Several factors affecting cell growth, Abstract: Cells are cultured in vitro and lose their regulation and control. Therefore, in addition to meeting the nutritional requirements, the cell survival environment must be close to the living environment. The external environment culture conditions such as temperature, osmotic pressure, pH, etc. Can affect the growth of cells. When the cells are cultured in suspension, the stirring speed should not be too fast or too slow. If it is too slow, the cells tend to agglomerate, sink and adhere, which is not conducive to the proliferation of cells in suspension. If it is too fast, it is easy to foam. The cells are easy to suffocate and die. At the same time, the strong agitation may cause the cells to rupture due to mechanical damage. Therefore, it is important to choose a suitable stirring speed. For example, the suitable speed of BHK-21 cells is 460-330r/min; lymphocytes (Raji cells) The strain has a fast growth rate at 200r/min and 400r/min (Namalva cells). It does not agglomerate at 80-1000r/min, and does not require anti-foaming agent. The cell growth is still fast. Suitable for various cells. The mixing speed is inconsistent, usually related to the characteristics and quality of the cells, and should be noted. Household Cleaner,Grease Cleaner Kitchen,Household Carpet Cleaner,Cleaning Spray Wuxi Keni Daily Cosmetics Co.,Ltd , https://www.wxkenidaily.com
Generally, the suitable temperature for in vitro culture of mammalian and avian cells is 37~38 °C. The temperature is too high or too low, which will affect the growth of cells. The ability of cells to withstand low temperature is stronger than that of heat resistance. At low temperature, cell metabolism Vitality and nuclear division are reduced. When the temperature is lower than 0 °C, although it affects cell metabolism, it has no harmful effect. When the cells are placed at 23~25 °C, the cells can still survive and grow, but the speed is slow, but the fish cells The suitable culture temperature is 23~25°C. If the temperature is too low, the cell will be damaged by extracellular water and cytoplasmic freezing when it falls below the freezing point, but if glycerol or dimethyl sulfoxide is added to the medium, (DMSO) and other protective agents, sealed in ampoules, placed in liquid nitrogen or low temperature refrigerator (-70 ° C), can play a protective role, at this time the cells can withstand temperatures below -70 ° C, can be stored for a long time, after thawing Cell resuscitation can still continue to grow and proliferate, and cell traits are not affected at all. This is the main means of preserving cells.
High temperature is not good for cell culture. Cells cultured at 39~40°C for 1h will be damaged, but they may recover, but they can't stand the temperature increase of 2°C for several hours, that is, culture at 41~42°C for 1h. The operation is severe. When the temperature reaches above 43 °C, most of the cells are killed. The high temperature mainly causes the inactivation of the enzyme, the destruction of the lipidoids, the destruction of the nuclear fission, the coagulation enzyme causes the cells to coagulate, and the protein is denatured. Therefore, the cells are cultured in vitro. Always avoid high temperatures.
Second, osmotic pressure:
In hypotonic solution in high-permeability solution, cells can immediately shrink or swell and rupture. Therefore, osmotic pressure is one of the important conditions for cell culture in vitro. Maintenance of osmotic pressure of mammalian and other animal tissue cells in vitro Mainly related to NaCl, but can not ignore the relationship between other electrolytes and osmotic pressure. The osmotic pressure is proportional to the number of molecules in the solvent per unit volume and the ion enthalpy. To this end, the ion balance in the medium is controlled to maintain the normal osmotic pressure. It is important not only to maintain cell tension, but also to regulate cell metabolism. Because extracellular ion transport and ion concentration alter the transport of other nutrients (such as amino acids, sugars, etc.), directly affecting the cell's basic synthesis system. .
The ideal osmotic pressure varies depending on the type and ethnicity of the cells. The human plasma osmotic pressure is 290 mmol/L, which is considered to be the ideal osmotic pressure for culturing human cells in vitro. The osmotic pressure of mammalian cells is generally 290-300 mmol/L. The embryonic lung fibroblasts are 250~325mmol/L, and the mouse cells are about 310mmol/L. In practical applications, the osmotic pressure of 260~320mmol/L can be suitable for most cells. The osmotic pressure of commonly used medium is shown in the table. 2-3.
Third, the gas environment and PH value:
In vitro culture of cells requires an ideal gas environment, one of the necessary conditions for the survival of oxygen and carbon dioxide cells.
1. Oxygen:
Oxygen participates in the cell's tricarboxylic acid cycle, producing energy for the growth, proliferation, and synthesis of various desired components. Some cells can obtain energy by glycolysis under hypoxic conditions, and most cells cannot survive under hypoxia. Oxygen tension is usually maintained slightly below atmospheric conditions, and oxygen partial pressure may be harmful to some cells if it exceeds the oxygen content in the atmosphere.
2, carbon dioxide:
When using open culture (disc or culture flask cap culture or culture plate culture), the cells are generally placed in a mixed atmosphere of 95% air plus 5% carbon dioxide. CO2 is both a metabolite of cells and a cell growth facility. The necessary ingredients are related to the pH of the maintenance medium. In the case of a low CO2 concentration in a closed bottle, the cells are easy to grow, generally not less than 1%, otherwise the cells are damaged. The increase in CO2 will lower the pH.
3. pH:
Most cells are suitable for growth at pH 7.2 ~ 7.4, below pH 6.8 or higher than pH 7.6 harmful to cells, even degeneration or death of various cells have different pH requirements. General primary The tolerance of the cultured cells to the pH of the base is poorer than that of the acid. The environment of the acidity is more favorable to the growth of the cells than the environment of the alkaline. In order to maintain a constant pH in the culture environment, the addition of phosphoric acid to the medium is often used. Method of buffer such as salt. NaHCO3 in phosphate buffer can supply CO2, but CO2 is easy to escape, so it is only suitable for closed culture. If open culture is still placed in a gas environment containing 5% CO2. To overcome the trouble of NaHCO3 adjustment, N-2-hydroxyethyl piperazine-N'ethanesulfonic acid (Hepes) can also be used, which is non-toxic to cells, and its main function is to prevent rapid pH changes, in open bottle aeration culture or Live cells can maintain a relatively constant pH when observed.
4. Non-toxic and sterile:
Non-toxic and sterile is the primary environmental condition for in vitro culture of cells. Cells are deeply in vivo, occasionally invaded by bacteria or harmful substances. Because of the powerful detoxification system and immune system in the body, they can be detoxified or eliminated without being damaged. However, in an isolated environment, the defense system is lost. Once the harmful substances invade or the bacteria are polluted, the cells may be killed and discarded. For example, culture bottles, culture dishes, supports, culture media, and cork are cytotoxic. During the culture process, cell death can occur. Therefore, when selecting these utensils and materials, be careful. If these items are not thoroughly disinfected, the bacteria or the process may cause contamination of the cells, which may lead to cell death. If cross-contamination occurs, the experiment can be performed. The original cell line of the room loses its original characteristics and should be given enough attention.
5. Radiation and ultrasound:
1. The wavelength of visible light and visible light is 390~780nm. Various colored light of visible light can cause cell degeneration, prolong the interphase of nuclear fission, and significantly reduce the ability of cells to attach to the wall. Therefore, cells cultured in vitro should avoid direct sunlight. It is best to cultivate in the dark or short-term storage.
2. The cells with strong tolerance under ultraviolet light and weak ultraviolet light have little change, but they are harmful to sensitive cells. When the ultraviolet rays are strong, the cells isolated show that they cannot undergo complete mitosis; when they are mitotic, they increase syneresis; in mitosis When the cytoplasmic foaming is reduced, the Elrlich ascites cancer cells are exposed to ultraviolet light and observed by laser confocal scanning microscopy to form blisters on the irradiated surface, and then the cells swell and the damage is severe.
3. Radiation X-rays have obvious damage to cells. B-rays can affect the division of the nucleus. R-rays reduce the number of mitotic divisions and cause abnormal nuclear division, which can cause cell death.
4. Under ultrasonic vibration, the cells will rupture quickly, starting with the cytoplasmic disorder, and the protoplast colloid structure also has obvious changes. If the ultrasonic vibration is stopped, it can be recovered. The cause of cell death is caused by cavitation. When the ultrasound is at 2.5W/cm2, the cells are damaged and the chromosomes are distorted. The first thing that is distorted is the nuclear chromosome.
6. Cell seeding density:
In vitro cultured cells, the number of cells inoculated has an effect on the growth of cells. The appropriate seeding density can promote cell proliferation. The density of inoculation is too low or too high, which is not conducive to cell growth and proliferation. If the volume ratio of medium to cell is greater than 2000:1, the growth material is dispersed to the extracellular space, which will make the intracellular concentration lower than the minimum concentration. At this time, the cells can no longer proliferate, the pH of the medium becomes alkaline, inhibits cell growth, and the cells become round and can not be attached. Even causing cell death, etc. If the seeding density is too high, the volume of the medium surrounding each cell falls below 0.007 mm3. As the diffusion rate decreases, the concentration of intracellular biomass increases, and the discharge or other metabolites are easily reached. Saturated, the energy source is also quickly depleted, so it will also prevent cell proliferation.
How to choose the appropriate inoculation concentration should be determined according to the metabolism and growth rate of the cells and the needs of the work. In general, cells with high metabolism and fast growth (such as tumor cells) should be inoculated at a low concentration; The growth is slow, the metabolism is not strong enough, and the inoculation concentration can be high; if it is for conservation or no need for emergency use, the inoculation can be lower; sometimes, in order to facilitate observation of the cell morphology, the inoculation concentration can be appropriately reduced.
7. Container rotation speed and suspension agitation speed:
Sometimes the cultured cells are rotated or agitated in a container for research purposes. For example, the adherent cells are cultured in a rotating tube, so that the rotation speed of the drum is increased, and the cell proliferation rate is increased. The reason may be that the rotation makes sufficient nutrition. Material flow and adequate oxidation.