File Name: plant cell and tissue culture .zip
Plant Tissue Culture, Third Edition builds on the classroom tested, audience proven manual that has guided users through successful plant culturing A. The included experiments demonstrate major concepts and can be conducted with a variety of plant material that are readily available throughout the year. This book provides a diverse learning experience and is appropriate for both university students and plant scientists.
It requires specific technical skills to carry out successfully. This chapter describes the essential techniques of animal cell culture as well as its applications.
This chapter describes the basics of animal cell culture along with the most recent applications. The primary aim is to progressively guide students through fundamental areas and to demonstrate an understanding of basic concepts of cell culture as well as how to perform cell cultures and handle cell lines. This chapter gives insights into types of cell culture, culture media and use of serum, viability assays, and the translational significance of cell culture.
Cell culture is the process by which human, animal, or insect cells are grown in a favorable artificial environment. The cells may be derived from multicellular eukaryotes, already established cell lines or established cell strains. In the mids, animal cell culture became a common laboratory technique, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century. Animal cell culture is now one of the major tools used in the life sciences in areas of research that have a potential for economic value and commercialization.
The development of basic culture media has enabled scientists to work with a wide variety of cells under controlled conditions; this has played an important role in advancing our understanding of cell growth and differentiation, identification of growth factors, and understanding of mechanisms underlying the normal functions of various cell types.
New technologies have also been applied to investigate high cell density bioreactor and culture conditions. Many products of biotechnology such as viral vaccines are fundamentally dependent on mass culturing of animal cell lines. Although many simpler proteins are being produced using rDNA in bacterial cultures, more complex proteins that are glycosylated carbohydrate-modified currently have to be made in animal cells.
At present, cell culture research is aimed at investigating the influence of culture conditions on viability, productivity, and the constancy of post-translational modifications such as glycosylation, which are important for the biological activity of recombinant proteins. Biologicals produced by recombinant DNA rDNA technology in animal cell cultures include anticancer agents, enzymes, immunobiologicals [interleukins, lymphokines, monoclonal antibodies mABs ], and hormones. Animal cell culture has found use in diverse areas, from basic to advanced research.
It has provided a model system for various research efforts:. The study of basic cell biology, cell cycle mechanisms, specialized cell function, cell—cell and cell—matrix interactions. The characterization of cancer cells, the role of various chemicals, viruses, and radiation in cancer cells. Production of viruses for use in vaccine production e. Today, mammalian cell culture is a prerequisite for manufacturing biological therapeutics such as hormones, antibodies, interferons, clotting factors, and vaccines.
The first mammalian cell cultures date back to the early 20th century. The cultures were originally created to study the development of cell cultures and normal physiological events such as nerve development. Ross Harrison in showed the first nerve fiber growth in vitro.
However, it was in the s that animal cell culture was performed at an industrial scale. It was with major epidemics of polio in the s and s and the accompanying requirement for viral vaccines that the need for cell cultures on a large scale became apparent. The polio vaccine from a de-activated virus became one of the first commercial products developed from cultured animal cells Table Tissue culture is in vitro maintenance and propagation of isolated cells tissues or organs in an appropriate artificial environment.
Many animal cells can be induced to grow outside of their organ or tissue of origin under defined conditions when supplemented with a medium containing nutrients and growth factors.
For in vitro growth of cells, the culture conditions may not mimic in vivo conditions with respect to temperature, pH, CO 2 , O 2 , osmolality, and nutrition. In addition, the cultured cells require sterile conditions along with a steady supply of nutrients for growth and sophisticated incubation conditions. An important factor influencing the growth of cells in culture medium is the medium itself.
At present, animal cells are cultured in natural media or artificial media depending on the needs of the experiment. The culture medium is the most important and essential step in animal tissue culture. This depends on the type of cells that need to be cultured for the purpose of cell growth differentiation or production of designed pharmaceutical products. In addition, serum-containing and serum-free media are now available that offer a varying degree of advantage to the cell culture.
Sterile conditions are important in the development of cell lines. Cells from a wide range of different tissues and organisms are now grown in the lab. Earlier, the major purpose of cell culture was to study the growth, the requirements for growth, the cell cycle, and the cell itself. At present, homogenous cultures obtained from primary cell cultures are useful tools to study the origin and biology of the cells.
There are three methods commonly used to initiate a culture from animals. Whole organs from embryos or partial adult organs are used to initiate organ culture in vitro.
These cells in the organ culture maintain their differentiated character, their functional activity, and also retain their in vivo architecture. They do not grow rapidly, and cell proliferation is limited to the periphery of the explant. As these cultures cannot be propagated for long periods, a fresh explanation is required for every experiment that leads to interexperimental variation in terms of reproducibility and homogeneity.
Organ culture is useful for studying functional properties of cells production of hormones and for examining the effects of external agents such as drugs and other micro or macro molecules and products on other organs that are anatomically placed apart in vivo.
Fragments exercised from animal tissue may be maintained in a number of different ways. The tissue adheres to the surface aided by an extracellular matrix ECM constituent, such as collagen or a plasma clot, and it can even happen spontaneously. This gives rise to cells migrating from the periphery of the explant.
This culture is known as a primary explant, and migrating cells are known as outgrowth. This has been used to analyze the growth characteristics of cancer cells in comparison to their normal counterparts, especially with reference to altered growth patterns and cell morphology.
This is the most commonly used method of tissue culture and is generated by collecting the cells growing out of explants or dispersed cell suspensions floating free in culture medium. Cells obtained either by enzymatic treatment or by mechanical means are cultured as adherent monolayers on solid substrate. Cell culture is of three types: 1 precursor cell culture, which is undifferentiated cells committed to differentiate; 2 differentiated cell culture, which is completely differentiated cells that have lost the capacity to further differentiate; and 3 stem cell culture, which is undifferentiated cells that go on to develop into any type of cell.
Cells with a defined cell type and characteristics are selected from a culture by cloning or by other methods; this cell line becomes a cell strain. The monolayer culture is an anchorage-dependent culture of usually one cell in thickness with a continuous layer of cells at the bottom of the culture vessel. Some of the cells are nonadhesive and can be mechanically kept in suspension, unlike most cells that grow as monolayers e. This offers numerous advantages in the propagation of cells.
Passaging is the process of subculturing cells in order to produce a large number of cells from pre-existing ones. Subculturing produces a more homogeneous cell line and avoids the senescence associated with prolonged high cell density. Splitting cells involves transferring a small number of cells into each new vessel.
After subculturing, cells may be propagated, characterized, and stored. Adherent cell cultures need to be detached from the surface of the tissue culture flasks or dishes using proteins. Proteins secreted by the cells form a tight bridge between the cell and the surface. A mixture of trypsin-EDTA is used to break proteins at specific places.
Trypsin is either protein-degrading or proteolytic; it hydrolyzes pepsin-digested peptides by hydrolysis of peptide bonds. EDTA sequesters certain metal ions that can inhibit trypsin activity, and thus enhances the efficacy of trypsin. The trypsinization process and procedure to remove adherent cells is given in Flowchart Quantitation is carried out to characterize cell growth and to establish reproducible culture conditions.
Cell counts are important for monitoring growth rates as well as for setting up new cultures with known cell numbers. The most widely used type of counting chamber is called a hemocytometer. It is used to estimate cell number. The concentration of cells in suspension is determined by placing the cells in an optically clear chamber under a microscope. The cell number within a defined area of known depth is counted, and the cell concentration is determined from the count. For high-throughput work, electronic cell counters are used to determine the concentration of each sample.
In some cases, the DNA content or the protein concentration needs to be determined instead of the number of cells. Cells propagated as a cell suspension or monolayer offer many advantages but lack the potential for cell-to-cell interaction and cell—matrix interaction seen in organ cultures. For this reason, many culture methods that start with a dispersed population of cells encourage the arrangement of these cells into organ-like structures.
These types of cultures can be divided into two basic types. Cell—cell interactions similar to tissue-like densities can be attained by the use of an appropriate ECM and soluble factors and by growing cell cultures to high cell densities. This can be achieved by a growing cells in a relatively large reservoir with adequate medium fitted with a filter where the cells are crowded; b growing the cells at high concentrations on agar or agarose or as stirred aggregates spheroids ; and c growing cells on the outer surface of hollow fibers where the cells are seeded on the outer surface and medium is pumped through the fibers from a reservoir.
To simulate heterotypic cell interactions in addition to homotypic cell interactions, cells of differentiated lineages are re-combined. Co-culturing of epithelial and fibroblast cell clones from the mammary gland allows the cells to differentiate functionality under the correct hormonal environment, thus producing milk proteins.
These cells are obtained directly from tissues and organs by mechanical or chemical disintegration or by enzymatic digestion. These cells are induced to grow in suitable glass or plastic containers with complex media. These cultures usually have a low growth rate and are heterogeneous; however, they are still preferred over cell lines as these are more representative of the cell types in the tissues from which they are derived.
The morphological structure of cells in culture is of various types: 1 epithelium type, which are polygonal in shape and appear flattened as they are attached to a substrate and form a continuous thin layer i. These cultures represent the best experimental models for in vivo studies.
They share the same karyotype as the parent and express characteristics that are not seen in cultured cells. However, they are difficult to obtain and have limited lifespans. Potential contamination by viruses and bacteria is also a major disadvantage. Depending on the kind of cells in culture, the primary cell culture can also be divided into two types.
These cells require a stable nontoxic and biologically inert surface for attachment and growth and are difficult to grow as cell suspensions. Mouse fibroblast STO cells are anchorage cells. These cells do not require a solid surface for attachment or growth. Cells can be grown continuously in liquid media. The source of cells is the governing factor for suspension cells.
Blood cells are vascular in nature and are suspended in plasma and these cells can be very easily established in suspension cultures. When primary cell cultures are passaged or subcultured and grown for a long period of time in fresh medium, they form secondary cultures and are long-lasting unlike cells of primary cell cultures due to the availability of fresh nutrients at regular intervals.
Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:. Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant totipotency. Single cells, plant cells without cell walls protoplasts , pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones. Preparation of plant tissue for tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet.
Recent Advances in Plant in vitro Culture. Optimal growth and morphogenesis of tissues may vary for different plants according to their nutritional requirements. Moreover, tissues from different parts of plants may also have different requirements for satisfactory growth [ 1 ]. Tissue culture media were first developed from nutrient solutions used for culturing whole plants e. Plant tissue culture media should generally contain some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or nitrogen supplements, source s of carbon, undefined organic supplements, growth regulators and solidifying agents. According to the International Association for Plant Physiology, the elements in concentrations greater than 0. It should be considered that the optimum concentration of each nutrient for achieving maximum growth rates varies among species.
The techniques of plant organ, tissue, and cell culture concentrated on Secondary Metabolites in Tissue Cultures. Front Matter. Pages PDF.
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Overview DOI: Plant tissue culture PTC is a set of techniques for the aseptic culture of cells, tissues, organs and their components under defined physical and chemical conditions in vitro and controlled environment Fig. PTC technology also explores. Antibody Data Search Beta. Loyola-Vargas 1 ,. Quiroz-Figueroa 3.
In biological research, tissue culture refers to a method in which fragments of a plant or animal tissue are introduced into a new, artificial environment, where they continue to function or grow. While fragments of a tissue are often used, it is important to note that entire organs are also used for tissue culture purposes. Here, such growth media as broth and agar are used to facilitate the process. Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For this method, explants tissue from the plant are obtained from an in-vitro derived plant and introduced in to an artificial environment, where they get to proliferate. In the event that a plant material is used directly for this process, then it has to be sterilized to prevent tissue damage and ensure optimum regeneration. Embryo culture is the type of tissue culture that involves the isolation of an embryo from a given organism for in vitro growth.
It requires specific technical skills to carry out successfully. This chapter describes the essential techniques of animal cell culture as well as its applications. This chapter describes the basics of animal cell culture along with the most recent applications. The primary aim is to progressively guide students through fundamental areas and to demonstrate an understanding of basic concepts of cell culture as well as how to perform cell cultures and handle cell lines. This chapter gives insights into types of cell culture, culture media and use of serum, viability assays, and the translational significance of cell culture.
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