cell culturing

cell culturing

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  1 Amjad Khan AfridiDate: 13th May, 2017 Basics of Cell Culture and Establishment of Cell Line • Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells. • Cell culture was first successfully undertaken by Ross Harrison in 1907 • Roux in 1885 for the first time maintained embryonic chick cells in a cell culture Major development’s in cell culture technology • First development was the use of antibiotics which inhibits the growth of contaminants. • Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel • Third was the use of chemically defined culture medium. Why is cell culture used for? Areas where cellculture technology is currently playing a major role. • Model systems for o Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies • Toxicity testing o Study the effects of new drugs • Cancer research o Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells • Virology o Cultivation of virus for vaccine production, also used to study there infectious cycle. • Genetic Engineering o Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles • Gene therapy o Cells having a functional gene can be replaced to cells which are having non-functional gene Tissue culture • In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture • Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells.
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  2 Amjad Khan AfridiDate: 13th May, 2017 Primary culture • Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture • Primary cells have a finite life span • Primary culture contains a very heterogeneous population of cells • Sub culturing of primary cells leads to the generation of cell lines • Cell lines have limited life span, they passage severaltimes before they become senescent • Cells such as macrophages and neurons do not divide in vitro so can be used as primary cultures • Lineage of cells originating from the primary culture is called a cell strain Continous cell lines  Most cell lines grow for a limited number of generations after which they ceases  Cell lines which either occur spontaneously or induced virally or chemically transformed into Continous cell lines  Characteristics of continous cell lines • smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio • Fast growth and have aneuploid chromosome number • reduced serum and anchorage dependence and grow more in suspension conditions • ability to grow upto higher cell density • different in phenotypes from donar tissue • stop expressing tissue specific genes Types ofcells On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into three.  Epithelial like-attached to a substrate and appears flattened and polygonal in shape  Lymphoblast like- cells do not attach remain in suspension with a spherical shape  Fibroblast like- cells attached to an substrate appears elongated and bipolar Establishment ofCell Line Introduction to Cell lines  A cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space.  A cell culture developed from a single cell and therefore consisting of cells with a uniform genetic make-up. Cell Culture  Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.
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  3 Amjad Khan AfridiDate: 13th May, 2017  The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. Primary culture  Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence).  At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth. Cell line or subclone  After the first subculture, the primary culture becomes known as a cell line or subclone.  The term cell line refers to the propagation of culture after the first subculture  Cell lines derived from primary cultures have a limited life span (i.e., they are finite), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. Cell strain  If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.
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  4 Amjad Khan AfridiDate: 13th May, 2017 Culture Conditions Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing the following:  a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals)  growth factors  hormones  gases (O2,CO2)  a regulated physico-chemical environment (pH, osmotic pressure, temperature)  Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture). Cryopreservation If a surplus of cells are available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed. Morphology ofCells in Culture Cells in culture can be divided in to three basic categories based on their shape and appearance (i.e., morphology). Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes,and grow attached to a substrate.
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  5 Amjad Khan AfridiDate: 13th May, 2017 Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches. Types ofCell Lines:  Finite cell lines  Continuous cell lines Finite Cell Lines :  Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite.  The cells normally divide 20 to 80 times (i.e. is 20-80 population doublings) before extinction. The actual number of doublings depends on the species, cell lineage differences, culture conditions etc.  The human cells generally divide 50-80 times, while murine cells divide 30-50 times before dying. Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface.
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  6 Amjad Khan AfridiDate: 13th May, 2017 Continuous Cell Lines or Immortal cell lines  When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.  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. Property Finite cell line Continuous cell line Growth rate Slow Fast Mode of growth Monolayer Suspension or Monolayer Yield Low High Transformation Normal Immortal, tumorigenic Ploidy Euploidy Aneuploid Anchorage dependence Yes No Contact inhibition Yes No Cloning efficiency Low High Serum requirement High Low Markers Tissue specific Chromosomal, antigenic or enzymatic Comparison ofproperties offinite and continuous cell lines
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  7 Amjad Khan AfridiDate: 13th May, 2017 Nomenclature ofCell Lines:  It is a common practice to give codes or designations to cell lines for their identification. For instance, the code NHB 2-1 represents the cell line from normal human brain, followed by cell strain (or cell line number) 2 and clone number 1.  While naming the cell lines, it is absolutely necessary to ensure that each cell line designation is unique so that there occurs no confusion when reports are given in literature.  Further, at the time of publication, the-cell line should be prefixed with a code designating the laboratory from which it was obtained e.g. NCI for National Cancer Institute, Wl for Wistar Institute. Selecting the Appropriate Cell Line  Consider the following criteria for selecting the appropriate cell line for your experiments: 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. Functional characteristics: What is the purpose of your experiments? For example, liver- and kidney- derived cell lines may be more suitable for toxicity testing. 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. Normal or transformed: Transformed cell lines usually have an increased growth rate and higher plating efficiency, are continuous, and require less serum in media, but they have undergone a permanent change in their phenotype through a genetic transformation. Growth characteristics: The following growth parameters need to be considered: i. Population doubling time ii. Ability to grow in suspension iii. Saturation density (yield per flask) iv. Cloning efficiency Stability: The stability of cell line with particular reference to cloning, generation of adequate stock and storage are important.
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  8 Amjad Khan AfridiDate: 13th May, 2017 Phenotypic expression: It is important that the cell lines possess cells with the right phenotypic expression. EXAMPLES OF ESTABLISHED CELL LINES May be derived from Normal or Tumor cells. Cell line Organism Origin Tissue HeLa Human Cervical cancer 293-T Human Kidney (embryonic) A-549 Human Lung carcinoma ALC Murine Bone marrow CHO Hamster Ovary HB54 Hybridoma Hybridoma FM3 Human Metastatic lymph node Animal cell lines and products Cell line Product Human tumour Angiogenic factor Human leucocytes Interferon Mouse fibroblasts Interferon Human Kidney Urokinase Transformed human kidney cell line, TCL-598 Single chain urokinase-type plasminogen activator (scu-PA) Human kidney cell (293) Human protein (HPC) Dog kidney Canine distemper vaccine Cow kidney Foot and Mouth disease (FMD) vaccine Chick embryo fluid Vaccines for influenza, measles and mumps Duck embryo fluid Vaccines for rabies and rubella Applications  Screening of the anti cancer drugs  Cell based bioassay  To determine the cytotoxicity  In vitro screening of severaldrugs  Production of antiviral vaccines  Cancer research,which required the study of uncontrolled cell division in cultures  Cell fusion techniques  Genetic manipulation  Study of the effects of toxins & pollutants using cell lines  Study of the function of nerve cells  Chromosome analysis of cells derived from womb