how much media in t25 flask?
Generally,5-6ml medium is added to the t25 culture flask. If the cells grow rapidly, change the medium every day, and if the cells are slow, change the medium every 2-3 days.Human cells and cells have ranged from cultured types and multiple cell types to animal cells and plant cells.
Documentary evidence
Tissue culture is to simulate the physiological environment in vivo in vitro, under sterile, appropriate temperature and certain nutritional conditions. To allow the tissue removed from the body to survive, grow, multiply and passage, and maintain the original structural and functional properties. Tissue culture in a broad sense is synonymous with in vitro culture. In vitro culture (Invitro) includes culture at all structural levels, namely: tissue culture, cell culture and organ culture.The so-called cell culture refers to the growth of cells including single cells in vitro.
The development history of tissue culture has been nearly a hundred years. The initial tissue culture is an extension of embryology and microbiology. It is based on the principle of sterility. Natural body fluids (such as fetal juice, plasma) are used to maintain tissue pieces cut from the whole body. Observation of cell morphology and function.
Through the great improvement and innovation of many scholars, the medium was changed from natural animal plasma to synthetic medium, and the cell growth-promoting substance was changed from fetal juice to animal serum. At present, there are more than 10,000 kinds of cells stored in the world. Tissue culture is a necessary technique not only for cell biology, but also for disciplines such as molecular biology, oncology, genetics, and immunology.
Basic Principles and Methods
Living conditions of in vitro cultured cells:
Nutrition
The nutrients required for in vitro cultured cells are the same as those in vivo, mainly including sugars, amino acids and vitamins. At present, synthetic media such as 1640, 199, etc. on the market contain enough amino acids, but when using synthetic media, some natural ingredients, such as human or animal serum, plasma and fetal juice, still need to be added. At present, serum is mainly used, mainly bovine serum. The biological effect of serum has long been proved, it contains a variety of cell growth factors, promoting adhesion factors and other active substances. Not only can promote cell growth but also help cells adhere to the wall. Different serums have different effects on cells. Calf serum is the best, followed by adult bovine and horse serum.Synthetic medium can maintain cell survival without serum, but cannot grow very well. Adding 5% serum can maintain cell immortality and slow growth for most cells. To make cells grow normally, it is generally necessary to add 10% serum. ~15% calf serum. Each batch of serum should be heat-treated before use, generally inactivated in a 56°C water bath for 30 minutes, and each batch of serum should be heat-treated before use, generally inactivated in a 56°C water bath for 30 minutes, shake every 5 minutes. 10% serum nutrient solution can promote cell proliferation called growth solution, 2~5% serum culture solution cannot make cells proliferate but can only maintain their survival as maintenance solution. The addition of serum is beneficial to cell growth, but the addition of unknown components makes it difficult to analyze the experimental results. Therefore, serum-free solutions without serum are being explored and some progress has been made.
surroundings
1. Sterility: Sterility is the primary condition to ensure the survival of cultured cells. The culture medium is not only high nutrients for cells, but also high nutrients for bacteria and molds. In cell culture, such as contaminated microorganisms, they multiply faster than cells and can produce toxins to cause cell death. Therefore, one of the keys to cell culture technology is to prevent contamination. . Contaminating microorganisms can originate from tissue culture fluids, utensils, the tissue itself, and the worker itself. Therefore, the air in the cultivation room should be thoroughly disinfected, and all operations should be strictly implemented aseptic operations. Various items should be disinfected before they are brought into the operation table, and the surface should be wiped with a spray of essence, and exposed to ultraviolet rays before use; the operator washes and soaks his hands. , Wipe your hands with alcohol, burn the bottle mouth on the flame to fix the dust before opening the culture tube, place the bottle and tube at an angle during operation, avoid contact with the bottle mouth when the straw is poured into the liquid, and do not speak during operation.2. Temperature: The optimum temperature for tissue culture is 35-37°C. Deviation from this temperature range will affect the normal metabolism and growth of cells, and even lead to death. Cultured cells are more tolerant to low temperatures than high temperatures. An increase in temperature of 2-3°C has adverse effects on cells, causing them to die within 24 hours. When the temperature is above 43°C, most cells are killed, while low temperature has little effect on cells. When cells are placed at 25-35°C, cells still remain. It can survive and grow, but the speed is slow. After being placed at 4°C for several hours and then cultured at 37°C, the cells can still continue to grow.
3. Gas: Gas is also one of the necessary conditions for cell survival. The gases required are mainly O2 and CO2. O2 participates in the tricarboxylic acid cycle to generate energy for cell growth, proliferation and synthesis of various components. CO2 is both a cellular metabolite and a required component of the cell. It is mainly directly related to maintaining the pH value of the culture medium.
4. pH value: The suitable pH value for most cells is 7.0-7.4, and deviations from this range can have harmful effects on cells. Different cells have different pH requirements, but in general, cells are more resistant to acid than to alkali. The buffer system in the culture medium is mainly bicarbonate/CO2. Various acids produced by cell metabolism decrease pH, while NaHCO3 in the culture medium produces CO2 and discharges into space to increase pH.
5. Treatment of the culture vessel: The quality of the treatment of the culture vessel has a great influence on the adherent growth of cells. Except for a few cells growing in suspension, most of the cells belong to anchorage-dependent cells or cultures. They can only grow, survive or maintain function when they are attached to surfaces that do not react chemically. At present, the commonly used utensils are mainly glass and plastic two categories. Glassware generally needs to be soaked in cleaning solution for 1 to 7 days, rinsed 20 times with clean water, then washed twice with distilled water, and dried for later use. Sterilize at 160°C for 2 hours before use. 96-well or 24-well plastic plates are generally soaked in 2% NaOH for 4 hours, rinsed with clean water, then soaked in 1N HCL for 4 hours, rinsed with clean water, rinsed with distilled water, dried at 37°C, and irradiated with ultraviolet light for disinfection; new rubber stopper It must be boiled with 1/2NnaOH for 15 minutes, rinsed and then boiled with 4% HCL for 15 minutes, rinsed with clean water, washed with distilled water for 5 times, boiled for 10 minutes for sterilization, or sent to autoclave.
In short, cells can proliferate rapidly under appropriate culture conditions, but if they encounter adverse conditions, cells will become round, stop growing, or even die. This is a protective mechanism for cells. To sum up, the basic conditions of cell culture mainly include nutrient solution, sterile conditions, pH, temperature, gas conditions and the cleanliness of culture vessels.
Commonly used cultivation methods
According to the characteristics of culture cell biology, tissue culture can be divided into primary culture and subculture. It can also be divided into static culture and dynamic culture according to different culture conditions and vessels. Static culture is the most common way, and the cells can be either adherent cells or suspended non-adherent cells.Stationary culture of passaged cells
The existing cells in our laboratory are all passaged cells. CNE-2Z is a tissue obtained from a nasopharyngeal carcinoma patient in the 1980s. After primary culture, subculture and serial passage in vitro, the growth is stable, and it has undergone a series of growth characteristics tests (such as Growth curve, division index, cell population doubling time, colony formation rate), chromosome analysis, allotransplantation, electron microscope observation, etc., and won the first prize of scientific research achievements of the Provincial Department of Higher Education and Health Department in 1983, and the Ministry of Health in 1984. The second prize of scientific research achievements. Since the establishment of the department 20 years ago, CNE-2Z has been widely used all over the world, and it is still preserved and used in our laboratory.Observation and passage of cultured cells: All in vitro cultured cells, including primary culture and various cell lines (strains), need to be passaged when the growth reaches a certain density. The frequency and interval of passages are related to the number of inoculated cells, cell biological characteristics and nutritional properties. When the number of inoculated cells is large, the number of cells saturates rapidly; the tumor cell line proliferates faster than the primary cultured cells, and when the serum content in the culture medium is large, the cell proliferation is faster. Generally, subculture is performed when the cells are overgrown with the walls of the flask, and the nutrition and pH are slightly reduced. According to the characteristics of the cells, a flask of cells is passaged 1:3 or 1:4. When the cells are full, they should be passaged as soon as possible, otherwise the cells will be poisoned, their morphology will change, or they will fall off the wall and die. Passaging too late (if there are signs of poisoning) can affect the functional state of the next generation of cells. So, how to achieve timely passage? To achieve timely passage, carefully observe the growth of cells every day to decide whether to change the medium or passage.Morphological observation is generally performed, and the morphology, cytoplasm and membrane of living cells are observed with an inverted microscope. Cells in good growth state have high transparency, few intracellular particles, no vacuoles, clear cell membranes, and strong refraction. The culture supernatant is clear and transparent, and suspended cells and debris cannot be seen. When cells are dysfunctional, vacuoles, lipid droplets and other particulate matter often appear in the cytoplasm, the space between cells increases, and the cell shape may become irregular or even lose its original characteristics. Only healthy cells can be used for experiments. Generally, after cell inoculation or passage, every day or at most 1-2 days, the cell morphology, cell growth, pH value of the culture medium and contamination should be observed, and the dynamic changes of the cells should be grasped at any time, so as to facilitate the medium change or passage treatment. If any abnormal situation is found, take timely measures. Under normal circumstances, the culture medium is pink. When cultured in a general incubator, the accumulation of carbon dioxide increases with the extension of cell growth time. Due to the existence of pH indicator in the medium, its color can indirectly reflect the growth state of cells. When it is orange, the cells generally grow well; if it is pale yellow, it may be that the culture time is too long, the nutrition is insufficient, and there are too many dead cells; if it is purple, it may be that the cell growth is not in a good state or has died.
Preservation and recovery of cultured cells
Before doing experiments, you must learn to cryopreservation, in case you can maintain the same source, stable and reliable cells in subsequent experiments, and can reduce contamination or other adverse effects.1. Factors affecting the viability of cryopreserved cells
(1) Cell cryopreservation agents: dimethyl sulfoxide (DMSO) and glycerol are commonly used, and the commonly used concentration is 5%-10% (90% calf serum). DMSO is less toxic to diploid cells than glycerol.
(2) The freezing speed of the cell suspension: during slow freezing, the freezing is formed outside the cells, so it will not damage the cells. Most cells were frozen at -20°C at a rate of 1°C per minute. Satisfactory results can be obtained.
(3) Storage temperature: liquid nitrogen tank, which can reach -196 °C, at this time, all physical and chemical activities are reduced to a minimum to ensure long-term preservation of cells.
(4) Resuscitation: Rapid thawing and resuscitation can prevent damage to cells. After the frozen cells are removed from the liquid nitrogen, they should be immediately placed in a water bath at 37°C to 43°C and thawed within 30 seconds to one minute.
2. Cryopreservation and recovery methods
Suitable for primary tissues, primary cells and cell lines (strains). A. The cells were digested with trypsin + EDTA and diluted to 2-5X106/ml with the freezing solution. B. Dispense into 2ml cryopreservation tubes with a volume of 1ml, seal it, mark it well, and tie it with several layers of gauze. C. There should be a gradual cooling process before the cryopreservation tube enters the liquid nitrogen, and the temperature is lowered at a rate of 1 °C/min. Generally, after hanging on the liquid nitrogen for 8 hours, it is put into the liquid nitrogen storage tank for long-term storage. D. To revive the cryopreserved cells, place the cryopreservation tube in a water bath at 37°C to 43°C, shake well to thaw it rapidly, and complete within 30 seconds to 1 minute. E. The cell suspension was centrifuged at a low speed, the protective additive in the supernatant was removed, the original growth medium was added, and the cells were cultured at 37°C.
Basic equipment and reagents
The environment requires clean, dry air and smoke free.
Basic reagents
Culture medium: ① Dissolve 1640 in freshly steamed three-distilled water, stir to fully dissolve, and filter and sterilize. ②The calf serum is inactivated (destroyed the complement) at 56℃ for 30 minutes before use, and stored at -20℃ for later use. ③The cyanine chain double antibody solution Fully dissolve 1,000,000 units of penicillin sodium salt and streptomycin sulfate with 100ml of triple-distilled water after high pressure for 2 times, and freeze them in aliquots and store at -20℃. Thaw before use. Add 1ml, that is, the final concentration is 100μg/ml containing P.S. (Note: P.S cannot be frozen and thawed multiple times, it should be packaged in small quantities and used up at one time). ④NaHCO3 solution is used to adjust the pH value. The usual concentration is 5-6%. For preparation, dissolve in triple distilled water, filter and sterilize or autoclave, and store in aliquots at 4°C. ⑤ Trypsin has the strongest force at pH 8.0 and temperature 37℃. ⑥EDTA solution
Attached:
Pancreatin Digestive Solution (500ml)
NaCl 4 g
KCL (19%) 1 ml
Na2HPO4 0.126g(12H2O)
Tris 1.5g (tris)
After the above drugs are fully dissolved, add water to 500 ml, then add 5 g of trypsin, and finally adjust the pH to 7.7 with 1N HCL, filter and sterilize, and then store at -20°C for future use.
EDTA liquid formula (0.2% 1000ml)
EDTA (versene) 2 g
NaCl 8 g
KCL 0.2g
Na2HPO4(12H2O) 2.9g
KH2PO4 0.2g
After the above drugs are also fully dissolved, add fresh three-distilled water to 1000 ml, sub-pack and then sterilize by high pressure 15 pounds for 30 minutes, and store at 4°C for later use.
Cell freezing medium: 90℅ of calf serum mixed with 10℅ of dimethyl sulfoxide (DMSO); stored in -20℃ refrigerator for later use. Or calf serum 30℅, 1640 medium 60℅, DMSO 10℅ mixed; - 20 ℃ refrigerator for future use.
