细胞培养基本知识（Cell culture basic knowledge）1 cell cultureCell culture is an important technology in biomedicine, and it is widely applied in various fields such as cell biology, biochemistry and clinical testing.1.1 source of cellsIt can be directly from human or animal tissues or indirectly from cell lines. The former is directly from the organism, and the biological indexes are closer to the body. However, the extraction process of normal cells is more complicated, and most cells can only be grown or can be transmitted from one to two generations, such as human vascular endothelial cells and mouse nerve cells. The advantages of cell line are more convenient, which can be purchased directly and used without separation process. Generally, it can be passed on a stable way, but its biological indicators are different from those in the body.1.2 laboratory requirements for cell cultureCell culture requires high cell culture, with the aim of enabling cells to survive in aseptic conditions. The surgical instruments that extract the cells should be sterilized with high temperature and high pressure before each use, and the water of the mixed culture medium is required to be used to double steam water and join the double resistance. It is better to use the culture bottle, centrifuge tube and other utensils. To operate in a sterile room. Before each operation, use theultraviolet lamp to illuminate the table 20-30min, then wash the hands clean, change the slippers into the sterile room, and use the new jure to extinguish the operation.Endothelial cells of human vascular endothelial cells were used to describe cell cultureBecause of the different nature of the cell, the separation and cultivation methods can be different. Its origin is usually fresh umbilical cord of umbilical vein (from) within 24 h after delivery, the separation of the specific method is: take neonatal umbilical cord blood (25 cm), and into the green, streptomycin each 100 u/mL of sterilization in PBS, 4 c preservation, experiment within 24 h.2.1. Materials and primary cultureOn both ends of the umbilical vein flatten each insert a syringe needle, hemostatic forceps clipping the umbilical cord in case needles sliding out, with a syringe needle inject PBS flushing of the blood vessels from heresy, no obvious blood flow until the PBS, the residual blood is rinsed clean, with PBS to fully exposed endothelial cells. Injection of 0.1? Collagenase solution (g/L) (prepared with PBS), after being residual PBS intravascular flow, sealed at the other end with a syringe needle, and continue to inject collagenase, filled with blood vessels, to digest the 20-30 min at room temperature, mastering digestion time, otherwise, the time is too short to digest is incomplete, cause waste; Too long, it may digest the smooth muscle cells together, causing biological contamination and interfering with the growth of endothelial cells. Infiltratethe digestive fluid into the sterile centrifuge tube, centrifugal (800r/min, 8min). Gently remove the upper cleaning fluid and add 5mL medium to the suspended cells in the centrifuge tube. Plus 2 mL fibronectin (1 mg/mL) in the culture bottle, evenly spread, and the remaining liquid absorption, placing for a while, the cells into a culture flask, CO2 incubation boxes to cultivate, in liquid after 24 h, 48 h after each change liquid once, 4 to 6 days between endothelial monolayer. Master the digestion time. If the time is too short, the digestion will not be complete, resulting in waste. The time is too long, and it will digest the smooth muscle cells together, causing biological pollution and interfering with the growth of endothelial cells.2.2. The batchesWhen endothelial cells are cultured in a bottle to form a dense single layer, carry out the generation. PBS rinse cells twice, plus 0.125? (g/L) trypsin / 0.02? EDTA (g/L)? Na2 1 ml, digestive cells was observed under the microscope cell shrinkage, immediately turn round, when observed that most of the cell falls off to join the culture, to end the role of the pancreatic enzyme, dropper percussion wall cells make them fall off and separation, centrifugal (800 r/min, 5 min), add broth, according to the proportion of 1:2 vaccination in culture bottle.2.3 cryopreservedEndothelial growth to a certain number, the temporary unused cells, to the frozen in liquid nitrogen in standby, namely: thecells with trypsin digestion down, add a small amount of nutrient solution, centrifugal (800 r/min, 5 min), abandon the clear liquid, add fluid cells cryopreserved (10?) (volume fraction) dimethyl sulfoxide broth, dispatch cell density to 106 cells/mL, put in cells cryopreserved tubes, 1 mL per tube,Plug in the foam box (to slow down the cooling rate), put in the -70c refrigerator overnight, and quickly transfer to liquid nitrogen for the second day. The frozen cells are recovered when used. When it comes to recovery, invest in 37? In C water bath, it is constantly vibrated to cause rapid melt, centrifugal (800r/min, 5min), and discard the cleaning fluid to remove the dimethyl sulfoxide. Add the appropriate amount of culture medium and cultivate it in a culture bottle. Cryopreservation and recovery, a simple generalization is to slow freeze-thaw.2.4 the otherDetermine which cells are to be cultured, depending on the object and purpose and the Angle of the problem. For example, the vascular endothelial cells should be cultured in order to study the vascular problems of atherosclerosis. In order to study the problem of senile dementia, the nerve cells should be further studied.Determine which cells are to be cultured, and then consider which properties or characteristics of the cells to study. The research method can be selected according to the cell surface marker or secretion function. Cell surface markers can be used for cell immuno-chemistry and immunofluorescence. Using cell secretion, ELISPOT and other methods can be used.Application of cell culture3.1 molecular biology researchDifferent organisms express some kind of material difference, and there are differences in the expression of different cells. Does the difference come from genetic levels or from protein levels? Further study can be done through reverse transcription PCR (rt-pcr) method. First of all, the cell culture for a sufficient number of cells, the cells of the total RNA extracted, then reverse transcription for cDNA, by PCR, differences in the levels of genes can be amplified, thus can clear differences from the gene mRNA (transcription) or from the protein level (translation). The differences in the expression of the glucocorticoid receptor (GR), as studied in the laboratory, were in the transcription level.3.2 gene therapyCell culture also has a big role in gene therapy. In this laboratory, the cultured SY5Y cells were transfected into SY5Y cells by ps-1. Then, rt-pcr was used to show that ps-1 mRNA transcription was present in cells with ps-1 gene transfected with the expression of ps-1 protein in monoclonal antibody. It can then be used to detect whether cells have an earlier appearance. In a similar way, we can study the calcium channels of cells and the membrane pathways of other drugs to guide clinical drug applications and study new drugs accordingly.The 2009-05-29 caught a replyWhite people do things home to doSeven fansCore member 6The third floorA liquid medium is kept in cold storage. Frozen?Refrigerate!! As the liquid medium is frozen and then dissolved, the pH of the solution will change, the solution is often alkaline, and the dissolution of certain components will also affect the growth of the cell. Therefore, the liquid medium must be stored in the refrigerator, and usually the liquid medium can be stored for 6 months to a year under refrigeration conditions.The function and use of glutamine in liquid medium.Almost all cells have high requirements for glutamine, which requires glutamine to synthesize proteins, and in the absence of glutamine, cells grow poorly and die. Therefore, a large amount of glutamine is found in various cultures. Glutamine is very unstable in solution, should be set to -20? C freeze preservation, use the former to add medium. A liquid medium with glutamine? C refrigerators should be rejoined to the original amount of glutamine when stored for more than two weeks. The center of basic medical cells has a high concentration (100 times) glutamine solution in its service project (1ml/branch).The final concentration was 2mM/ml medium for each 1ml of glutamine added to a complete 99ml complete medium. The package is pink, -20? C.3. Effect of pH on cell growthSince most cells are suitable for a pH of 7.2 ~ 7.4, deviations from this range will have a detrimental effect on the cell.The requirements for pH of various cells are not exactly the same, and the original culture cells are generally tolerant to pH changes, and the infinite cell lines have strong tolerance.But in general, the cells are more acidic than alkaline, and the acid environment is more conducive to cell growth. Therefore, we can slightly adjust the pH of the liquid to a slightly more acidic solution when preparing the liquid. When the liquid is filtered through a 0.10 um or 0.22 um filter membrane, the pH of the solution will float upwards of 0.2.4. The higher the pancreatic enzyme concentration, the better?Not really! Because the digestion of trypsin solution is related to the pH, temperature, pancreatic enzyme concentration, and whether the solution contains Ca2 +, Mg2 + ion and serum. Normally, pH 8.0, temperature 37? C is the most powerful. In addition, calcium, magnesium ion and serum can greatly reduce their digestion. Must be used before every time we extend the digestion, D - Hanks rinsed repeatedly cell culture fluid or PBS fluid bottle, serum containing medium is rinsed clean, can be so it can greatly improve the digestiveability of digestive juices.Usually the concentration of digestive juices is:(1) 0.05 % of trypsin + 0.02 % EDTA(2) 0.25 % of the enzyme + 0.03 % EDTA(3) 0.25 % trypsinSolution pH8.0 ~ 9.0; Use D - Hank's liquid preparation. Most cells can be digested using 0.05 % of trypsin + 0.02 % EDTA.5. Problems that should be noted during the use of media:(1) the medium should be 37 before use. C preheat or re-use at room temperature.(2) in the process of cell culture, the sucker that has been absorbed through the culture medium can no longer be burned by the flame, preventing the culture of the residue remaining in the straw from coking and bringing harmful substances into the culture medium.(3) minimize the exposure time of various liquids and cells.(4) avoid cross-contamination between liquids and cells.(5) when the complete medium is made, it is best to use it in two weeks.6. Related problems of serum:Newborn bovine serum: new born cattle are made from blood in 5 daysCalf serum: mavs are made from blood collection within 16 weeks of birth.Fetal bovine serum: (import serum) requires a laparotomy.Domestic manufacturers often cannot do, often take birth 2 days inside the blood is called fetal bovine serum.According to the production process of serum and the contents of hemoglobin and endotoxin are divided into:(1) the serum of the special fetal bovine: 40 nanometer filtration, the content of endotoxin is less than 10EU, and the content of hemoglobin is less than 10mg/dl.(2) superior fetal bovine serum: after 3 100 nm filtering, the content of endotoxin was less than 25EU/ml, and the hemoglobin content was less than 25mg/ml.(3) standard fetal bovine serum: 3 times 100 nanometer filtration, low endotoxin, low hemoglobin content.(4) activated carbon/dextran treatment serum: the hormone content is greatly reduced.(5) dialysis fetal bovine serum: the content of hypoxanthine and thymic nucleotide were significantly reduced.7. Other cell culture solution: except medium, serum, glutamine, other kinds of liquid are also necessary and must not be omitted.(1) double resistance: 200 times, (1ml/branch) its final concentration is penicillin 100U/ml; Streptomycin 100ug/ml, -20? C.(2) l-glutamine: 100 times, (1ml/branch), final concentration of 2mM, -20? C.(3) sodium pyruvate: 50mM, 4ml/branch, and final concentration of 1mM/ml, -20? C.(4) HEPES fluid: a mixture of 1M (5ml/branch), a HEPES added to the 200ml full medium, the final concentration of 25mM/ml, room temperature preservation.8. Mycoplasma pollution and detection:(1) mycoplasma pollution is the most common and difficult to be found in cell culture, but mycoplasma pollution can significantly affect the results of cell function interference. Mycoplasma is the smallest microorganism that is currently known to live independently of the bacteria and virus. It has no cell wall and is highly pleomorphic, with a minimum diameter of 0.2 um, which can pass through the filter.At present, the situation is not optimistic through the detection of more than 100 plants and cells in China. The pollution rate is 30% ~ 60%. The study group introduced cell lines from abroad and the pollution of mycoplasma often appeared. After the mycoplasma is contaminated, it inhibits cell growth by affecting the cell's DNA, RNA synthesis and the rapid consumption of amino acids in the medium, which can reduce the rate of cell fusion. Therefore, in the application of various cell lines for experimental study, it is important to prove that the cell has the pollution of mycoplasma.(2) after the mycoplasma contamination, the medium is not turbid, and the cell lesion is slight or not obvious, therefore it is difficult to detect. Currently, there are several methods of mycoplasma detection.(a) phase contrast microscope observation; (b) low-tension lichen-dyeing method; (c) fluorescence staining method; (d) enzyme labeling method;(e) PCR method: basic medical cell center used this method for detection.Advantages: high sensitivity, short test time, small sample size (only 50ul cell culture solution).Disadvantages: high cost of primer and preparation.。
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