Cell culture refers to a method that simulates the internal environment (sterility, suitable temperature, pH and certain nutritional conditions) in vitro to make it survive, grow, reproduce and maintain its main structure and function.Cell culture is also calledCell cloning technologyThe formal term in biology is cell culture technology.Whether for the wholebioengineering technology, or one of themBiological cloning technologyCell culture is an indispensable process, and cell culture itself is a large-scale cellclone。Cell culture technology can transform a cell into a simple single cell or a few differentiated cells after a large number of culturesmulticellular This is an essential part of cloning technology, and cell culture itself is cell cloning.Cell culture technology is an important and commonly used technology in cell biology research methods. Through cell culture, we can not only obtain a large number of cells, but also study cell signal transduction, cell anabolism, cell growth and proliferation.[1]
Among all cell cultures in vitro, the most difficult isAnimal cell culture。Here are the special conditions it requires.
⑴ Serum: Serum is often required for animal cell culture in vitro.The most commonly used is calf serum.Serum provides essential growth factors such as hormones, trace elements, minerals and fats.Here, the serum is equal toAnimal cellNatural nutrient solution for in vitro culture.
⑵ Support: Most animal cells have the habit of adhering to the wall.In vitro culture, glass and plastic are often used as supports.
⑶ Gas exchange:carbon dioxideThe ratio of oxygen to oxygen should be constantly adjusted during cell culture to maintain the required gas conditions.[2]
Plant cell culture
(1) Light: cultured in vitroplant cellLight conditions are not very strict, because the materials needed for cell growth are mainly supplied by the culture medium.But light is not only related to photosynthesis, but also related tocell differentiationFor example, photoperiod can regulate sex cell differentiation and flowering, so earlyPlant cell cultureIn the process, light conditions are particularly important.In the process of obtaining important substances, such as drugs, by means of plant cell culture in vitro, most plant cellsReactorinsuspension culture 。
(2) Hormone: it is especially needed for the division and growth of plant cellsPhytohormoneRegulation, growth promoting auxin and promotioncell divisionMitogen is the most basic hormone.Plant cell division, growth, differentiation and individual growth cycle are all regulated by corresponding hormones.Compared with animal cells, the principle of hormone requirements for plant cell culture in vitro has been understood, and its application technology has also been quite mature. There is already a set of culture media that can be used.At the same time, it solves the problem of plant cells' influence on water, nutrients, hormonesosmotic pressure, pH, trace elements, etc.[2]
Microbial cell culture
Microbes are mostly single celled organisms, and the wild living conditions are relatively simple.Therefore, the conditions for artificial cultivation of microorganisms are much simpler than those of animal and plant cells.amongAnaerobic microorganismCulture ratioAerobic microorganismComplex, because strict anaerobic needs to maintain carbon dioxide and other non oxygeninert gasConcentration, while aerobic microorganisms only need to provide sterile oxygen through continuous stirring.Microbes have less stringent requirements on culture conditions than animal and plant cells. Corn syrup, peptone, wort, yeast extract, etc. become good microorganismsNatural culture medium。For the nutritional requirements of some special microorganisms, they can be added on the basis of these natural culture media.[2]
Environment and conditions
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environmental factor
1．Sterile environment
Non toxicity and sterility are the primary conditions for cell culture in vitro.Cells in vivo, detoxification system andimmune systemIt can resist the invasion of microorganisms or other harmful substances. However, during the process of in vitro culture, cells lack the protection of the immune system of the body and lose the ability to defend microorganisms and detoxify harmful substances.In order to ensure that cells can grow and reproduce in an in vitro environment, aseptic working areas, good personal hygiene, aseptic reagents and media, and aseptic operations must be ensured.
commonMicrobial contaminationThere are mycoplasma, bacteria and fungi.Mycoplasma has no lethal toxicity, can coexist with cells for a long time, and has potential impact on cells, but it is small and difficult to identify. It can be detected by lichen red or Hoechst33342 staining.Bacteria proliferate quickly, and can expand in a short time, and produce toxins to kill cells.There are many kinds of fungi, which can be seen by the naked eye, floating on the culture liquid surface, and can be filiform, tubular or dendritic.
twoSuitable temperature
Generally, the suitable temperature for mammalian and poultry cells to be cultured in vitro is 37~38 ℃, and the unsuitable ambient temperature will affect the growth of cells.The tolerance of cells to low temperature is stronger than that to high temperature. Under low temperature, the metabolic activity and mitotic ability of cells are reduced.If the temperature is not lower than 0 ℃, although the cell metabolism is affected, there is no damage;At 25~35 ℃, the cells grew slowly;However, if it is kept at 40 ℃ for several hours, it is not conducive to the survival and growth of cells, and may even lead to their death.
threeAppropriate osmotic pressure
Hypertonic solution orHypotonic solutionIt will cause cells to wrinkle, swell and break.Therefore, osmotic pressure is one of the important conditions for cell culture in vitro.Most cells cultured in vitro have a certain tolerance to osmotic pressure. In practical application, 260~320mmol/L osmotic pressure can be applied to most cells.
4．Gas environment and pH
The cell culture in vitro needs an ideal gas environment. Oxygen and carbon dioxide are necessary conditions for cell survival.Oxygen participates in the tricarboxylic acid cycle of cells, providing energy for cell survival, metabolism and synthesis;Carbon dioxide is not only a metabolic product of cells, but also an essential component for cell growth, and is related to maintaining the pH of the culture medium.The suitable pH range for most cells is usually 7.2~7.4.In open culture, 5% of carbon dioxide is suitable.[1]
Nutritional conditions
1. Culture medium
Cell culture mediumContains various nutrients needed for cell growth, includingcarbohydrateAmino acids, inorganic salts, vitamins, etc.There are many kinds of nutrition requirements for different cellsSynthetic mediumAvailable for selection, such as EBSS, Eagle, MEM, RPMll640, DMEM, etc.
2. Other added ingredients
In addition to providing basic nutrients in various synthetic culture media, other ingredients, such as serum and factors, should be added according to different cells and different culture purposes.
Serum providesExtracellular matrix, growth factors and transferrinfetal bovine serum。The proportion of serum added should be determined according to different cells and different research purposes.10%~20% serum can maintain the rapid growth and proliferation of cells, which is called growth culture medium;To maintain the slow growth or immortality of cells, add 2%~5% serum, which is called maintenance culture medium.But there are also substances harmful to cell growth and reproduction in the serum, such as complementimmunoglobulinAnd some growth inhibitors;Unclear components affect the analysis of results;The serum activity of different animals and different batches varies greatly, which affects the stability of the culture effect.
glutamineIt is an important nitrogen source for cell growth and plays an important role in the process of cell growth and metabolism. However, because glutamine is very unstable and easy to degrade in solution, it can decompose about 50% after being placed at 4 ℃ for 7 days, so glutamine needs to be added before use.
To prevent pollution, a certain amount of antibiotics should be added to the culture medium. The name, concentration and sensitivity of common antibiotics are shown in the table below.[1]
Rubber products (preferably silicon products) are used as stoppers and covers for various bottles or test tubes.
4、 Metal equipment
Scissors, forceps, scalpels, scalpels, vascular forceps, tissue forceps, ophthalmic forceps and various types of needles
5、 Other items
Gauze, syringes and needles[3]
General process
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96 well cell culture aspirate
1、 Preparatory work
The preparation work is extremely important for cell culture, and the workload is also large, so enough attention should be paid to it. The negligence of a certain link in the preparation work may lead to the failure or failure of the experiment.Preparations include cleaning, drying and disinfection of utensils, preparation, subpackaging and sterilization of culture medium and other reagents, cleaning and disinfection of sterile room or ultra clean table, inspection and commissioning of incubator and other instruments. For details, please refer to relevant literature.
2、 Material acquisition
Take some from the body in a sterile environmentHistiocyte(depending on the purpose of the experiment). After a certain treatment (such as digestion and dispersion of cells, separation, etc.), it is connected to the culture vessel. This process is called taking materials.In the case of expanded culture of cell lines, there is no process of obtaining materials.The first culture of tissue cells taken out of the body is calledPrimary culture。In theory, all tissues in various animals and human bodies can be used for culture. In fact, young tissues (especially embryonic tissues) are easier to culture than adult tissues, tissues with low differentiation are easier to culture than those with high differentiation, and tumor tissues are easier to culture than normal tissues.The material should be treated immediately after being taken and cultured as soon as possible. If it cannot be cultured immediately for some reason, the tissue block can be cut into small pieces as big as soybeans and stored in the 4 ℃ culture medium.The organization shall be strictly maintainedsterileAt the same time, avoid contacting other harmful substances.Take pathological tissue, skin and digestive tractepithelial cellsIt is easy to carry bacteria, and can be treated with antibiotics to reduce pollution.The method of separating and dispersing cells from tissues can be referred to relevant literature.
3、 Cultivation
The process of connecting the obtained tissue cells into the culture bottle or culture plate is called culture.If it is tissue block culture, directly connect the tissue block to the bottom of the culture vessel. After a few hours, the tissue block can be firmly attached to the bottom, and then add the culture medium.In case of cell culture, cells shall be counted before being connected to the culture vessel, and a certain amount (expressed in cells per milliliter) shall be connected to the culture vessel as required and directly added to the culture medium.After the cells enter the culture vessel, put them into the incubator immediately to make the cells enter the growth state as soon as possible.The cells being cultured should be observed at regular intervals, including whether the cells grow well, whether the morphology is normal, whether there is pollution, and whether the PH of the culture medium is too acidic or too alkaline (byPhenol red indicatorIn addition, the culture temperature and CO2 concentration should also be checked regularly.commonlyPrimary cultureAfter entering the culture, there is a incubation period (from several hours to tens of days). In the incubation period, cells generally do not divide, but can stick to the wall and travel.After the incubation period, cells enter a vigorous division and growth period.After the cells grow at the bottom of the bottleSubcultureThe cells in one bottle were digested and suspended, and then divided into two or three bottles for further culture.eachpassageOnce called "generation".Diploid cellGenerally, it can only be passed on for dozens of generations, while the transformed cell line or cell line can be passed on indefinitely.Transformed cellIt may be malignant, or it may only be immortal without malignancy.Culturing growing cells is a good material for various biomedical experiments.
4、 Freeze storage and recovery
In order to preserve cells, especially mutant cells or cell lines that are difficult to obtain, cells should be frozen.The freezing temperature is generally - 196 ℃ of liquid nitrogen. Cells are collected into a cryopreservation tube and added with a medium containing a protective agent (usually dimethyl sulfoxide or glycerol). They are frozen at a certain cooling rate and finally stored in liquid nitrogen.At extremely low temperatures, the time of cell preservation is almost unlimited.Rapid thawing method is generally adopted for recovery, that is, after taking out the cryopreservation tube from liquid nitrogen, put it into 37 ℃ water immediately to make it melt rapidly within one minute.Then the cells were transferred into culture vessels for culture.The selection of protective agent, cell density, cooling rate, temperature during resuscitation, and melting rate all affect cell viability during cryopreservation.[4]
Specific steps
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1、 Cell resuscitation
After the cryopreserved cells were taken out of liquid nitrogen, they were shaken in a 37 ℃ water bath to promote their melting.Transfer into 15ml centrifuge tube, add 10ml preheated DMEM complete culture medium, gently blow well, centrifuge, 2000rpmX2min, and discard the supernatant.Add 10ml DMEM medium for cleaning and discard the supernatant.Add 10ml DMEM complete culture medium, gently blow, inoculate in 10cm plate, and add 5% COtwoIn the cell incubator.
2、 Cell passage
When the cell density reaches 80%~90%, remove the culture medium and wash it twice with 10ml PBS.Add 3ml with 0.25% EDTATrypsinAnd put it into the cell incubator for 3min.Add 1ml DMEMComplete mediumStop trypsin digestion and transfer to 15ml centrifuge tube.Add 10ml PBS to clean the cell culture plate, transfer to 15ml centrifuge tube, 2000rpmX2min, and discard the supernatant.Add 10ml PBS (autoclaved, stored at 40C), blow well, suck 10 μ L for counting, and count as per 1 × 10five/Plate inoculation with 5% COtwoContinue to culture in the cell incubator.
3、 Cell cryopreservation
When the cell density reaches 80%~90%, remove the culture medium and wash it twice with 10ml PBS.Add 3ml trypsin containing 0.25% EDTA and put it into the cell incubator for 3min.Add lmlDMEM complete culture medium to terminate trypsin digestion and transfer to 15ml centrifuge tube.Add 10ml PBS to clean the cell culture plate, transfer to 15ml centrifuge tube, 2000rpmX2min, and discard the supernatant.Add 1ml of cryopreservation solution (90% serum, 10% DMSO), put it into a cryopreservation box (isopropanol is in the box to ensure the speed of temperature reduction), put it into an 80 ℃ refrigerator immediately, overnight, and put it into liquid nitrogen the next day, which can be stored for at least two years; if not, it can be stored for three months.
Preparation of frozen solution: 70% complete medium+20% FBS+10% DMSODMSO should be added slowly and shaken while dropping.[5]
matters needing attention
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(1) The first time you start to culture a certain cell, you must do it in WWWSearch the name of the cell on ATCC. ORG to get detailed information about the cell, including the required culture medium, serum, additives, general digestion time, passage time, etc.For specific cells (such as primary cultured cells), it is necessary to consult relevant literature to obtain more accurate culture methods.
(2) When entering the intercellular space to start cell culture, the following steps must be strictly followed:
① Make sure that all the solutions and consumables for cell operation have been disinfected and tested without problems. Do not use uncertain solutions and consumables. Do not borrow other people's solutions except under special circumstances.
② Make sure the cuffs of the clothes have been rolled up or the cuffs of the white coats have been fastened.
③ Determine the amount of alcohol in the alcohol lamp, and supplement it if necessary.
④ Make sure that all solutions and consumables needed are within reach.In order to open the bottle caps with one hand, all bottle caps can be loosened before the experiment.
⑤ Try not to pour the solution directly unless the bottle mouth is not burned.If the pouring fails and the solution sticks to the bottle mouth, please spray it75% alcoholClean the area around the bottle mouth carefully (do not touch the bottle mouth) and then burn it on the flame.
⑥ If the consumables are not sure to be clean during operation, they must be replaced in time.
⑦ Clean up in time after the experiment, keep the work area clean and tidy, and finally clean the table with 75% alcohol.
(3) Prevention of cell pollution
① The laboratory supplies shall be protected from pollution.The reagents, consumables and equipment used for cell culture should be thoroughly cleaned and sterilized, and all solutions should be carefully sterilized. They can only be used after the sterility test is negative.The operation room and the remaining sterile equipment shall be cleaned, sterilized and sterilized regularly.
② Prevent pollution during operation.
③ Wearing clothes that are easy to generate static electricity or absorb dust must be replaced with white coats before entering the cells.
④ Before the experiment, you need to make sure that the gloves you wear are OK, as long as you have touched themBiosafety cabinetThe gloves must be disinfected in time for other articles.
⑤ After entering the cell culture room, close the door, sit down and walk as little as possible to avoid affecting the wind curtain of the biosafety cabinet.Wipe hands and bottle caps with 75% alcohol cotton ball before work.Strictly check the equipment, solution and cells used in advance. Do not bring contaminated or unsterilized articles into the sterile room, and do not use them casually to avoid large-scale pollution.
⑥ Cell operation should be light. The bottle mouth must be opened in the sterile area around the flame, and the bottle mouth should be placed around the flame for simple rotation and burning. Be careful not to let the flame burn the plastic bottle mouth.
⑦ During experimental operation, the partition of the biosafety cabinet should be lowered as far as possible to minimize conversation. When sneezing or coughing, it should never face the working area to avoid unnecessary pollution.
⑧ Bottle caps should be placed upside down away from themselves to avoid contamination caused by misoperation.
⑨ Do not pass over the open container mouth to avoid cell pollution caused by unknown objects falling from clothes.
⑩ Pay attention to timely replacement during experimental operationPasteur straw. The nozzle and pipette of the pipette gun should not be made from one pipe to the end.Once it is found that it is in contact with the unclean or can not be determined that it is clean, it must be discarded directly.After the experiment, clean up in time, keep the laboratory clean and tidy, and finally clean the table with 75% alcohol.
(4) Prevent cross contamination of cells
① When conducting various cell culture operations, the instruments used should be strictly distinguished, and it is better to mark them for easy identification.Operate in sequence. Only one cell is processed at a time. When multiple cells and operations are performed together, confusion is likely to occur.
② During the fluid change or passage operation, the tip of the pipette gun and pipette with cells should not touch the mouth of the reagent bottle, so as not to bring cells into the culture medium to contaminate other cells.
③ Once all cells are purchased, introduced from other places, or established by themselves, they must be preserved and frozen in a timely manner. In case of pollution, cells can be revived and cultured again·[6]
