Cell and Tissue Culture PDF

CELL AND TISSUE CULTURE BY Prof. Dr. Hany Mohamed Samy Hassan Clinical Nutrition Program Faculty of Home Economics 4th Grade  A single cell is the building block for life  Cell A cell is the smallest structural and functional un...

CELL AND TISSUE CULTURE BY Prof. Dr. Hany Mohamed Samy Hassan Clinical Nutrition Program Faculty of Home Economics 4th Grade  A single cell is the building block for life  Cell A cell is the smallest structural and functional unit of life. Therefore it is referred to as a fundamental unit of life. The term cell was first coined in the year 1665 by an English scientist Robert Hooke. Among all the living organisms, some organisms are unicellular, consisting of only one cell, which is capable of performing all the life functions. These unicellular organisms include amoeba, bacteria, Protista. Multicellular organisms consist of different types of cells which have specialized functions. Plants, animals, human, and birds are examples of multicellular organisms. There are two different types of cells, the prokaryotic cells and the eukaryotic cells and these differences are mainly based on the presence and absence of the nucleus in their cell.  Tissue Tissues are groups of similar cells, working together to perform a specific function. The word tissue is mainly derived from “tissue” – a French word which is the past participle of the tisser (verb), “to weave”. In the plant kingdom, tissues are divided into two different types: Meristematic tissue and Permanent tissue. In the animal kingdom, tissues are divided into four different types: Connective Tissue: Blood, bone, cartilage, adipose, and lymph are examples of Connective Tissue. Muscle Tissue: Skeletal muscle, Cardiac muscle, and Smooth muscle are examples of Muscle Tissue. Nervous Tissue: Nervous tissue is seen in the brain, spinal cord, and nerves Epithelial Tissue: The surface of the skin, the reproductive tract, the airways, and the inner lining of the digestive tract are examples of Epithelial Tissue.  CELL TISSUE Cells are the smallest structural and functional units of an organism, which are characteristically Tissues are clusters of cells, specialized cells. microscopic. Found in both unicellular and multicellular Found only in multicellular organisms. organisms. Comprise of different cellular organelles, including Comprise of similar types of cells, specialized for the nucleus, mitochondria, lysosomes, Golgi a unique function. apparatus, etc. Four main types of tissue- Epithelial tissue, Two types of cells – Eukaryotic cells and Connective tissue, Muscular tissue, and Nervous prokaryotic cells. tissue. Has its own unique function. A group of similar cells combines together to perform a similar Functions include Growth, metabolism, and function. Example – Nerve cells of the nervous reproduction. system are involved in different functions of the nervous system. How do plant and animal cells differ? Both plant and animal cells are eukaryotic, so they contain membrane-bound organelles like the nucleus and mitochondria. However, plant cells and animal cells do not look exactly the same or have all of the same organelles, since they each have different needs. For example, plant cells contain chloroplasts since they need to perform photosynthesis, but animal cells do not. Both animal and plant cells have mitochondria, but only plant cells have chloroplasts. Plants don’t get their sugar from eating food, so they need to make sugar from sunlight. This process (photosynthesis) takes place in the chloroplast. Once the sugar is made, it is then broken down by the mitochondria to make energy for the cell. Because animals get sugar from the food they eat, they do not need chloroplasts: just mitochondria. Both plant and animal cells have vacuoles. A plant cell contains a large, singular vacuole that is used for storage and maintaining the shape of the cell. In contrast, animal cells have many, smaller vacuoles. Plant cells have a cell wall, as well as a cell membrane. In plants, the cell wall surrounds the cell membrane. This gives the plant cell its unique rectangular shape. Animal cells simply have a cell membrane, but no cell wall. Tissiue culture Tissue fragments from animals or plants are transplanted to an artificial environment where they can continue to thrive and function as part of the biological research technique known as tissue culture. One cell, a group of cells, an entire organ, or a portion of an organ may make up the cultured tissue. In culture, cells can divide, alter their shape, size, or function, engage in specialised activity or collaborate with other cells History Of Plant Tissue Culture The concept of tissue culture originated from the idea of totipotent—the ability of any plant cell to grow into a whole plant—a property of plant cells. Let’s have a look at the successive timeline for the development of the tissue culture technique: 1902: Gottlieb Haberlandt proposed the theoretical basis of plant tissue culture. He is known as the father of plant tissue culture. He experimented with isolated photosynthetic leaf cells but was unsuccessful in inducing any growth. However, he predicted that one can obtain artificial embryos from vegetative cells using this culturing technique and established the concept of totipotency. 1904: Henning isolated embryos of some crucifers and successfully grew on mineral salts and sugar solutions 1922: WJ Robbins and W. Kotte independently cultured small root tips of peas and maize. This led to the development of the concept of Organ culture. 1934: Gautheret cultured cambium cells of Salix caprea, and Populus nigra on Knop’s solution containing glucose and cysteine hydrochloride. The cultures survived a few months. 1939: Gautheret obtained the first established continuously growing tissue cultures from carrot root cambium. At this same point, P. R. White Demonstrated indefinite culture of tomato roots on subculturing in a liquid medium. 1941: J Van Overbeek demonstrated that coconut milk is essential for the growth and development of very young Datura embryos. 1942: P.R. White and A.C Braun began studying crown galls and tumor formation in plants. 1952: G. Morel and Martin C demonstrated that virus-free plants can be recovered from infected plants using shoot meristem culture. 1957: F. Skoog, and C.O. Miller proposed that a particular auxin-cytokinin ratio can regulate shoot and root initiation in cultured callus. 1959: G. Melchers and Bergmann L cultivated haploid tissues other than pollen for the first time. 1960: E.C Cocking isolated and cultured protoplasts after digesting the cell walls enzymatically and showed new cell wall regeneration on tomato fruit locule protoplasts. This same year L. Bergmann first obtained callus by transferring cells from suspension cultures on to solid medium and G. Morel developed a method of producing virus-free Cymbidium progenies through meristem culture. 1966: S G Guha and S C Maheshwari cultured anthers and pollen and produce haploid embryos. 1973: I. Potrykus attempted the first chloroplast and nucleus transfer from Petunia hybrida into albino protoplasts of the same species. 1974: J P Nitsch cultured microspores of Datura and Nicotina, doubling their chromosome number and harvesting seed from homozygous diploid plants within five months. At the same time, Mursahige developed the concept of developmental stages in cultures in vitro: Stage I: Establishment; Stage II: Multiplication; and Stage III: Rooting and hardening. 1975: G. Morel established cold storage of regenerated plants for a year. 1978: A. Zelcer, D. Aviv, and E. Galun devised a method for transferring organelles from one plant to another called Donor - Recipient protoplast fusion. 1981: P. J. Larkin and W. R. Scowcraft developed the concept of somaclonal variation. At the same time D. Wilson, G. Patnaik, G., and E. C. Cocking regenerated a whole plant from a single free cultured tobacco protoplast. 1986: J. D. Hamill, A. J. Parr, R. J. Robins, and M. J. C. Rhodes established hairy root cultures of Beta vulgaris and Nicotiana rustica following infection with Agrobacterium rhizogenes. Compared to in vitro roots of the same variety, the transformed cultures synthesized their characteristic secondary products. 1991: C. Sautter, H. Waldner, and G. Neuhaus developed a novel method for the acceleration of microprojectiles, called micro-targeting. Historical background for animal tissue culture Tissue culture involves the in vitro maintenance and propagation of cells in optimal conditions. Culturing animal cells, tissue or organs in a controlled artificial environment is called animal tissue culture. The importance of animal tissue culture was initially realized during the development of the polio vaccine using primary monkey kidney cells (the polio vaccine was the first commercial product generated using mammalian cell cultures). These primary monkey kidney cells were associated with many disadvantages such as: - Chances of contamination with adventitious agents (risk of contamination by various monkey viruses is high). - Most of the cells are anchorage-dependent and can be cultured efficiently only when they are attached to a solid or semi-solid substrate (obligatorily adherent cell growth). T- he cells are not well characterized for virus production. A scarcity of donor animals as they are on the verge of extinction. The foundation of animal tissue culture can be considered to have occurred in 1880: Arnold showed that leukocytes can divide outside the body. Then, in the beginning of the 19th century:Jolly investigated the behavior of animal cells in serum lymph. 1907 : Harrison developedment animal tissue culture commenced after the breakthrough frog tissue culture technique. Due to this effort Harrison is considered as the father of tissue culture. In his experiment he introduced tissue from frog embryos into frog lymph clots and showed that not only did the tissue survive, but nerve fibers grew out from the cells. During the mid-20th century, human diploid fibroblast cells were established by Hayflick and Moorhead. They named this cell line MRC-5 (a cell line of fibroblasts derived from lung tissue). (1964): Wiktor et al explored the utilization of this cell line in the production of rabies virus for vaccine production. After a couple of years they suggested a large- scale production protocol along with a method for the assessment of purified rabies vaccine immunogenicity. Losee and Ebeling cultured the first cancer cells and after a few decades the first continuous rodent cell line was established by Earle (1943). In 1951, Gay established that human tumor cells can give rise to continuous cell lines. The cell line considered as the first human continuous cell line was derived from a cancer patient, Henrietta Lacks, as mentioned above, and HeLa cells are still used very widely. Continuous cell lines derived from human cancers are the most extensively used resource in the modern laboratory.  Advantages of Plant Tissue Culture There are several advantages to using the tissue culture process. We already mentioned its effectiveness in helping developing countries to increase food production, but what are some other advantages that may be relevant to you? - The new plantlets can be grown in a short amount of time. - Only a small amount of initial plant tissue is required. - The new plantlets and plants are more likely to be free of viruses and diseases. - The process is not dependent on the seasons and can be done throughout the year. - You need only a relatively small space to perform the process (ten times the plants in one-tenth of the space). - On a larger scale, the tissue culture process helps to supply the consumer market with new subspecies and variety. - People looking to cultivate challenging plants such as specific breeds of orchid find more success with the tissue culture process than traditional soil. Disadvantages of Plant Tissue Culture - Tissue culture can require more labor and cost more money in building the facility and equipping the lab with all the instruments and chemicals. - There is a chance that the propagated plants will be less resilient to diseases when grown in outside conditions due to the type of environment they are grown in. - It is imperative that, before being cultured, the material is screened; failure to pick up any abnormalities could lead to the new plants being infected. - While the success rate is high if the correct procedures are followed, success with the tissue culture is not a guarantee. That's why accurate protocols are necessary to grow plants in tissue culture setting, which can be laborious when you try to create one working protocol by yourself. - Contamination is the major issue in tissue culture setting. Plants can get infected by bacteria, fungi, and viruses. That's why all measures should be taken and PPE kit should be used while performing tissue culture in your lab. - Tissue culture is an advanced technique and require some advanced knowledge and practice for anyone to get started in the field. Advantages of using animal cell culture - Homogeneity of cells can be maintained more easily by using selective media. - Physiological conditions (nutrient and hormone levels) as well as physio-chemical environment in the culture (temperature, pH, level of dissolved gases, and osmolarity) can be precisely controlled. - Cell-to-cell interactions, regulation of matrix, cell substrate attachment, and other micro-environment components of the cell can be controlled. - It is possible to store cell cultures in liquid nitrogen for a long period of time using an appropriate cryopreservation medium. - Various cell quantification techniques can be used to easily quantify cells in the culture. - Immune-staining or cytological techniques can be used to easily characterize cells in the culture. - Researchers can test the potential toxicity of drugs and other compounds using in vitro cytotoxicity studies, which use the cell culture technique. - Cell cultures can be used to study the effects of concentration and time on drugs, toxins, and pharmacologically active molecules. - Using cell culture techniques significantly reduces the use of animals in scientific experiments. - Monoclonal antibodies can be produced using cell culture with hydridoma technology. - Aids in the study of molecular pathways inside cells. - Helps researchers visualize various molecular and physiological events in the cells. Disadvantages of animal cell culture: - It can be challenging to maintain sterile aseptic conditions in vitro. - There are challenges involved in maintaining proper concentration of nutrients and serum, and standardization of medium as they vary among different cell types and their origin. - There is a high possibility of microbial and chemical contamination as well as cross contamination among different types of cells in culture. - There is a high possibility of cells undergoing chemical, physical and physiological changes, induced by the micro-environment inside the culture container. - Chances of genetic variation within a single cell population are very high because of the rapid growth rate of cells in artificial culture. - It is often difficult to identify cell types as the expression of marker proteins are often insufficient under in vitro conditions. - Setting up a cell culture requires a very high capital investment. - Proper maintenance of the cells requires considerable experience and expertise. - There are a limited number of passages in most of the primary cells in culture.