secondary cell culture or cell line.pptx

secondary cell culture or cell line.pptx

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  Secondary Cell Cultureand Cell Lines Presented By - Heera V. Karemore Assistant Professor M.pharm
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  Secondary Cell Culture PrimaryCell Culture CELL LINE Sub - Culture  When Primary Culture is Sub – cultured it become secondary culture or cell line.
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  CELL LINES The developmentand various other aspects of primary culture are described above. The term cell line refers to the propagation of culture after the first subculture. In other words, once the primary culture is sub-cultured, it becomes a cell line. TYPES : 1. Finite cell lines 2. Continuous cell lines
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  FINITE CELL LINES The cells in culture divide only a limited number of times, before their growth rate declines and they eventually die.  The cell lines with limited culture life spans are referred to as finite cell lines. The cells normally divide 20 to 100 times (i.e. is 20-100 population doublings) before extinction.  The actual number of doublings depends on the species, cell lineage differences, culture conditions etc.  The human cells generally divide 50-100 times, while murine cells divide 30-50 times before dying.
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  CONTINUOUS CELL LINES A few cells in culture may acquire a different morphology and get altered. Such cells are capable of growing faster resulting in an independent culture.  The progeny derived from these altered cells has unlimited life (unlike the cell strains from which they originated).  They are designated as continuous cell lines. The continuous cell lines are transformed, immortal and tumorigenic.  The transformed cells for continuous cell lines may be obtained from normal primary cell cultures (or cells strains) by treating them with chemical carcinogens or by infecting with oncogenic viruses.
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  The most commonlyused terms while dealing with cell lines are explained below. Split ratio: The divisor of the dilution ratio of a cell culture at subculture. For instance, when each subculture divided the culture to half, the split ratio is 1: 2. Passage number: It is the number of times that the culture has been sub-cultured. Generation number: It refers to the number of doublings that a cell population has undergone.
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  NOMENCLATURE OF CELLLINES a) A code e.g. NHB for Normal Human Brain. b) A cell line number – This is applicable when several cell lines are derived from the same cell culture source e.g. NHB1 , NHB2. c) Number of population doublings, the cell line has already undergone e.g. NHB2/2 means two doublings.
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  SELECTION OF CELLLINES Several factors need to be considered while selecting a cell line. Some of them are briefly described. 1. Species: In general, non-human cell lines have less risk of biohazards, hence preferred. However, species differences need to be taken into account while extrapolating the data to humans. 2. Finite or continuous cell lines: Cultures with continuous cell lines are preferred as they grow faster, easy to clone and maintain, and produce higher yield. But it is doubtful whether the continuous cell lines express the right and appropriate functions of the cells. Therefore, some workers suggest the use of finite cell lines, although it is difficult. 3. Normal or transformed cells: The transformed cells are preferred as they are immortalized and grow rapidly.
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  4. Availability: Theready availability of cell lines is also important. Sometimes, it may be necessary to develop a particular cell line in a laboratory. 5. Growth characteristics: The following growth parameters need to be considered.  Population doubling time  Ability to grow in suspension  Saturation density (yield per flask)  Cloning efficiency. 6. Stability: The stability of cell line with particular reference to cloning, generation of adequate stock and storage are important. 7. Phenotypic expression: It is important that the cell lines possess cells with the right phenotypic expression.
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  MAINTENANCE OF CELLCULTURES For the routine and good maintenance of cell lines in culture (primary culture or subculture) the examination of cell morphology and the periodic change of medium are very important. Cell morphology: The cells in the culture must be examined regularly to check the health status of the cells, the absence of contamination, and any other serious complications (toxins in medium, inadequate nutrients etc). Replacement of medium: Periodic change of the medium is required for the maintenance of cell lines in culture, whether the cells are proliferating or non-proliferating. For the proliferating cells, the medium need to be changed more frequently when compared to non-proliferating cells. .
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  The following factorsneed to be considered for the replacement of the medium. 1. Cell concentration: The cultures with high cell concentration utilize the nutrients in the medium faster than those with low concentration; hence the medium is required to be changed more frequently for the former. 2. A decrease in pH: A fall in the pH of the medium is an indication for change of medium. Most of the cells can grow optimally at pH 7.0, and they almost stop growing when the pH falls to 6.5. A further drop in pH (between 6.5 and 6.0), the cells may lose their viability. 3.Cell type: Embryonic cells, transformed cells and continuous cell lines grow rapidly and require more frequent sub-culturing and change of medium. This is in contrast to normal cells, which grow slowly. 4. Morphological changes: Frequent examination of cell morphology is very important in culture techniques. Any deterioration in cell morphology may lead to an irreversible damage to cells. Change of the medium has to be done to completely avoid the risk of cell damage.
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  THANK YOU !