Cell culture environment
1. Laboratory design Cell culture is a sterile operation technique that requires that the working environment and conditions must be free from microbial contamination and from 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. The design principle of the cell culture chamber is generally that the aseptic operation area is located on the inner side of the room where the movement is less, and the conventional 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 called clean bench, it is divided into three categories: side flow type, direct current type and external flow type.
(2) Aseptic operation room: generally consists of three parts: locker room, buffer room and operation 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: ordinary incubator, centrifuge, water bath, fixed clock, ordinary balance and daily analytical processing
(4) Scrubbing and disinfection room: oven, disinfection pot, distilled water processor and acid tank.
(5) Analytical room: microscope, computer and printer.
3, culture vessels commonly used cell culture vessels have culture bottles, culture plates, petri dishes and so on. The amount of regular preparation is three times the amount used. The utensils should be made of materials with good transparency, non-toxicity, cell adhesion and growth, and commonly used disposable polystyrene products or neutral hardness glass products. The commonly used utensils are as follows.
(1) Liquid storage bottle: It is used to store various prepared culture liquids, serum and other liquids. The common specifications are 500 ml, 250 ml, 100 ml and so on.
(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
1 cm, allowing the straw to reach any part of the bottle. The specifications are 200 ml, 100 ml, 50 ml, 25 ml, 10 ml, etc.
(3) Petri dishes: for open culture and other uses. It is divided into several diameters of 30 mm, 60 mm and 120 mm.
(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 1 ml and 10 ml. 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 50 ml,
30 ml, 15 ml; the latter is mostly 10 ml and 5 ml.
(6) Others: such as a triangular flask, a beaker, a measuring cylinder, a funnel, a syringe, and the like.
4, cell culture temperature
To maintain the vigorous growth of cultured cells, there must be a constant and appropriate temperature. Different types of cells have different culture temperature requirements. 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 are generally damaged, only a small percentage may recover; at 41-42 ° C for 1 hour, the cells are severely damaged, most of the cells die, and some cells still recover; when the temperature is 43 ° C All the cells died within 1 hour. On the contrary, when the temperature is not lower than 0 °C, it has an effect on cell metabolism, but it has no harmful effect; when the cells are placed at 25-35 °C, the cells can still survive and grow, but the speed is slow; put at 4 °C After an hour, return to 37 ° C to culture, the cells can continue to grow. Cellular metabolism slows as temperature decreases. When the temperature drops below freezing, cells can die due to impaired cytoplasmic icing. However, if a certain amount of cryoprotectant (dimethyl sulfoxide or glycerol) is added to the culture solution, it can be stored for a long period of time at a low temperature such as -80 ° C or -196 ° C (liquid nitrogen).
5. Suitable gas environment
Gas is one of the necessary conditions for mammalian cell culture to survive. The required gases are mainly oxygen and carbon dioxide. oxygen
The gas participates in the tricarboxylic acid cycle, producing energy for supplying cell growth and proliferation and various components required for the growth of synthetic cells. In open culture, cells are typically 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 medium.
pH value. The optimum pH for most cells is 7.2-7.4, and deviating 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. However, some cells also prefer to grow in an alkaline environment, such as fibroblasts, which are most suitable for pH 7.4-7.6. Each cell has its optimum pH.
Cell culture aseptic operation basic technology
The aseptic technique is divided into three parts: the working environment and the surface treatment, the treatment of the glass and plastic products used for cell culture, and the treatment of the culture medium and the cultured cells.
The processing of the working environment is the most cost-effective means of using a laminar flow clean bench. When the ultra-clean workbench is working normally, the downward airflow can block outside air pollutants from entering the clean bench.
(1) Before the experiment, the sterile room and the aseptic table are sterilized by ultraviolet light for 30-60 minutes, the aseptic table is wiped with 70% alcohol, and the aseptic operation fan is turned on for 10 minutes before starting. Experimental operation. Only one cell is treated per operation to avoid cross-contamination of cells. After the experiment is over, the experimental items are taken out of the workbench. If you need to proceed to the next experiment, wipe the aseptic table with 70% alcohol and let the aseptic table fan run for 10 minutes before proceeding to the next experiment.
(2) The aseptic operation work area should be kept clean and spacious. The necessary items, such as test tube racks, pipettes or pipette tips, can be temporarily placed. Other laboratory supplies should be removed in time after use to facilitate gas circulation. Laboratory supplies should be wiped with 70% alcohol before being brought into the aseptic table. The experimental procedure should be carried out in the sterile area in the center of the station, not in the non-sterile area at the edge.
(3) Carefully remove the sterile laboratory supplies to avoid contamination. Do not touch the suction tube and the tip of the tip or the mouth of the container. Do not operate the experiment directly above the open container. After the container is opened, hold the bottle cap by hand and hold the bottle body, and use it at an angle of about 45°. Try not to put the cap on the table.
(4) The staff should pay attention to their own safety. They must wear the lab coat and gloves before conducting the experiment. Special care should be taken for cell lines derived from human or viral infections and an appropriate level of sterile workstation (at least two levels) should be selected. During the operation, avoid the generation of aerosols, beware of toxic reagents such as DMSO and TPA, and avoid sharp objects from injuring people.
(5) Regularly check the following items: CO2 pressure in CO2 cylinder; CO2 concentration, temperature, and water tray in CO2 incubator; whether the air pressure in the aseptic operating station is normal, regular replacement of UV lamp and HEPA filtration Filter, pre-filter (300 hours / pre-filter, 3000 hours / HEPA).
Culture cell growth process : incubation period → exponential proliferation period → stagnation period
Latent phase
After inoculation, the cells are first suspended in a suspension state in the culture medium. At this time, the cytoplasm retracts and the cell body is rounded. Then the cells are attached to the surface of the carrier, and the cells are attached to the wall, and the suspension period ends. It is closely related to cell types, medium components, and physical and chemical properties of the carrier. In general, primary cultured cells are slow to adhere to 10-24 hours or more, while passaged cell lines adhere quickly, usually 10 to 30 minutes. After the cells are attached to the wall, they need to go through a latent stage before entering the growth and proliferation period. The primary culture cells have a long incubation period of about 24-96 hours or longer, and the continuous cell line and tumor cells have a short incubation period of only 6-24 hours.
(2) logarithmic growth phase
This is the most vigorous stage of cell proliferation, and the number of dividing cells increases. The number of cell division phases in the exponential proliferative phase can be used as an important marker to determine whether cell growth is strong. It is usually expressed as the Mitotic index (MI), which is the number of dividing phases per 1000 cells in the cell population. Generally, the cell's division index is between 0.1% and 0.5%, and the primary cell division index is low, while the continuous cell and tumor cell division phase index can be as high as 3% to 5%. The best period of cell viability in the exponential phase is the best time to carry out various experiments and the best time to cryopreserved cells. When the number of cells inoculated is appropriate, after the exponential proliferation period lasts for 3-5 days, as the number of cells increases and the growth space decreases, the cells finally contact each other to form a synthetic sheet. Normal cells can inhibit cell movement after contact with each other. This phenomenon is called contact inhibition. However, the malignant tumor cells have no contact inhibition and can continue to move and proliferate, causing the cells to expand into a three-dimensional space and cause the cells to pile up. After the cells are exposed to the synthetic tablets, although the contact inhibition occurs, as long as the nutrition is sufficient, the cells can still undergo proliferation and division, and thus the number of cells is still increasing. However, when the cell density is further increased, the nutrient content in the culture solution is reduced, and the metabolites are increased, the cells are stopped due to nutrient depletion and metabolites, and this phenomenon is called Density Inhibition.
(3) Stagnate phase
After the number of cells reaches the saturation density, if they are not passaged in time, the cells will stop proliferating and enter the cessation period. At this time, the number of cells is flat, so it is also called the Plateau phase. Although cells do not proliferate during stagnation, they still have metabolic activity. If the separation is not carried out, the cells will be poisoned due to factors such as nutrient depletion, accumulation of metabolites, and decreased pH in the culture solution. The morphological changes will occur, and the adherent cells will fall off and severe death will occur. Therefore, they should be passaged in time.
Cultivating basic morphology of 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. Cells often have 1-2 nucleoli during the growth phase. When the cell function is poor, the cell contour is enhanced and the contrast is increased. If granules, de-drip and cavities appear in the cytoplasm, it indicates that the cells are poorly metabolized.
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 support when cultured. 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 may be referred to as fibroblasts when their morphology is similar to that of fibroblasts. This type of cell is named after the morphology of the fibroblasts in the body. The cells grow in a fusiform or irregular triangle on the surface of the support. The center of the cell has an ovate nucleus with cytoplasm extending outwards by 2-3 cm. Different lengths and lengths, except for true fibroblasts, tissue cells originating from the mesodermal mesenchyme often grow in this form.
(2) Epithelial cells This type of cells grow on a culture vessel support with a flat irregular polygonal shape with a circular nucleus in the center of the cell, and 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 and are 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.
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