Primary cell culture method
Primary cell culture is also called primary culture. It is the first culture of tissue cells obtained from donors in vitro. It is the first step in establishing cell lines and is a basic technology. Since the primary cells have just been separated from the tissue, the biological characteristics have not changed greatly, and the original genetic characteristics are retained. The closest to and reflect the growth characteristics in vivo, it is suitable for experimental studies such as drug sensitivity test and cell differentiation.
Primary cells often have a variety of cell components that are more confounding, even though they are morphologically of the same type (epithelial or fibroblast-like), but there are still large differences between cells. If the donors are different, even if the tissue type and location are the same, the individual differences still exist, and the primary cell biological characteristics are still unstable. If more rigorous comparative experimental research is needed, short-term subculture should be carried out.
1. Tissue block culture method Tissue block culture is a common culture method with common, simple, and high success rate. It is also a method for early cultivation of cells, so it was originally called tissue culture. The method comprises: injecting the cut small tissue mass into a culture bottle (or a dish), the bottle wall may be pre-coated with a thin layer of collagen to facilitate adhesion of the tissue block to the bottle wall, so that the surrounding cells can be outward along the bottle wall Growth, simple method, is conducive to culture, some kinds of tissue cells swim around the tissue block after 24 hours of adherence, and then gradually extend, grow into a halo that can be observed by the naked eye, tissue block after 5-7 days The central tissue cells gradually necrotic and floating, and the floating pieces can be discarded with the change of the fluid. The adherent cells extending around the tissue block also gradually form a layer, which can be observed under a microscope and used for experimental research. The cultivation method is as follows:
(1) The material was taken according to the aforementioned method, and the tissue was cut or cut into small pieces of 1 mm3 size, and a little medium was added to wet the tissue.
(2) Apply small pieces evenly to the bottle wall. The spacing between each small piece is 0.2 cm~0.5cm. Generally, it is suitable to inoculate 20~30 small pieces in a 25mL culture bottle (bottom area is 17.5cm2). After the small pieces are placed, Gently flip the flask so that the bottom of the flask is facing up, then add the appropriate amount of medium to the stopper and place the stopper in a 37 ° C incubator.
(3) After culturing for 2~4 hours, after the small pieces are attached, the culture bottle is slowly flipped and placed flat, and the culture is allowed to stand still. The movement is light, and it is strictly prohibited to shake and oscillate back and forth to prevent the small pieces from floating due to the impulse. If the culture fails, if the tissue block is not easy to adhere to the wall, a thin layer of serum, fetal juice or rat tail collagen may be applied to the bottle wall in advance. When starting the culture, the medium should not be too much, so that the tissue block can be kept moist. After the culture for 24 hours, the liquid should be replenished. During the initial movement and observation, it should be handled lightly. Try not to move for a few days to facilitate adherence and growth. When the culture is carried out for 3 to 5 days, the liquid can be exchanged, on the one hand, the nutrition is supplemented, and on the other hand, the toxic effects of metabolites and floating small pieces are removed.
Primary cell culture-2
2. Digestion culture method This method uses the above-described digestion and dispersion method to remove cell interstitial substances (including matrix, fiber, and the like) which hinder cell growth, disperse cells to form a cell suspension, and then culture in a divided bottle.
The digestion methods and procedures for certain types of cells, such as endothelial cells and bone cells, will be described in the relevant sections.
3. Suspension cell culture method For suspension growth cells, such as leukemia cells, lymphocytes, bone marrow cells, pleural fluid and ascites, cancer cells and immune cells do not need to be digested, and can be centrifuged at low speed, directly cultured, or stratified by lymphocytes. After the liquid was separated, the cells were inoculated.
4. Organ culture Organ culture refers to the cultivation of organs or tissue blocks from donors without direct tissue separation under specific environmental conditions in vitro. Its characteristics still maintain the organization and connection of the original organ cells and can survive. The purpose and technique of organ culture are different from those of single-layer cell culture, but organ culture can be used to observe the growth changes of organ tissues in vitro. And observe the effects of different culture conditions on organ tissue. Organ culture maintains the relative integrity of organ tissues and can be used to focus on the intercellular connections, alignments and interactions, as well as the biomodulation of the local environment. In vitro organ culture creates convenient conditions for clinical organ transplantation. Organ culture conditions are different from cell culture and require special requirements.
1. Special requirements for organ culture
(1) Special culture conditions are required Because the organs are cultured in vitro, their nutrients and oxygen are supplied only by natural infiltration. Therefore, in order to ensure that the center does not suffer from oxygen deficiency and nutrients, the thickness or diameter should not exceed 1 mm.
(2) The cells inside the organ need to have enough oxygen to penetrate. The following two methods are often used:
1 Place the organ tissue block on the gas-liquid surface of the medium to facilitate gas exchange.
To increase the partial pressure of oxygen in the medium, it is generally necessary to add pure oxygen, and it is necessary to maintain 5% CO2 when increasing the partial pressure of oxygen to maintain the pH.
2. Method of organ culture
(1) A stent made of a stainless steel mesh was placed in a petri dish at a height of 1/2 dish, and a 0.5 mm aperture filter was placed on the surface.
(2) Add the medium to the plate so that the medium is just in contact with the filter, but do not allow the filter to float.
(3) Place the organ tissue to be cultured flat on the filter membrane. Generally, the thickness should not exceed 200 μm, and the horizontal area should not exceed 10 mm 2 . If the liver and kidney are not more than 1 mm 2 .
(4) Place the culture in a CO2 incubator and add oxygen to adjust the partial pressure of oxygen to 90%. Keep the liquid level and the membrane at a consistent level during the culture.
(5) The above-mentioned organs can be used for experiments and tests after 1 to 3 weeks (changing every 2 to 3 days).
Primary cell culture and maintenance
(1) Primary cell culture
1. Resting adherent cells (including culture of semi-adherent cells)
The cells from the solid tissue treated by the digestive juice should be thoroughly rinsed to remove the toxicity of the digestive juice. The concentration of the cells should be slightly larger, at least 5×108 cells/L, and the medium can be used with Eagle (MEM) or DNEM. Culture, calf serum concentration of 10% ~ 80%, conditional should be cultured in a 37 ° C 5% CO2 incubator, in the first 2 days to minimize oscillation, to prevent the newly attached cells from falling off, float. If the primary adherent cells are not used for long-term culture, but are used for isolation and propagation or for measuring viruses, the cell concentration can be increased, and the layers should be thickened as much as possible to improve the titer of the virus and the measurement results (such as plaques). Obvious and accurate, until the cells are basically attached to the wall and gradually form a network. If the pH changes significantly at this time, the primary cells should be changed, that is, the old liquid should be poured out and replaced with fresh medium to remove aging. Dead cells and old media allow the adherent cells to get enough nutrients.
If the suspension cells in bone marrow or peripheral blood are cultured for 1 week, a small amount of myofibroblastic stromal cells or stromal cells may begin to adhere to the wall, in order to facilitate the adherence and growth of the adherent cells. After changing the liquid, the cell suspension should be centrifuged at a low speed, and the old solution should be discarded by adding half of the liquid to the new solution. Then the cell suspension is placed in the original bottle to continue the culture. After repeated fluid changes, the adherent muscle The sample cells gradually form a network. At this time, the original suspension cells and the culture medium can be transferred to another new bottle, and then the fresh medium is added separately (the new liquid is also added in half by the half-change method), and the original culture is continued. The adherent cells of the bottle gradually grow into a single layer, and secondary adherent cells appear in the new bottle. After several changes, the cells gradually grow into a single layer. In the case of such cells, the medium often needs to be added. A small amount of vitamin D3, bFGF, dexamethasone, calf serum (concentration should be more than 20%), in order to facilitate cell adhesion growth.
2. Culture of suspension cells All lymphocytes, hematopoietic stem cells and leukemia cells from peripheral blood, chest and ascites, spleen, lymph nodes, bone marrow, and red blood cells should be removed as much as possible in primary culture. If the short-term culture is applied to the test, it can be cultured in RPMI1640 medium containing 10% calf serum, and the cell concentration can be in the range of 5-8 x 109/L, and then subjected to a vial test. In order to long-term culture of lymphocytes and leukemia cells, growth factors should be added to the lymphocytes, and a small amount of the original patient serum should be added to the leukemia cells to facilitate cell growth, and the cells should start to proliferate or even form small clumps in the medium. The pH becomes sour, indicating that the cells grow well and reproduce. Generally, half of the liquid is changed every 3 days (the cells should not be lost when changing the liquid). When the cell proliferation is accelerated, the concentration is obviously increased, and the pH changes significantly, this may be considered. pass on. However, you must not rush to pass on, you must wait until the cell density is high, in order to prevent the failure of passage.
(2) Maintenance of primary cells
1. Adherent cells (including fluid exchange of semi-adherent cells)
When the adherent cells grow into a network or a basic monolayer, due to lack of nutrients, the metabolites increase, the pH becomes acidic, and the cells are not suitable for cell growth. At this time, the cells have not grown into a single layer, and the saturation density is not yet reached, and the culture needs to be continued. Therefore, a liquid exchange method is needed to update the nutrients to meet the needs of the cells to continue to grow and multiply. The liquid exchange method is relatively simple, that is, the old liquid is discarded, and the same amount of complete medium as the original culture liquid is added. If you want the cells to survive for a long time, but do not need to proliferate, replace them with a maintenance solution containing 2% calf serum.
2. Suspension cells are cultured and suspended in cell culture medium without adhering to the cells. The morphology and growth of the cells should be observed under the inverted microscope. The short-term culture of lymphocytes does not require liquid exchange, but is added to the growth. After factor or mitotic sources (PHA, PMA, COnA, PWM, LPS, etc.), the cells not only transform but also divide and multiply. At this time, the nutrients in the medium can not maintain the nutrient requirements of the cells, and the metabolic products increase. The pH becomes sour, the cells are not suitable for growth, and a liquid exchange is required. When leukemia cells or lymphoma cells are cultured in vitro for a long time, they need to be exchanged for culture, and can be passaged until saturation density is reached. When changing the liquid, only half the amount of liquid change can be used. Never change the liquid by adding the old liquid to the new liquid. The method of changing the amount of half is as follows:
The original culture flask is erected. If the cells sink to the bottom of the bottle within 30 minutes, half of the supernatant can be gently discarded by a pipette, and an equal amount of fresh complete medium is added. If the cells can not sink to the bottom of the bottle, the cell suspension can be aspirated. Half of the supernatant is discarded by low-speed centrifugation (1000r/min 10min), and the same amount of fresh complete medium is added. After mixing, transfer to the original bottle and continue to culture.
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