Jinma analysis of cell culture technology basic information - Database & Sql Blog Articles

I. Cell culture medium The concept of cell culture refers to a culture technique in which the cells in the body tissues are taken out from the body tissues, and the environment is grown, propagated, and maintained in a sterile, appropriate temperature, pH and certain nutrient conditions. . The culture of the cell culture is a single cell or a population of cells. The most widely used in medical genetics research are peripheral blood lymphocytes, skin or fibroblasts, and various cell lines that can grow in vitro for a long time. Peripheral blood lymphocyte culture has the advantages of short time, simple technology, and reusable materials. It is the most widely used in clinical chromosome analysis. In vitro cultured cell lines can be transformed spontaneously or under external action during the culture process, becoming a permanent cell line, or can be directly constructed into a permanent cell line, and the permanent cell line can be passaged and grown in vitro. Permanent cell lines typically have a non-identical feature of aneuploid cells and karyotypes of individual cells. However, this feature of cell cloned cell lines may not be apparent. Second, the environmental cells of the cell culture require the same conditions in vitro as the cells in the body. 1. Non-polluting environment The culture environment is non-toxic and sterile, which is the primary condition for ensuring cell survival. When the cells are placed in vitro, the cells lose their defense against microorganisms and toxic substances compared to the body, and once contaminated or accumulated by their own metabolic substances, they can cause cell death. Therefore, in the culture, maintaining the cell living environment without pollution, timely removal of metabolites, etc., is the basic condition for maintaining cell survival. 2. Constant temperature maintains vigorous growth of cultured cells and must have a constant and appropriate temperature. The standard temperature for human cell culture is 36.5 ° C ± 0.5 ° C. Deviating from this temperature range, the normal metabolism of cells will be affected or even die. The tolerance of cultured cells to low temperature is stronger than that of high temperature. When the temperature rise does not exceed 39 °C, cell metabolism is proportional to temperature; human cells can be damaged at a temperature of 39-40 ° C for 1 hour, but it is still possible to recover; At 40-41 ° C for 1 hour, the cells will be generally damaged, only a small half may recover; 1 hour at 41-42 ° C, the cells are severely damaged, most of the cells die, individual cells still recover; when the temperature is 43 ° C All of the cells died within 1 hour. 3, gas environment gas is one of the necessary conditions for human cell culture survival, the main gas is oxygen and carbon dioxide. Oxygen participates in the tricarboxylic acid cycle, producing the energy required to supply cell growth and proliferation and the various components required for the growth of synthetic cells. In open culture, the cells are generally placed in a mixture of 95% air plus 5% carbon dioxide. Carbon dioxide is both a cellular metabolite and a component required for cell growth and reproduction. Its main role in cell culture is to maintain the pH of the medium. The optimum pH for most cells is 7.2-7.4, and deviations from this range will have a detrimental effect on cell culture. However, cell acid resistance is greater than alkali resistance and is more conducive to cell growth in a fatty acid environment. Some data show that the original amniotic fluid cells are optimal for PH6.8. The most common method for adjusting the pH concentration of cell culture fluids is to add NaHCO3. Because NaHCO3 is available for CO2, but dioxane is easy to escape, it is most suitable for closed culture, and hydroxyethylpiperazine ethanesulfonic acid (HEPES) is It is non-toxic to cells, also acts as a buffer, and has the property of preventing rapid changes in pH. It is used in open cell culture technology, and its greatest advantage is that it maintains a relatively constant pH value in open culture or cell observation. 4. Cell culture medium is the basic material for supplying cell nutrition and promoting cell reproduction and proliferation in cultured cells, and also the living environment for culturing cell growth and reproduction. There are many kinds of culture media, and they are divided into semi-solid medium and liquid medium according to their material state; they are divided into synthetic medium and natural medium according to their sources. (1) Synthetic medium: The synthetic medium is strictly prepared according to the kind and quantity of the substances required by the cells. Contains carbohydrates, amino acids, lipids, inorganic salts, vitamins, micronutrients and cell growth factors. Although the cells used alone survive but do not grow well. (2) Natural medium: The most common natural medium used is serum, which is most commonly found in calf serum. Serum contains a variety of cell growth factors, pro-adhesive factors and their multiple active substances. In combination with the Synechic Medium, the cells can be proliferated and grown. The most common use is 5-20%. Third, cell culture facilities and basic conditions 1, laboratory design Cell culture is a sterile operation technology, the work environment and conditions must be guaranteed to be free of microbial contamination and other harmful factors. The cell culture chamber and design principles are designed to prevent microbial contamination and harmful effects, requiring a clean working environment, fresh air, dry and smokeless dust. Cell culture work includes: working fluid preparation, aseptic processing (sampling), incubation, aseptic processing, storage of cells and supplies, and the like. The design principle of the cell culture chamber is generally that the aseptic operation area is set on the inner side of the room where the movement is less, the routine operation and the closed culture are in one chamber, and the washing and disinfection is performed in the other chamber. 2, common facilities and equipment (1) ultra-clean workbench: also known as purification workbench, divided into three categories of side-flow, DC and outflow. (2) Aseptic operation room: generally consists of three parts: the dressing room, the tempering room and the operating room. Place a clean bench and carbon dioxide incubator, centrifuge, inverted microscope, etc. in the operation room. Refrigerators, refrigerators, and sterile sterile items can be placed in the buffer room. (3) Operation room: common incubator, centrifuge, water bath, fixed clock, ordinary balance and daily analysis and processing items. (4) Scrubbing and disinfection room: oven, disinfection pot, distilled water processor and acid tank. (5) Analytical room: microscope, computer and printer. 3, culture vessels Cell culture is based on glassware, often prepared to use the most three times the most. Vessels should be made of transparent, non-toxic, neutral hardness glass products. There are several types of commonly used glassware. (1) Liquid storage bottle: used to store various prepared culture fluids, serum and other liquids, often replaced by 500ml, 250ml, 100ml saline or plasma bottles. (2) Culture flask: Different culture flasks have different shapes depending on the type of cultured cells. The cells used for cell subculture need uniform bottle wall thickness, which is convenient for cell adherence growth and observation. The size of the bottle mouth should be the same, and the caliber is generally not Less than 1cm, the straw can be inserted into any part of the bottle. The specifications are 200ml, 100ml, 50ml, 25ml, 10ml and so on. Common 10ml common round bottle for peripheral blood culture. Both culture bottles are required to be made of high quality glass. (3) Petri dishes: for open culture and other uses. Divided into several diameters of 30mm, 60mm, 120mm and so on. (4) Straw: Commonly used are long straws and short straws, long straws are also called scale straws. The modified tube has a spherical scale on the upper part of the improved straw, and a graduated straw is used to move the liquid. It is commonly used in 1ml and 10ml. The short straw is also called the dropper, which is divided into elbow and straight. (5) Centrifuge tubes: Centrifuge tubes are the most widely used vessels in cell culture. Depending on the application, the centrifuge tubes are commonly used in cell culture. There are two types of centrifuge tubes: large abdominal tipped centrifuge tubes and common pointed bottom centrifuge tubes. The former is 50ml, 30ml, 15ml; the latter is mostly 10ml and 5ml. (6) Others: such as a triangular flask, a beaker, a measuring cylinder, a funnel, a syringe, and the like. 4. Cultured cell morphology In vitro cultured cells can be classified into two types, patch-type growth and suspension-type growth, depending on whether they can be attached to the support on the culture vessel. The attached cells can be attached to the surface of the branch during growth. For example, amniotic fluid cells are adherent cells, often expressed as fibroblasts and epithelial cells. Suspension cells are grown in suspension in culture. 1. Fibroblasts Cells in culture can be called fibroblasts when they are similar in morphology to fibroblasts. This type of cell is named after the morphology of the fibroblasts in the body. The cells grow in the shape of a fusiform or irregular triangle on the surface of the support. The center of the cell has an ovate nucleus with cytoplasm extending 2-3 cm outward. Different lengths and lengths, except for the real fibroblasts, the tissue cells originating from the mesodermal mesenchyme often grow in this form. 2, epithelial cells This type of cells grow on the culture vessel support with flat irregular polygonal features, the center of the cell has a circular nucleus, the cells are closely connected to a single layer of membrane-like growth. Epithelial morphological growth occurs in tissue cells derived from endodermal and ectodermal cells such as skin, epidermal derivatives, and gastrointestinal epithelial cells. 3, migratory cells This type of cells are scattered on the support, generally not connected into pieces. The cytoplasm often protrudes from a pseudopod or protuberance, exhibiting active migration or deformation, and is fast and irregular. This type of cell is not very stable, and sometimes it is difficult to distinguish it from other types of cells. Under certain conditions, after the cell density is increased, the film may be in the form of a polygonal shape or a fine fiber. Common in the early stage of amniotic fluid cell culture. V. Analysis of cultured cells The cultured cells vary in shape depending on the shape of the attached support, and the most common one is attached to the planar support cells. The cells in the normal light microscope are homogeneous and transparent, and the structure is not obvious. When cells have 1-2 nucleoli in the growth phase, when the cell function is poor, the cell contour will increase and the contrast will increase. If granules, de-drip and cavities appear in the cytoplasm, it indicates that the cells are poorly metabolized. 6. Cleaning and Disinfection of Culture Products At present, the cell culture vessels in China mainly use glassware that can be used repeatedly. The main purpose of cleaning is to remove impurities and microorganisms, so that no ingredients affecting cell growth remain in the vessel. Therefore, cleaning and disinfecting in tissue cell culture is an extremely important part. (a) Cleaning In tissue culture, in vitro cells are very sensitive to any harmful substances. Microbial products are accompanied by sundries, and the last cell residues and non-nutrient chemicals can affect the growth of cultured cells. Therefore, the newly used and reused culture vessels should be thoroughly and thoroughly cleaned, and different cleaning methods should be selected according to the composition of the vessels. 1, glassware cleaning tissue cell culture, the largest use of glassware, so the most important work is the cleaning of glassware. Generally, the cleaning of glassware includes four steps of soaking, brushing, invading and rinsing. The cleaned glassware only requires clean, clear, oil-free traces, and no material remains. (1) Soaking: The glassware after initial use and culture should be soaked with water first to soften the deposit or be dissolved by the solution. The new glassware used for the first time has a large amount of dry dust on the surface of the glass during production and transportation, and the surface of the glass is often alkaline and has some harmful substances to the cells. The new bottle should be simply brushed with tap water before use and then soaked in dilute hydrochloric acid (5) overnight to neutralize the alkaline material. Reusable glassware is often accompanied by a large amount of freshly used protein. It is not easy to be washed off after drying. Therefore, it should be immersed in water immediately after use, and it is required to be completely immersed, and no air bubbles or floating on the liquid surface. (2) Brushing: The glassware after soaking should be brushed with a brush and detergent to remove the relatively tight impurities on the surface of the vessel. Brushing should be moderate and excessive will damage the surface gloss of the vessel. (3) Pickling: The cleaning solution is prepared by a certain proportion of potassium dichromate, concentrated sulfuric acid and distilled water, and the treatment process is called pickling. The cleaning solution has no corrosive effect on the glassware, and its strong oxidation can remove trace impurities that cannot be washed away. The cleaning solution has a strong decontamination ability. It is a key part of the cleaning process. When soaking, the vessel should be filled with cleaning liquid. Do not leave air bubbles or utensils to expose the cleaning liquid. The soaking time is generally overnight and should not be less than 6 hours. The cleaning solution can be made into different strengths according to the needs. The following three types are commonly used: potassium dichromate (g) concentrated sulfuric acid (ml) distilled water (ml) (A) strong cleaning liquid 63 1000 200000 B) secondary strong cleaning liquid 120 200 1000 (C) Weak cleaning liquid 100 100 100 Cleaning liquid should be prepared with care. Wear acid-resistant gloves and apron, and protect the exposed parts of the face and body. Potassium dichromate can be dissolved in water during the preparation process, and then concentrated sulfuric acid is slowly added. Stirring with a glass rod is carried out to volatilize the generated heat. Potassium dichromate can be dissolved in water during the preparation process, and then concentrated sulfuric acid is slowly added. Stirring with a glass rod, the generated heat is volatilized, and the preparation solution should be selected from plastic products. After the preparation, the cleaning liquid is generally brownish red. (4) Flushing: After use, the glassware must be thoroughly rinsed with water after brushing and soaking. Keep it as free as possible from any contamination or cleanliness. It is preferred to use a washing device for rinsing. That is to save effort and good results. If it is operated by hand, it needs to be rinsed more than ten times. The water should be filled and cleaned every day. It is best to wash it with distilled water for 3-5 times and dry it for later use. 2. Cleaning of rubber plugs The rubber products used in cell culture are mainly stoppers. The newly purchased cork has a large amount of talcum powder and impurities. It should be washed first with tap water and then routinely. The conventional cleaning method is: soak it in water immediately after each use, then boil it with 2% NaOH or washing powder 10- 20 minutes to remove the protein in the culture. After washing with tap water, soak it in 1% dilute hydrochloric acid for 30 minutes or rinse with distilled water, then boil for 10-20 minutes, and let it dry for use. 3, plastic products cleaning plastic self-products are now non-toxic and has been specially processed packaging, open the package can be used, mostly disposable items. If necessary, soak overnight with 2% NaOH, rinse thoroughly with tap water, soak for 30 minutes with 5% hydrochloric acid solution, rinse with tap water and distilled water, and dry for use. (2) The biggest danger of disinfection cell culture is the contamination of bacteria, fungi and viruses caused by culture. The pollution is mainly caused by the negligence of the operator. Common causes are uncleanness in the operation room or surrounding space, culture vessels and If the culture solution is unqualified or incomplete, the failure of each link in the culture can lead to failure of the culture. Therefore, every step of the cell culture should strictly abide by the operating routine to prevent contamination. There are three types of disinfection methods: (A) Physical sterilization (UV, damp heat, slag, etc.). (B) Chemical sterilization (various chemical disinfectants). (C) Antibiotics. (1) UV disinfection: used for air, surface of the console and cannot be disinfected and cultured using other methods. Direct ultraviolet radiation is convenient and effective. After a certain period of time, most of the bacteria in the air can be eliminated. The ultraviolet light in the culture room should not exceed 2.5 meters from the ground, and the disinfected items should not be shielded from each other. Less than disinfection. Ultraviolet rays can produce ozone, pollute the air, reagents and culture fluids have adverse effects, and also cause damage to human skin. It is not suitable for near-irradiation experiments. (2) Warm disinfection: high pressure steam disinfection is the most widely used and best disinfection method. When warming and disinfecting, the disinfectant items should not be overfilled to prevent gas clogging in the sterilizer and millions of dangers, ensuring the circulation of gas inside. Before heating and boosting, first open the exhaust air to discharge the cold air in the sterilizer. After the cold air is exhausted, close the exhaust valve and check that the safety valve is free to move, then start to increase the pressure. When the required pressure is reached, Start to calculate the disinfection time. During the disinfection process, the operator cannot leave the job, and should regularly check the pressure and safety to prevent disinfection and epidermal accidents. Disinfection pressure and time of common items: culture liquid, rubber products, 10 pounds and 10 minutes; cloth, glass products, metal equipment, 18 pounds and 20 minutes. The top two are the most common physical disinfection methods. (3) Chemical disinfection method: The most common is 70% alcohol and 1 ‰ clean and dry, the former is mainly used for the operator's skin, the surface of the console and the wall of the sterile room. The latter is mainly disinfected by soaking of the instrument and wiping the skin of the skin and the operating room. The chemical disinfection method is simple, convenient and effective. (4) Antibiotic disinfection: It should be recorded as antibiotic sterilization, mainly used for sterilizing culture liquid or preventing culture pollution.