Basic requirements of cell culture medium Cell recovery Cell passaging Cell price
Cells cultured in vitro live directly in the medium, so the medium should be able to meet the cell's requirements for nutrients, growth factors, hormones, osmotic pressure, pH, etc.
1. Amino acids: All cells need 12 kinds of essential amino acids: valerian, liang, isoliang, su, lai, color, styrene, egg, group, casein, sperm, cystine.
2. Monosaccharides: Six-carbon sugar is the main energy source and the raw material for the synthesis of certain amino acids, fats, and nucleic acids. The cells have the highest ability to absorb glucose and the lowest galactose. When cultivating animal cells in vitro, glucose is used as an indispensable energy substance in almost all culture media or culture fluids.
3. Vitamins: Biotin, folic acid, nicotinamide, pantothenic acid, pyridoxine, riboflavin, thiamine, and vitamin B12 are all common ingredients in the culture medium.
4. Inorganic ions and trace elements: In addition to basic elements such as sodium, potassium, calcium, magnesium, nitrogen, and phosphorus, cell growth requires trace elements, such as iron, zinc, selenium, copper, manganese, molybdenum, and vanadium.
Growth Promoting Factors and Hormones Various hormones and growth factors play a very important role in maintaining the function of cells and maintaining the state of cells (differentiated or undifferentiated). Some hormones have a growth-promoting effect on the growth of many cells, such as insulin, which can promote the use of glucose and amino acids by cells. Some hormones can obviously promote a certain type of cells, such as hydrocortisone can promote the growth of epidermal cells, prolactin can promote the growth of breast epithelial cells.
Osmotic cells must live in an isotonic environment, and most cultured cells have a certain tolerance to osmotic pressure. The osmotic pressure of human plasma is 290mOsm / kg, which can be regarded as the ideal osmotic pressure for culturing human cells. The osmotic pressure of rat cells is around 320mOsm / kg. For most mammalian cells, the osmotic pressure is suitable in the range of 260-320mOsm / kg.
pH and gas are also one of the necessary conditions for the survival of cells. The abundance of gases required is oxygen and carbon dioxide. Oxygen participates in the tricarboxylic acid cycle, producing energy for cell growth, proliferation, and synthesis of various components. Some cells can obtain energy through glycolysis under hypoxia, but most cells cannot survive without hypoxia. In open culture, cells are generally cultured in a mixed gas environment of 95% air and 5% carbon dioxide. Carbon dioxide is both a cell metabolite and a component required by the cell. It is mainly directly related to maintaining the pH of the culture medium. Most animal cells require slightly alkaline conditions, with a pH of about 7.2 ~ 7.4 Â° C. During cell growth, as the number of cells increases and metabolic activity increases, carbon dioxide is continuously released and the culture fluid becomes acidic. The PH value changes. Since NaHCO3 is easily decomposed into carbon dioxide, it is very unstable, making it difficult to control the buffer system accurately. Therefore, this buffer system is suitable for closed culture. HEPES combined with sodium bicarbonate can provide a more effective buffer system, mainly to prevent the pH value from changing rapidly, but the biggest disadvantage is that it is difficult to maintain a normal pH value during open culture or observation. The pH fluctuations are mainly caused by metabolic CO2. In the closed cultivation process, CO2 combines with water to produce carbonic acid, and the pH of the medium quickly drops. To solve this problem, the NaHCO3-CO2 buffer system is used in the synthetic medium, and open culture is used to allow the CO2 generated by cell metabolism to overflow the culture bottle in time, and then the CO2 concentration (5%) in the incubator is stably adjusted and cultured The NaHCO3 in the base is in equilibrium.
Non-toxic and pollution-free
Cells grown in vitro have no resistance to microorganisms and some harmful toxic substances, so the culture medium should be free of chemical pollution, no microbial pollution (such as bacteria, fungi, mycoplasma, viruses, etc.), and no other biological activities that damage the cells Material contamination (eg antibodies, complement). For natural culture media, the pollution mainly comes from the material extraction process and the biological material itself, and materials should be selected and operated strictly. For the synthetic medium, the pollution mainly comes from the preparation process. The water used for the preparation should be very clean, and the preparation should be strictly filtered and sterilized. |||
Cell recovery and cryopreservation
1. Turn on the water bath and preheat to 37 degrees
2. Remove the frozen cells from the liquid nitrogen tank and quickly dissolve them in a 37-degree water bath
3. In the ultra-clean table, transfer the lysed cells into a centrifuge tube (first add about 2mL of culture medium to the centrifuge tube). Centrifuge at 1000RPM for 5 minutes
4. Discard the supernatant, add 1ML medium, pipette evenly, and transfer into the culture bottle ((4-5ML medium is added to the culture bottle first)) Cultivate the cells in the 5% CO2 incubator at 37 degrees 1. Digest the cells, transfer to a centrifuge tube and centrifuge, discard the supernatant 2. Proportion of frozen storage solution: 50% FBS + 40% medium + 10% DMSO (now used)
3. Add 1.5 cryopreservation solution to the centrifuge tube, pipette the cells evenly, and move into the cryopreservation tube.
Put it in the -20 degree refrigerator for 2-4 hours. 3. Put it into the -80 degree refrigerator overnight, put it into the liquid nitrogen tank the next day Add 1.5 cryopreservation solution to the centrifuge tube, pipette the cells evenly and move into the cryopreservation tube.
Freezing time. Place in a 4 degree refrigerator for 10 minutes. Put it in the -20 degree refrigerator for 30 minutes to 1 hour. No more than 1 hour. Finally put it into -80 degree refrigerator overnight. The cryopreservation solution put into liquid nitrogen the next day is 20% FBS + 10% DMSO + 70% medium.
Equipment and reagents Dry powder type culture medium, trypsin, penicillin, streptomycin. Pure water system, electronic balance, PH meter, magnetic stirrer and other specific steps
The cell culture water must be very pure and free of ions and other impurities. Fresh double distilled water, triple distilled water or purified water is required.
2. PBS (can also be used for the preparation of other BSS, such as: Hanks, D-Hanks solution):
Mainly used for rinsing and dissolving volume of tissues or cells during immunohistochemical staining: Pour medicine (NaCl 8.0g, KCl 0.2g, Na2HPO4? H2O 1.56g, KH2PO4 0.2g) into a beaker containing double distilled water, Stir the glass rod to dissolve it fully, then pour the solution into a volumetric flask to make the volume accurately to 1000ml, shake it to make a freshly prepared PBS solution. Adjust the pH to 7.4 with HCl or NaOH.
Transfer to the solution bottle for sterilization: Pour PBS into the solution bottle (large needle bottle), cover with a rubber cap, and insert the needle into the pressure cooker to sterilize for 8 minutes for 20 minutes. Note that after autoclaving, use sterile distilled water to replenish the evaporated water.
3. Trypsin solution:
The role of trypsin is to hydrolyze the proteins between cells to separate the cells. Different tissues or cells respond differently to pancreatic enzymes. The activity of pancreatin dispersing cells is also related to its concentration, temperature and action time. At a pH of 8.0 and a temperature of 37 Â° C, the action ability of the pancreatin solution is the strongest. When using pancreatin, the concentration, temperature and time should be grasped to avoid cell damage caused by excessive digestion. Since Ca2 +, Mg2 + and serum and protein can reduce the activity of pancreatin, BSS without Ca2 + and Mg2 + should be used when preparing pancreatin solution, such as D-Hanks solution. When the digestion is stopped, the effect of pancreatin on the cells can be terminated with serum culture medium or pancreatin inhibitor.
Weighing trypsin: According to the concentration of trypsin solution is 0.25%, use an electronic balance to accurately weigh the powder into the double-distilled water dissolved in a small beaker (if using double-distilled water need to adjust the pH to about 7.2) or PBS (D-hanks) In the liquid. Stir and mix and place at 4 Â° C overnight.
Sterilization by suction filtration with an injection filter: the prepared pancreatin solution should be filtered and sterilized with an injection filter (0.22 micron microporous filter membrane) in the ultra-clean table. Then dispense into vials and store at -20 Â° C for use.
4. 0.05% trypsin-0.02% EDTA solution
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