of animal cell tissue culture.

    Animal Cell Tissue Culture

    Animal cell tissue culture refers to the process by which cells are grown in a controlled artificial environment. Cells can be maintained in vitro outside of their original body by this process which is quite simple compared to organ and animal cell tissue culture. In a cell culture technique, cells are removed from an animal or a plant and grown subsequently in a favourable environment. 

    For animal cell tissue culture the cells are taken from the organ of an experimental animal. The cells may be removed directly or by mechanical or enzymatic action. The cells can also be obtained by previously made cell line or cell strain. Examples of cells used to culture are fibroblast, lymphocytes, cells from cardiac and skeletal tissues, cells from liver, breast, skin, and kidney and different types of tumour cells.

    Types of animal cell culture

    Based on the number of cell division, animal cell tissue culture can be classified as primary cell culture and cell lines. Cell lines can undergo finite or infinite cell divisions.

    1. Primary cell culture

    This is the animal cell tissue culture obtained straight from the cells of a host tissue. The cells dissociated from the parental tissue are grown on a suitable container and the culture thus obtained is called primary cell culture. 

    Such animal cell tissue culture comprises mostly heterogeneous cells and most of the cells divide only for a limited time. However, these cells are much similar to their parents. Depending on their origin, primary cells grow either as an adherent monolayer or in a suspension.

    a. Adherent cells

    These cells are anchorage dependent and propagate as a monolayer. These cells need to be attached to a solid or semi-solid substrate for proliferation. These adhere to the culture vessel with the use of an extracellular matrix which is generally derived from tissues of organs that are immobile and embedded in a network of connective tissue. Fibroblasts and epithelial cells are of such types.

    When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, these are known as monolayer cultures. Majority of continuous cell lines grow as monolayers. As being single layers, such cells can be transferred directly to a cover slip to examine under a microscope.

    b. Suspension cells

    Suspension cells do not attach to the surface of the culture vessels. These cells are also called anchorage independent or non-adherent cells which can be grown floating in the culture medium. Hematopoietic stem cells (derived from blood, spleen and bone marrow) and tumor cells can be grown in suspension. These cells grow much faster which do not require the frequent replacement of the medium and can be easily maintained. 

    These are of homogeneous types and enzyme treatment is not required for the dissociation of cells; similarly these cultures have short lag period.

    c. Confluent Culture

    After the cells are isolated from the tissue and proliferated under the appropriate conditions, they occupy all of the available substrate i.e. reach confluence. For a few days, it can become too crowded for their container and this can be detrimental to their growth, generally leading to cell death if left for a long time. 

    The cells thus have to be subculture i.e. a portion of cells is transferred to a new vessel with fresh growth medium which provides more space and nutrients for the continual growth of both portions of cells. Hence subculture keeps cells healthy and in a growing state.

    A passage number refers specifically to how many times a cell line has been sub-cultured. In contrast with the population doubling level in that the specific number of cells involved is not relevant. It simply gives a general indication of how old the cells may be for various assays.

    2. Secondary cell culture

    When a primary culture is sub-cultured, it is known as secondary culture or cell line or sub-clone. The process involves removing the growth media and disassociating the adhered cells (usually enzymatically).

    Sub-culturing of primary cells to different divisions leads to the generation of cell lines. During the passage, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. However, as they are sub-cultured serially, they become different from the original cell.

    a. Finite cell lines

    The cell lines which go through a limited number of cell division having a limited life span are known as finite cell lines. The cells passage several times and then lose their ability to proliferate, which is a genetically determined event known as senescence. Cell lines derived from primary cultures of normal cells are finite cell lines.

    b. Continuous cell lines

    When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line. Such transformation/mutation can occur spontaneously or can be chemically or virally induced or from the establishment of cell cultures from malignant tissue. Cell cultures prepared in this way can be sub-cultured and grown indefinitely as permanent cell lines and are immortal.

    These cells are less adherent, fast growing, less fastidious in their nutritional requirements, able to grow up to higher cell density and different in phenotypes from the original tissue. Such cells grow more in suspension. They also have a tendency to grow on top of each other in multilayers on culture-vessel surfaces.

    Animal Tissue Culture Principles and Applications

    Cell line development methods

    1. Growth Requirements

    The culture media used for cell cultures are generally quite complex, and culture condition widely varies for each cell type. However, media generally include amino acids, vitamins, salts (maintain osmotic pressure), glucose, a bicarbonate buffer system (maintains a pH between 7.2 and 7.4), growth factors, hormones, O2 and CO2. To obtain best growth, addition of a small amount of blood serum is usually necessary, and several antibiotics, like penicillin and streptomycin are added to prevent bacterial contamination.

    Temperature varies on the type of host cell. Most mammalian cells are maintained at 37⁰C for optimal growth, while cells derived from cold-blooded animals tolerate a wider temperature range (i.e. 15⁰C to 26⁰C). Actively growing cells of log phage should be used which divide rapidly during culture.

    2. Process to Obtain

    Primary cell cultures are prepared from fresh tissues. Pieces of tissues from the organ are removed aseptically; which are usually minced with a sharp sterile razor and dissociated by proteolytic enzymes (such as trypsin) that break apart the intercellular cement. The obtained cell suspension is then washed with a physiological buffer (to remove the proteolytic enzymes used). 

    The cell suspension is spread out on the bottom of a flat surface, such as a bottle or a Petri dish. This thin layer of cells adhering to the glass or plastic dish is overlaid with a suitable culture medium and is incubated at a suitable temperature.

    Aseptic techniques

    Bacterial infections, like Mycoplasma and fungal infections commonly occur in cell culture creating a problem to identify and eliminate. Thus, all cell culture work is done in a sterile environment with proper aseptic techniques. Work should be done in laminar flow with the constant unidirectional flow of HEPA filtered air over the work area. All the material, solutions and the whole atmosphere should be of contamination-free.

    Cryopreservation

    If a surplus of cells is available from sub-culturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C until they are needed.  

    This stores cell stocks and prevents original cell from being lost due to unexpected equipment failure or biological contaminations. It also prevents finite cells from reaching senescense and minimizes risks of changes in long term cultures.

    When thawing the cells, the frozen tube of cells is warmed quickly in warm water, rinsed with medium and serum and then added into culture containers once suspended in the appropriate media.

    Examples of common Cell Lines

    HeLa cell line

    HeLa cells are one of the first continuous culture human cell lines with the help of cells of the cervical carcinoma.

    These cells are used for processes like virus cultivation and preclinical drug evaluation.

    b. HL 60 (Leukemia)

    c. MCF-7 (breast cancer cells)

    Applications

    Vaccines Production

    One of the most important uses of animal cell tissue culture is in research and production of vaccines. The ability to grow large amounts of virus in animal cell tissue culture eventually led to the creation of the polio vaccine, and cells are still used today on a large scale to produce vaccines for many other diseases, like rabies, chickenpox, hepatitis B, and measles.

    In early times, researchers had to use live animals to grow poliovirus, but due to the development of animal cell tissue culture, they were able to achieve much greater control over virus production and on a much larger scale which eventually develop vaccines and various treatments.

    However, continuous cell lines in animal cell tissue culture are not used in virus production for human vaccines as these are derived from malignant tissue or possess malignant characteristics.

    Virus cultivation

    Cell culture is widely used for the propagation of viruses as it is convenient, economic, easy to handle compared to other animals. It is easy to observe cytopathic effects and easy to select particular cells on which the virus grow as well as to study the infectious cycle.

    Cell lines are convenient for virus research because cell material is continuously available. Continuous cell lines have been extremely useful in cultivating many viruses previously difficult or impossible to grow.

    Molecular biology

    Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of different toxic compounds on the cells, and mutagenesis and carcinogenesis. The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

    Cancer Research

    Normal cells can be transformed into cancer cells by methods including radiation, chemicals, and viruses. These cells can then be used to study cancer more closely and to test potential new treatments.

    Gene therapy

    Cells having a functional gene can be replaced to cells which are having non-functional gene, and for which the cell culture technique is used.

    Immunology studies

    Cell culture techniques are used to know the working of various immune cells, cytokines, lymphoid cells, and interaction between disease-causing agents and the host cells.

    Others

    Cell lines are also used in in-vitro fertilization (IVF) technology, recombinant protein, and drug selection and improvement.

    Advantages

    The following are some of the advantages of animal cell tissue culture;

    1. Animal cell tissue culture is superior to other similar biotechnological approaches because it allows for the modification of various physiological and physiobiological conditions such as temperature, pH, and osmotic pressure.

    2. Animal cell tissue culture allows for studies on cell metabolism and the understanding of cell biochemistry.

    3. Animal cell tissue culture also allows for the study of the effects of various compounds, such as proteins and drugs, on various cell types.

    4. When a single cell type is used in animal cell cultures, the results are consistent.

    5. Animal cell tissue culture also allows for the identification of different cell types based on the presence of markers such as molecules or karyotyping.

    6. The use of animal cell culture in testing and other processes eliminates the need for animals to be used in experiments.

    7. Animal cell culture can be used to produce large quantities of proteins and antibodies, which would otherwise necessitate a significant investment.

    Disadvantages

    1. Despite the fact that animal cell tissue culture is a technologically advanced method, there are some drawbacks to this approach.

    2. Animal cell tissue culture is a specialised technique that necessitates the use of trained personnel as well as aseptic conditions. The technique is an expensive process because it necessitates the use of expensive equipment.

    3. In comparison to the original strain, the subsequent subculture of the animal cell tissue culture may result in differentiated properties.

    4. Animal cell tissue culture yields a negligible amount of recombinant proteins, which adds to the process’s costs.

    5. Mycoplasma contamination and viral infection are common and difficult to detect and treat.

    6. Because of the occurrence of aneuploidy chromosomal constitution, the cells produced by animal cell tissue culture are unstable.

    Further Readings

    Reference

    1. What is Animal Cell Culture? Definition, Types & Process – Biology Reader
    2. Animal cell culture – Online Biology Notes
    3. site.iugaza.edu.ps/siwini/files/2010/02/animal_culture_text_book_2.pdf
    4. Animal Cell Culture – Life Sciences Questions and Answers – Sanfoundry

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