(1) Setting of cell culture laboratory
The main difference between tissue cell culture technology and other general laboratory work is the requirement to maintain aseptic operation and avoid the effects of microorganisms and other harmful factors. The widespread use of ultra-clean benches has greatly facilitated tissue cell culture and has made it possible for some routine laboratories to perform cell culture.
Cell culture laboratories should be able to perform six tasks: aseptic processing, incubation, preparation, cleaning, sterilization, and storage.
1. Sterile operation area
(1) Aseptic operation room: The sterile operation area is limited to the area of ​​cell culture and other aseptic operations, and it is best to be isolated from the outside world, not to walk through or interfere with other interference. The ideal aseptic operating room should be divided into three parts:
a) Dressing room - for changing clothes, shoes and wearing hats and masks.
b) Buffer room – located between the dressing room and the operating room, in order to ensure a sterile environment in the operating room, and to place a constant temperature incubator and some necessary small instruments.
c) Sterile operation room - dedicated to aseptic processing, cell culture. The size should be appropriate, and the top should not be too high (not more than 2.5m) to ensure the effective sterilization effect of ultraviolet rays; the wall is smooth and has no dead ends for cleaning and disinfection. The workbench should not be placed against the wall. The countertop should be smooth pressed and molded into a surface, painted white or gray to facilitate the observation of the anatomical structure and phenol red.
Air disinfection in aseptic rooms:
Ultraviolet light - produces ozone, and the indoor temperature and humidity are high, which is not conducive to the health of workers.
Air-filtered constant temperature and humidity device - best.
Ozone-free UV sterilizer
Electronic Disinfector--Under the action of high-voltage electric field, the internal and external electrodes of the electron tube undergo strong electron bombardment, which ionizes the air and converts the oxygen in the air into ozone. Ozone is a strong oxidant, which can react with the bacterial membrane and the enzyme protein hydrogen sulfide to oxidize and decompose, so that the ozone gas diffuses diffusely to achieve the purpose of sterilization, and there is no dead angle when disinfecting. The residual ozone in the space after disinfection can be reduced to oxygen by itself for 30-40 minutes, the air does not leave odor, and the surface of the disinfected object is not left with residual poison.
(2) Purification workbench: simple operation, convenient installation, small space occupation and good purification effect. There are two main types of purification workbench used in general bacterial culture rooms: a) lateral flow or vertical b) external flow or horizontal laminar flow.
The working principle of the purification workbench (substantially the same) - usually the indoor air is filtered through the coarse filter, pressed into the static pressure box by the centrifugal fan, and then filtered by the high-efficiency air filter, so that the clean air flow is sent to a certain Uniform cross-section wind speeds through the sterile field to create a dust-free, sterile, high-clean work environment.
Side-flow workbench—The airflow after air purification flows from the left or right side through the work surface to the opposite side, and also from the top to the bottom or from the bottom to the opposite side, forming an airflow barrier to keep the working area sterile. The workbench structure is closed.
Outflow (horizontal) workbench—The purified air flows toward the operator, so the external airflow does not mix into the operation, but the hazardous substance test operation is unfavorable to the operator. The workbench structure is open (already used).
Purification workbench application note:
(1) The purification workbench should be installed in a separate sterile room or a clean and dust-free room to prevent the dust from clogging, reducing the purification effect and shortening the service life.
(2) Newly installed or long-term unused workbench, vacuuming or non-fiber-forming tools must be cleaned on the workbench and the surrounding environment before work, and then sterilized by drug sterilization or UV sterilization. deal with.
(3) Before using the purification workbench, the tabletop should be scrubbed with 75% alcohol, and the microorganisms accumulated in the purification work area should be treated by ultraviolet disinfection lamp for 30-50 minutes in advance. After turning off the sterilization lamp, the fan should be started to operate for two minutes before the cultivation operation.
(4) Unnecessary items should not be stored in the purification work area to keep the clean air flow pattern undisturbed.
(5) Pay attention to the change of airflow in the purification zone. Once the airflow is weakened, such as the flame of the alcohol lamp does not move, if the motor voltage is still not changed, the filter has been blocked and should be replaced in time. In general, the high efficiency filter is replaced once every three years. Replace the high filter and ask the professional to keep it sealed. The filter cloth (non-woven fabric) in the coarse filter should be cleaned and replaced regularly, and the time should be determined according to the cleanliness of the working environment, usually once every 3-6 months.
(6) When the cleaning workbench is used, the items on the work surface should be cleaned in time and the table top should be cleaned with alcohol to keep it clean.
(7) The purification workbench shall perform functional tests on a regular basis to check whether the work indicators of the purification workbench meet the requirements, such as conducting a sterility test, and regularly checking whether the cleanliness of the countertop air is up to standard.
Aseptic inspection of ultra-clean workbench After the disinfection treatment, the number of colonies in the air should be checked before and during the sterility test; take a plate with a diameter of about 90mm, and ignite the alcohol lamp in the ultra-clean workbench. Next to the alcohol lamp, aseptically, the plate is half-opened into a solubilized nutrient agar medium for about 20 ml, and made into a plate, pre-incubated at 30-35 ° C for 48 hours, and it is proved that the plate is sterile after 3 pieces, in a sterile manner. Bring into the left, middle and right of the ultra-clean workbench; open the lid of the plate and put it on. After the plate is exposed to the air for 30 minutes, cover the plate, set it at 30-35 °C for 48 hours, take out the inspection, level 100. Cleanliness requirements: The number of colonies growing on 3 dishes should not exceed one.
The ultra-clean workbench should regularly check the cleanliness of the relevant departments. It should reach level 100 (usually dust particle counter). The number of particles with a particle size of ≤5祄 should not exceed 3.5/liter; the air flow should be controlled at 0.75~ 1.0m3/s; the average number of bacterial colonies is <1, and the filter can be replaced if necessary according to the sterility.
2. Incubation area
Although the requirements for sterility in this area are not stricter than the aseptic area, they still need to be clean and dust-free, so they should also be placed in areas where there is less interference than walking through. Incubation can be carried out in an incubator or in a temperature-controlled greenhouse, which is expensive and generally used in incubators for labor.
3. Preparation area
In this zone, preparation of a culture solution, a liquid for culture, and the like is mainly performed.
4. Storage area
It mainly stores all kinds of refrigerators, drying boxes, liquid nitrogen tanks, sterile culture liquids, culture bottles, etc. This environment also needs to be clean and dust-free.
5. Cleaning and disinfection area
The cleaning and disinfection area should be separated from other areas, mainly for cleaning, preparation, disinfection and preparation of three steamed water for all cell culture vessels.
(2) Equipment for cell culture laboratory
Tissue cell culture chambers In addition to the general equipment in general laboratories, there are some special needs equipment. Basically, it can be divided into two categories: the first category is a common basic device; the second category is a higher-level special device.
1. Commonly used basic equipment
(1) Instrument:
a) Microscope: Inverted microscope - is one of the routine work routines necessary for tissue cell culture chambers, so that it is easy to grasp the growth of cells and observe the presence or absence of pollution. - If available, a high-quality phase contrast microscope with a photographic system, a dissecting microscope, a fluorescence microscope, a video system, or a time-lapse movie camera can be configured to observe, record, and photograph cell growth at any time.
b) Incubator: In vitro cultured cells, like cells in vivo, need to survive at a constant temperature. In most cases, the optimum temperature is 37 ° C, the temperature difference should generally not exceed ± 0.5 ° C, the cell temperature rises 2 At °C, it can not be tolerated for several hours, and will die soon after 40 °C. Therefore, there is a need for an incubator capable of controlling temperature, such as a constant temperature incubator with a high sensitivity and a CO2 incubator.
Constant temperature incubator - should choose the water-proof or transistor self-controlled temperature incubator, this type of incubator has high sensitivity and stable temperature control. The general constant temperature incubator is cheaper, and the disadvantage is that it is only suitable for closed culture.
CO2 incubators - Most cell culture chambers are currently in widespread use. The advantage of the CO2 incubator is that it can provide a certain amount of CO2 (usually 5%) required for cell culture, and it is easy to stabilize the pH of the culture solution, and is suitable for open or semi-open culture. The culture vessel can be used in a culture dish, a culture plate or a culture bottle; when the culture bottle is used, the bottle cap can be slightly loosened to keep the inside of the culture bottle ventilated. Since the culture method is connected to the outside of the culture vessel, the air in the incubator must be kept clean and should be periodically irradiated with ultraviolet rays or alcohol. At the same time, the incubator should be placed with a sink containing sterile distilled water to prevent the culture solution from evaporating, so that the relative humidity in the box is always maintained at 100%.
c) Drying box: Some instruments and utensils used in cell culture incubators need to be dried before they can be used. Glassware should be dry and heat sterilized. When dry heat disinfection, the electric oven has a higher temperature rise, generally needs to reach above 160 °C. A blower type electric drying oven is usually used. The advantage is that the temperature is uniform and the effect is good, and the disadvantage is that the heating process is slow. When heating up, the temperature should not be raised first, but the blast should start at the same time as the temperature rises. At 100 °C, the blast should be stopped. The paper or cotton in the package should be avoided. The charred debris can affect the growth of the cells. After disinfection, the door cannot be opened immediately to avoid sudden cooling and damage to the glassware. Wait until the temperature naturally drops below 100 °C to open the door.
d) Purified water device: Cell culture requires high quality of water, and the water used for cell culture and liquid associated with cell culture work must be strictly purified beforehand. An ion exchange unit or a distiller can be used for water purification. Ion exchange of pure water is not effective in removing organic matter, so the water needs to be distilled again. Three times of distilled water is required for the preparation of various culture solutions and reagents for cell culture: even for the rinse of glassware, more than two times of distilled water should be used. At present, the domestic use of more automatic double water distiller (quartz tube heating), easy to use, safe, fast distillation speed (1600ml / h).
Note:
It is not possible to use ordinary tap water distillation to avoid the formation of scale in the distiller to affect the distillation effect. At the same time, it is necessary to thoroughly clean the distiller periodically according to the scale of the distiller. The correct method of use is to add distilled water distilled through a metal distiller to a glass distiller for re-distillation.
Generally, the water used for the preparation of the culture solution should be distilled before the liquid preparation. It is not suitable to use the three steamed water for several days to avoid affecting the quality of the culture water. Currently available on the market are pure water systems containing various grades and types of one or more purification methods that purify ordinary water to pure water and ultrapure water. For example: a flexible design that can be wall-mounted or bench-top, or a water storage tank or a pure water device that can be directly used with a liquid separation gun. It can also be equipped with effective sterilization or removal of DNase and RNA according to various experimental water requirements. Enzymes, proteases, etc., can even effectively remove the heat source and endotoxin ultrafiltration type water purifier.
e) Refrigerator: Cell culture room must be equipped
a common refrigerator or freezer
- Store culture materials, physiological saline, Hanks liquid reagents and other culture items and short-term preservation tissue samples.
b low temperature refrigerator (-20 ° C), ultra low temperature refrigerator
- For the storage of preparations that require cryopreservation of biological activity and storage over a longer period of time, such as enzymes, serum, etc.
——The refrigerator in the cell culture room should be dedicated. It should not store substances that are harmful to cells, such as volatile and flammable, and should be kept clean.
f) Cell cryopreservation: The reservoir is usually a liquid nitrogen container. According to the needs of use, it is divided into different types and various specifications.
When purchasing a liquid nitrogen container, it is necessary to consider the volume, the ease of use and the three factors of liquid nitrogen volatilization (economic). The size of the liquid nitrogen container can be from 25L to 500L, and can store about 12.5 ampoules of 250 to 15,000. The liquid nitrogen temperature can be as low as -196 ° C and should be protected from frostbite during use. As the liquid nitrogen is continuously volatilized, attention should be paid to the observation of the residual liquid nitrogen, and the liquid nitrogen should be replenished regularly to avoid excessive evaporation and damage to the cells.
At present, new types of cell cryopreservation have been widely used in various domestic laboratories. The various new cell cryopreservation stores available in the market are characterized by excellent performance and ease of use. E.g:
——Automatically freeze and store the liquid nitrogen level and sample temperature through advanced electronic controllers to ensure that the sample temperature is always at the set temperature point;
——Equipped with advanced alarm system to alarm liquid nitrogen level, temperature, battery, voltage, power supply and other abnormal conditions:
——There is also a hot gas bypass system to prevent warm air above -130 °C from entering the liquid nitrogen tank, thus more effectively protecting the sample and preventing temperature rise.
In addition, the advanced liquid nitrogen transportation of various specifications, the supply tank series is not only easy to move, but also can supply liquid nitrogen to the storage tank through the connecting pipe to improve work efficiency and ensure sample safety.
g) Centrifuge: When performing cell culture, it is customary to prepare cell suspensions, adjust cell density, wash, collect cells, etc., usually using a centrifuge.
a Generally, a domestic desktop centrifuge of 4000 rpm can be conventionally arranged, for example, cell sedimentation can be carried out using a centrifuge of 80 to 100 g, and excessive centrifugal force may cause cell damage.
b In addition, other types of centrifuges, such as large capacity or adjustable temperature, can be added as needed.
c For special applications, such as the separation and preparation of certain cells to be subjected to gradient centrifugation, separate centrifuges with other special functions required for the experiment are required.
h) Balance: Commonly used are torque balances, precision balances and various electronic balances.
The sensitivities of the analytical balance were 0.1 mg, 0.01 mg and 0.001 mg. According to the requirements of weighing the material and weighing accuracy, choose the balance of the appropriate level. Precision timing is required. When the sampling amount is greater than 100 mg, the sensitivity is 0.1mg balance, the sensitivity is 0.01mg balance at 100-10 mg, and the sensitivity is 0.001 mg balance less than 10mg.
i) Sterilizer: Items that come into direct contact with cells directly or indirectly need to be sterilized. Commonly used:
a. Portable high pressure steam sterilizer
b. At present, the high-pressure sterilization device suitable for various vessels, liquids and culture media is in circulation (such as pulse vacuum sterilizer) with high efficiency, safety and convenience. It can be used to monitor the pressure and temperature in the sterilization container while sterilizing, to ensure the quality and safety of sterilization, and to change various parameters (such as sterilization, exhaust, heating, etc.) through the memory (storage) support system. ), the set parameters can be retained even if a power failure occurs during sterilization
j) Filter: At present, the culture liquid used in cell culture work, including synthetic culture medium, serum, and trypsin for digestion, often contain vitamins, proteins, peptides, growth factors, etc., which are easy to irradiate under high temperature or radiation. Degeneration or loss of function occurs, so the above liquids are often filtered and sterilized to remove bacteria.
Filters commonly used today include Zeiss filters, glass filters, and microporous filters, and various filters have their principles and characteristics (described in detail later).
(2) Culture utensils:
a) culture vessel: a vessel for inoculation, growth, etc., which can be made of transparent, non-toxic neutral hard glass or non-toxic, transparent and smooth.
Made of plastic.
(a) The advantage of the glass culture vessel is that most cells can grow, are easy to clean, disinfect, can be used repeatedly, and are transparent and easy to observe; the disadvantage is that it is fragile and laborious to clean.
(b) The advantage of plastic culture vessels is that they are used once and the manufacturer has sterilized and sealed the package, which can be used for cell culture operations.
Commonly used culture vessels:
Culture flask: made of glass or plastic. Mainly used for culturing and breeding cells. When the culture is carried out, the bottle mouth is covered with a screw cap or a rubber stopper, and the rubber plug is mostly used for sealing culture.
The specifications of domestic culture flasks are expressed in terms of capacity (ml), such as 250ml, 100ml, 25ml, etc.; imported culture flasks are mostly expressed in bottom area (cm2).
Petri dish: Made of glass or plastic for the purpose of taking, separating, processing tissue or performing cytotoxicity, colony formation, single cell separation, isotope incorporation, cell propagation and other experiments. Commonly used culture dishes have specifications of 10cm, 9cm, 6cm, 3.5cm, and the like.
Porous culture plate: a plastic product. It can be used in various assays such as cell cloning and cytotoxicity. The advantage is that it saves samples and reagents, can test a large number of samples at the same time, and is easy to perform aseptic operations. The culture plate is divided into various specifications, and the commonly used specifications are: 96 holes, 24 holes, 12 holes, 6 holes, 4 holes, and the like.
The number of cells available for the growth of various monolayer-grown cells in the culture vessel depends primarily on the bottom surface area of ​​the vessel and the size of the cell volume. Common culture vessels and the number of cells available.
b) Culture related equipment:
a liquid storage bottle: mainly used for storing or preparing various culture liquids such as culture liquid, serum and reagents. The liquid storage bottle is divided into various specifications, such as 1000ml, 500ml, 250ml, 100ml, 50ml, 5ml and the like.
b straw: mainly divided into scale pipette, no scale pipette. The scale pipette is mainly used for sucking and transferring liquid. The commonly used specifications are 1ml, 2ml, 5ml, 10ml and so on. The non-scaling straw is divided into a straight straw and an elbow straw. In addition to the suction and transfer of liquid, the elbow tip is also commonly used for blowing, mixing and passage of cells.
c applicator (pipette): used to draw, move liquid or drop samples. The amount of the amount can be adjusted as needed, and the suction amount is accurate and convenient. In particular, the micro-sampler ensures that the sample (or reagent) is accurate and repeatable. At present, high-temperature disinfection, multi-channel pipettes are available for users to ensure accurate, fast, convenient and sterility requirements.
c) Other supplies: There are still centrifuge tubes for collecting cells, reagents or tubes for temporary insertion of straws, glass or stainless steel containers for sterilizing, for storing small pieces of cultured articles for autoclaving. Or sump, rubber tip on the top of the straw, closure of various bottles, tube plugs, lids, ampoules or cryotubes for cryopreserved cells, syringes of different sizes, beakers and graduated cylinders, and funnels, ultra-clean work The alcohol lamp used in the station is used for cleaning and disinfecting the micro watering can with alcohol or other disinfectant before the experimenter operates.
(3) Instruments: mainly used for anatomy, material extraction, shearing tissue and holding objects during operation. Commonly used are: scalpels or scalpels, surgical scissors or anatomical scissors (bending and straight shearing), used to dissect animals, separate and cut tissues, prepare primary culture materials; ophthalmic iris small scissors (bending or straightening) Shear), used to cut tissue material into small pieces; vascular clamps and tissue sputum, ophthalmology sputum (bend, straight), used to hold sterile objects (such as small coverslips) to hold tissue; dental probe Or a substitute for placing a small piece of tissue in a primary culture.
2, special equipment
In addition to the above-mentioned common basic equipment, the cell culture laboratory may, if available, add special or advanced equipment to perform laboratory work more efficiently, accurately, and deeply. E.g:
(1) Enzyme-linked immunosorbent assay - can be used for immunological assays and cytotoxicity, drug sensitivity testing, and the like.
(2) Ultra-low temperature refrigerator (-85 ° C) - easy to store certain reagents and specimens.
(3) Rotating incubators - cultures used for certain special cells or for harvesting large numbers of cells.
(4) Fluorescence microscopy - observation of fluorescent staining samples.
(5) Flow cytometry - more accurate and rapid detection of cells.
(6) Various instruments for detecting cell culture conditions, such as a multifunctional cell culture analyzer designed to rapidly analyze major or key nutrients, metabolites, and gas contents in a cell culture medium, a portable CO2 concentration meter, etc. .
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