Shanghai Hengyuan_Cell Culture Technology
The culture is defined as a single cell or cell population. Cells must live in an artificial environment when they are cultured. Due to changes in the environment, the movement of cells or the influence of some other factors, the cultivation time is longer, and the passage leads to the singularization of cells. Widely used advantages in modern medicine and biological science research: 1. Direct observation of the morphological structure and life activities of living cells. It is used in cytology, genetics, immunology, experimental medicine and oncology. 2. Direct observation of changes in cells can facilitate photography. 3. Study cell types from low to high to human, embryo to adult, normal tissue to tumor. 4. It is easy to use various techniques: phase contrast, fluorescence, electron microscopy, histochemistry, isotope labeling and other methods to observe and study the cell status. 5. It is the research object of molecular biology and genetic engineering and its main component. 6. Easy to apply the experimental research of physical and chemical biology. 7. It is easy to provide a large number of experimental objects with similar biological properties, and it is more economical with less cost. 8. Become a material source for the production of monoclonal antibodies and genetic engineering of biological products. Disadvantages: Tissues and cells live independently in an artificial culture environment after being isolated. Although they mimic the in vivo environment, there are still great differences. Therefore, when using cultured cells for experiments, it should not be considered that the cells in the body are exactly the same. The results of the experiment are guessed in the body, and it is easy to draw a conclusion equivalent to the body. Cell types Cell types are divided into: Attached suspension type (1) Attached cells grow on the surface of the support, and cells that only depend on attachment to grow are called attached types. The phenomenon of cells (Anchorrage-dependent cells) is related to cell differentiation. Typing of attached cells 1. Fibroblasts: (fibroblast) From mesoderm Features: Similar in shape to fibroblasts in the body, with fusiform or irregular triangles in the cell body, round nucleus in the center, and cytoplasm extending outward 2 to 3 protrusions with different lengths. When the cells grow, they are radial, swirling or flaming. Origin: The cells come from mesoderm mesenchymal tissue. In addition to true fibroblasts, heart muscle, smooth muscle, osteoblasts, and vascular endothelial cells. Note: It is a common name for cells to become fibroblasts in culture, which is different from cells in vivo. 2. Epithelial cell type (epithlium cell type) Features from ectoderm: flat irregular polygon, round nucleus, cells are closely connected to form an increased number of cell proliferation, the entire epithelial membrane moves with it. Marginal cells rarely detach from the cell population and move independently to "pull the net" phenomenon is related to the origin of endoderm tissue. Tissue: Skin epidermis and its derivatives (sweat glands, sebaceous glands) digestive tract epithelium, liver, pancreas, alveolar epithelium. 3. Wandering cell type (Wandering cell type) characteristics: scattered on the support, not connected into a piece, the cytoplasm protruding pseudopods or protrusions are active wandering or deforming movement, fast and irregular, high density connection It is not easy to distinguish from other types of cells in a polygonal shape. 4. Polymorphic cell type (polymorphic cell type) characteristics: irregular morphology, such as nerve cells. Suspended cell type (suspended cell type) Features: Not attached to the growth of the support, the cell body is round, the growth space in the culture medium is large, it can grow for a long time, and the reproduction is vigorous to facilitate the study of cell metabolism. S180 sarcoma, white blood cells in the blood, K562, HL-60. Purpose of typing: It is convenient to describe the morphological changes of cells during the cultivation process. When the culture conditions are good, the cells are relatively stable, which can reflect the difference between the origin and normal abnormality, and serve as an indicator to determine the biological characteristics of the cell. Emphasis: The general form is not a reliable indicator to be affected by all aspects. Such as: repeated opening and closing of the thermostat, temperature, CO2 concentration, medium alkali, mycoplasma, pH value. The cells were in a triangle-shaped state at the time of inoculation. After a certain period of culture, the cells had formed epithelial cells before passage. Cell growth and proliferation The living environment of cultured cells is culture flasks, utensils or other containers, and the living space and nutrition are limited. When the cells proliferate to a certain density, a part of the cells are separated and the nutrient solution is renewed to make the cells survive better. This process is called "passage" (passage or subculture). It is worth noting that each passage of cells is affected to some extent in terms of growth and proliferation. The life span of culture cells refers to the time during which cells continue to proliferate and grow during culture. Generally depends on the type of cell, traits and the age of the original donor. For example, diploid fibroblasts can be transmitted for 30 to 50 generations without cryopreservation and repeated passage, which is equivalent to 150 to 300 cell proliferation cycles, can maintain a year of life, and cells begin to apoptosis. ). During the survival process, the cells undergo the following three phases: 1. Primary culture period (primary culture) The time from tissue to first passage is about 1 to 4 weeks. Cell culture characteristics: Cells move actively, divide, but are not vigorously more than double Body karyotype. The morphological structure and functional activities of primary tissues and internal tissues are basically similar. The genetic traits of each cell are not related to each other, and the cells are highly interdependent. If this dilution is dispersed into single cells, the cloning efficiency of the cells cloned in soft agar medium decreases, indicating that the cells have poor independent viability. 2. The primary cultured cells in the passage (passage) are called the cell line (cell line) after passage. Features: The cells proliferate vigorously and maintain the diploid karyotype, also called the diploid cell line. To preserve the nature of diploid cells, the cells should be stored frozen in the first generation or early passage. Generally, cells are frozen within 10 passages. Cryopreservation formula: Add protective agent dimethyl sulfoxide (DMSO) or glycerin to the culture broth and seal it in the ampoule. The temperature in liquid nitrogen can reach -196 ℃, which can be stored for a long time. If the cells recover after thawing, they can still continue to proliferate and grow, and the cell traits are not affected, becoming the most important means of preserving cells. 3. Characteristics of recession phase: the cell is still alive, proliferation is slow, non-proliferation profile is enhanced, and apoptosis is reduced For example, the virus may be contaminated, the temperature and pH may be unstable, and the cells may undergo spontaneous transformation; the cells may acquire immortality or malignancy. Cells acquire persistent proliferative capacity. Such a cell population is called a continuous cell line. After the cells became undead, most of the karyotypes became heteroploid (heteroploid) contact inhibition disappeared. One-Generation Cell Lifetime "One Generation" refers to the time it takes for cells to be seeded until they are separated and then cultured. Generation with cell multiplication is not a meaning. In the first generation of cells, cells can be doubled 3 to 6 times and go through three stages: cell proliferation 1. Latent phase: the inoculation period (the cytoplasm retracts and the cell body is round) adheres to the wall after inoculation. It takes 10 to 24 hours or more for primary culture cells. Continuous cell lines and cancer cell lines are attached after 10 to 30 minutes. The attachment phenomenon is very complicated and affected by many factors. Affects cell attachment: The substrate surface is not clean, which affects cell attachment. Facilitates cell attachment: positive substances and special substances on the surface of the substrate: fibronectin (Fibronectin FN), cell surface protein (cell surface proteinCSP) Some of these substances exist on the cell surface, and some come from serum. Characteristics of incubation period: The length is related to cell seeding density, type, and the nature of the medium used. (1) The cells enter the incubation period after attachment, and the cells do not proliferate. The split phase is rare, and the cells have motor activity. (2) The incubation period of primary cultured cells is about 24-96 hours or longer, and the incubation period of continuous cell lines and tumor cells is about 6-24 hours. (3) The cell inoculation density is large and the incubation period is short. (4) When the division phase of the cell increases, it marks the cell enters the exponential hyperplasia phase. 2. Exponential growth phase (logarithmic growth phase): Features: the most prosperous phase of cell proliferation, the division phase increases. Cell division index (mitotie index MI): indicates the number of division phases per 1000 cells. Conditions: The number of split phases is related to cell type, culture components, and pH incubator temperature. Cell division index: Primary cells: 0.1% to 0.5%, continuous dividing cells and tumor cells: 3 to 5%. The exponential hyperplasia period is the best period for the cell generation and the best and most important stage for conducting experiments. The exponential hyperplasia period lasts for 3 to 5 days. After the number of cells increases, the growth space becomes smaller, and the cells can be connected to each other to form a piece. Normal cells: Cells in contact with each other can inhibit cell movement. This phenomenon is called contact inhibition. Tumor cells: There is no such phenomenon, and it can be used as one of the marks to distinguish normal cells from tumor cells. When tumor cells reach a certain density, they develop into a three-dimensional space, causing the cells to pile up. As cells continue to proliferate and divide, the number of cells continues to increase, the nutrient content of the culture fluid decreases, and metabolic products increase. Cells are affected by nutrient depletion and metabolites. Density inhibition occurs, causing cell division to stop. 3. Stagnate phase (stagnate phase) The cell volume and density reach saturation, and the cells stagnate and proliferate. It shows that the cells have entered the stagnation phase, and the number of cells is flat, also known as plateau. Features: The cells do not proliferate and have metabolic activities. The nutrients in the culture medium have been gradually depleted, the accumulation of metabolic products has increased, and the pH has been reduced. At this time, passages are required, otherwise the cells will be poisoned, morphological changes will occur, and the cells will fall off the substrate and die. Note: Passing too late will affect the growth of the next generation of cells, at least one or two passages will be passed, and dead cells and lightly damaged cells will be eliminated by changing the medium. Use it after all the cells have recovered.
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