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The cell room is generally a special laboratory responsible for the inspection of monoclonal antibodies, HIV, somatic cells and other therapeutic preparations and diagnostic reagents, production cells and bovine serum; and includes the technical review, review, revision or drafting of the national drug standards, Class I new drugs and imported drug standards of the corresponding varieties; responsible for the development, calibration and distribution of the national standard substances of the corresponding varieties; and conducting research on corresponding technical methods and technical personnel training.
The cell room of the pathology department mainly serves the clinic, providing clinicians with timely and accurate cytological examination results. It can reduce the pain of patients during examinations and obtain direct and objective results through non-invasive or minimally invasive methods.
(Figure 1: Cell compartment)
How to set up the cell room
The setting and facilities of the conventional cell room
1. Aseptic operation cell room
1. It consists of three parts: the dressing room, the buffer room and the operating room.
2. The dressing room: located at the outermost part of the culture room, used to change clean clothes, shoes and masks. Place sterilized protective clothing, wardrobes, and shoe cabinets.
3. Buffer room: located between the dressing room and the operating room, the purpose is to ensure the sterile environment of the operating room. Place refrigerators, liquid ammonia tanks, sterilized sterile items, etc.
4. Operation room: located inside, dedicated to aseptic operation and cell culture. Place a Clean bench and carbon dioxide incubator, centrifuge, inverted microscope, etc.
Room requirements: independent room, built according to the requirements of clean workshop, and need to keep the air disinfected and clean and dust-free:
Ultraviolet lamps produce ozone, and the indoor temperature and humidity are high, which is not conducive to the health of staff.
Constant temperature and humidity device for air filtration is best.
2. Preparation room
Often set outside the sterile cell extraction room, mainly for the preparation of culture fluid and related culture liquids. Main settings: centrifuge, water bath, timer clock, ordinary balance and daily analysis and processing.
3. Washing and disinfection room
To be separated from other areas, preferably an independent room, mainly used for cleaning, preparation and disinfection of various culture vessels, often set up: oven, high-pressure steam sterilizer, deionized water or distilled water processor, acid cylinder, water brush, etc.
Basic equipment commonly used in cell rooms
1. Instruments
Clean bench - commonly used side flow (vertical flow) bench, cleanliness should reach Class 100. Domestic products can generally meet the standards.
Microscope: Inverted fluorescence microscope. If conditions permit, it can also be equipped with a high-quality phase contrast microscope with a camera system, a dissecting microscope, a video system or a time-lapse film shooting device, etc., so as to observe, record and photograph cell growth at any time.
Drying oven: The electric drying oven has a high temperature rise, generally needing to reach above 160C.
Water purification device: Deionized water is produced, which can be replaced by triple distilled water.
Refrigerator: Low-temperature refrigerator (-20℃℃) can choose domestic, ultra-low temperature refrigerator (-70,80C)
Liquid ammonia tank.
Centrifuge: Low-speed centrifuge is sufficient, no low-temperature freezing is required. Used for cell sedimentation, high speed is not good.
Precision Electronic balance: precision required: analytical balance has a sensitivity of 0.1mg, 0.01mg and 0.001mg.
Sterilizer: high pressure steam sterilizer.
Filter: used to filter culture medium.
(Figure 2: Cell chamber equipment)
2. Commonly used culture vessels
1. Culture bottle: made of glass or plastic. Mainly used for culturing and propagating cells. When culturing, the mouth of the culture bottle is covered with a screw cap or a rubber plug, and the rubber plug is mostly used for sealing culture. The specifications of domestic culture bottles are expressed in capacity (ml), such as 250ml, 100m, 25ml, etc.: imported culture bottles are mostly expressed in bottom area (cm2).
2. Culture dish: made of glass or plastic, used for holding, separating, processing tissues or conducting experiments such as cytotoxicity, colony formation, single cell separation, isotope incorporation, cell propagation, etc. Commonly used culture dish specifications are 10cm, 9cm, 6cm, 3.5cm, etc.
3. Multi-well culture plate: a plastic product. It can be used for various detection experiments such as cell cloning and cytotoxicity. Its advantages are saving samples and reagents, being able to test a large number of samples at the same time, and being easy to perform aseptic operations. Culture plates are divided into various specifications, and the commonly used specifications are: 96-well, 24-well, 12-well, 6-well, 4-well, etc.
4. Utensils related to culture operations.
Liquid storage bottle: mainly used to store or prepare various culture liquids such as culture fluid, serum and reagents. Liquid storage bottles are divided into various specifications, such as 1000ml, 500ml. (250ml, 100ml, 50ml, 5ml, etc.)
Pipette: mainly divided into graduated pipette and ungraduated pipette. Graduated pipette is mainly used for aspirating and transferring liquids, and commonly used specifications are 1ml, 2ml, 5ml, 10ml, etc. Ungraduated pipettes are divided into straight-headed pipettes and elbow-headed pipettes. In addition to being able to aspirate and transfer liquids, elbow-tipped pipettes are also commonly used for blowing, mixing and cell passage.
Sampler (pipette): used to aspirate, move liquids or drip samples. The amount can be adjusted according to the needs, and the amount can be accurately and conveniently sucked. In particular, the micropipette can ensure that the content of the experimental sample (or reagent) is accurate and the repeatability is good. At present, high-temperature sterilizable, multi-channel pipettes are available for users to choose, which can ensure that the sample addition is accurate, fast, convenient and meets the sterility requirements.
Other supplies: There are centrifuge tubes for collecting cells, test tubes for placing reagents or temporarily inserting pipettes, glass or stainless steel containers for holding pipettes for disinfection, aluminum lunch boxes or storage tanks for storing small culture items for high-pressure disinfection, rubber pipette tips on the top of the pipette, rubber plugs and lids for sealing various bottles and tubes, ampoules or cryopreservation tubes for freezing cells, syringes, beakers and measuring cylinders of different specifications, and funnels, alcohol lamps for ultra-clean workbenches, and micro-spray bottles filled with alcohol or other disinfectants for laboratory personnel to clean and disinfect their hands before operation, etc.
Cell Room Management Rules and Regulations
The cell room is a clean-grade laboratory for various cell cultures. All personnel entering the laboratory must abide by the relevant laboratory rules and regulations and accept the management of laboratory managers.
Chapter 1 General Provisions
Article 1 The cell room is mainly used by relevant scientific researchers within the team. In principle, it is not open to the public. If other personnel really need to use the cell room, they must obtain the consent of the laboratory director and the cell room director in advance.
Article 2 Experiments in and out of the cell room need to be strictly registered. If there is any non-compliance with laboratory work and biosafety requirements, the laboratory director and cell room staff have the right to order rectification.
Article 3 Users must participate in special cell room use specification training and pass the assessment before they are allowed to enter the cell room for experiments. It is strictly forbidden to enter the cell room or use related equipment without permission.
Article 4 During the normal use of the cell room, personnel should enter and exit through the buffer room and air shower; instruments and samples entering and leaving the laboratory must pass through the transfer window, and it is strictly forbidden to bring them in. Experimental supplies should be brought in at one time as much as possible. It is strictly forbidden to bring items unrelated to the experiment into the laboratory. Microorganisms and other easily contaminated items shall not be cultured in the cell room, and any high-risk biochemical experiments are prohibited.
Article 5 Experimenters should take good care of laboratory instruments, use them according to the rules and keep the equipment clean. Expensive equipment in the experiment shall not be turned on or off without permission. Precision instruments must be operated after special training. The preset parameters of the equipment and instruments shall not be changed without permission. If the equipment and instruments fail or an accident occurs, it should be reported to the laboratory director in time and professional personnel should be arranged for maintenance.
Chapter 2 Regulations
Article 1 Routine Operation
1. Wear special laboratory coats. Experimenters must change the special laboratory coats for the cell room in the buffer of the cell room. It is strictly forbidden to enter the cell room without wearing laboratory coats or directly wearing ordinary laboratory coats.
2. Wear special slippers. Change your shoes in the preparation room and try to avoid walking and staying in the buffer room.
3. The cell room can be used by up to 6 people at the same time.
4. During the experiment, it is strictly forbidden to make noise, chat, and repeatedly enter and exit the cell room.
Article 2 Rules for using the incubator
1. Before taking items out of the incubator, clean your hands (or gloves) with alcohol, and try to shorten the time and frequency of opening the door. Note: The air in the incubator is filtered Clean air. Leaving the incubator open for a long time or opening and closing the incubator frequently can easily cause contamination.
2. When taking cells out of the incubator, be gentle and quick, and close the incubator door tightly. Without permission, it is forbidden to look at or move other people's cells or samples. If there are special needs, please contact the laboratory representative for coordination.
3. Before placing the culture flasks and dishes in the incubator, disinfect the surface with alcohol, and wait for the alcohol to evaporate before placing them in order to avoid too much ethanol vapor in the incubator.
4. The placement of cell cultures in the incubator must be neat and orderly for easy search. At the same time, the efficiency of the incubator should be improved as much as possible. Teacher Fu Mai can enable or stop the use of empty incubators according to the experimental situation to save resources.
5. For cells in ordinary culture, unless there is a special need for the experiment, each bottle/plate of cells only needs to observe the growth status once a day. For cells shared by more than 2 people, you can make an appointment to observe together.
Note: Frequently taking cells out for observation can easily cause contamination to the incubator, and will also affect the stability of cell growth conditions.
6. Primary cells must be placed in a dedicated incubator for primary culture and must not be placed in other incubators. If there is no primary cell experiment for a period of time, the person in charge can coordinate and arrange the incubator for other cell cultures.
7. Experimenters should always pay attention to checking whether the incubator temperature and CO2 gas volume are consistent with the set values. Pay close attention to the humidification plate in the incubator, regularly replace the sterile water and disinfect it. Pay close attention to the situation in the incubator. If there is toxic change, plaque, mycoplasma and chlamydia infection or other obvious bacterial stains, the administrator and other users should be notified immediately.
Article 3 Microscope Usage Rules
1. Before using the inverted phase contrast microscope, wipe the stage from the middle to the periphery with alcohol cotton.
2. When leaving the cell room or not in use for a long time, turn off the power in time to extend the life of the bulb. Try to avoid frequent switching of the microscope power.
3. After a cell experiment is completed, register in the microscope usage record book.
4. Fluorescence microscopes are valuable instruments and must follow the valuable instrument reservation and use regulations.
Article 4 Clean bench related operation rules
1. Clean bench use follows the reservation principle. Make an appointment at the entrance of the cell room and fill in the use registration in the cell room.
Those who have no appointment need to use it during the appointment time of others. They must first obtain the consent of the appointment person, otherwise they should unconditionally give up the workbench to avoid delaying other people's experiments.
Use a marker to make an appointment for the next week on the appointment form in advance. Please note that Saturday and Sunday refer to the weekend of the previous week.
Please include the appointment content: time range, name, if there is a special experiment, please indicate it.
(Figure 4: Clean bench)
2. Before using the clean bench, sterilize it with ultraviolet light for 15 minutes. Before and after use, wipe the work area with alcohol cotton balls for disinfection. All items should be disinfected before being placed in the clean bench. It is strictly forbidden to stack too many items in the clean bench to affect the balance of the air path. Frequent exchange of items in different clean benches is strictly prohibited.
3. Before lighting the alcohol lamp, check whether there is enough alcohol in the bottle. The liquid level should account for 1/3-2/3 of the bottle height.
Note: 75% alcohol is used for disinfection, and more than 95% alcohol is used for alcohol lamps. Please pay attention when adding alcohol to the spray pot or lamp.
4. A waste liquid cup should be placed in the clean bench for temporary storage of waste. After the experiment, add NaOH solution to the cup for a while, pour the waste liquid garbage into the pool and dilute it with water at the same time, and pour the solid garbage into the special trash can for biological samples.
Note: The waste liquid tank should be handled in time, and it is strictly forbidden to leave it for a long time to avoid leaving a large amount of culture fluid to breed bacteria.
5. After the cell experiment, any culture bottles, centrifuge tubes, pipettes, etc. containing cell culture dirt must be taken out of the cell room in time.
Article 5 Rules for cleaning instruments
1. Recyclable pipettes, centrifuge tubes, culture bottles, etc. should be soaked in a plastic bucket filled with clean water. There should be enough clean water in the bucket to cover the soaked items. Note: If necessary, recyclable utensils should be rinsed before being put into the bucket, especially when there is a large amount of high-nutrient liquid residue in the culture bottle or serum tube, which is easy to cause bacteria to grow in the clean water in the bucket. Ensure that the soaked bottles and tubes are completely filled with clean water, rather than being pressed to the bottom of the bucket when there is a large amount of air.
2. After rinsing with clean water, soak them in the acid tank in time. The user should clean and dry them in time for use. Please wear rubber gloves and operate carefully.
Note: It is forbidden to soak in clean water for a long time. At this time, the microbes may have grown in the bucket and released endotoxins. Microbe endotoxins are difficult to remove through conventional wet heat sterilization steps, and even dry baking cannot guarantee their complete inactivation.
Endotoxin has a great impact on cell growth and various experiments. Washing procedure: rinse with clean water more than ten times to remove heavy metal residues, then rinse with deionized water 5 times, and then with ultrapure water 5 times, and invert in the oven to dry. And wrap and sterilize in time.
Article 6 Refrigerators, reagents, etc. Related operating rules
1. Take and put items in the refrigerator quickly, and check whether the door is closed when closing it. Store reagents by category. It is strictly forbidden to open the refrigerator door for a long time.
2. The culture medium, biochemical reagents, and samples stored in the refrigerator by each person must indicate the name, preparation date, and sample name. Special reagents must be placed with the consent of the cell room manager. Other people's culture medium or other biochemical reagents must not be used.
3. The packaged serum is stored at -80℃ for a long time and pre-thawed at 4℃ before use. In principle, all thawed serum should be prepared into serum-containing culture medium for standby use. If there is any surplus, it should be temporarily stored at 4℃ and used as soon as possible. Try not to use serum opened by others to avoid cross contamination.
4. The refrigerator space in the cell room is limited. Try to place only reagents with high frequency of use. Do not place some idle reagents or contaminated reagents in the cell room for a long time.
5. The staff will clean the refrigerator regularly. If any illegal items such as unmarked and expired reagents are found, they will be removed, and the person who configured the reagents will be found and warned.
Article 7 Control of items entering the cell room
1. All items entering the cell room must pass through the transfer window, and try to be exposed to ultraviolet light and disinfected with alcohol surface.
2. It is strictly forbidden to bring experimental items that may contain pollutants or pathogens into the cell room.
3. Only a small amount of necessary consumables for experiments can be placed in the cell room. The whole box shall not be moved in, resulting in the accumulation of items in the clean room.
4. The placement of items in the lockers in the cell room shall be arranged by the relevant management personnel and marked.
(Figure 5: Cleaning the clean room)
Article 8 Cleaning Rules for the Cell Room
1. The weekly duty time starts on Saturday and ends on the next Friday.
2. Daily maintenance: pay attention to the normal display of the incubator, the normal use and maintenance of the microscope, refrigerator, and clean bench. Timely cleaning of the special trash cans for biological samples in the cell room and at the door.
3. Weekly cleaning: Clean the cell room with Sanisol every Friday at 5 pm.
Cleaning order: Wipe the clean bench (with alcohol cotton balls); use Sanisol to wipe the table, incubator, clean bench and the outer wall of the refrigerator, as well as the instruments on the table, the wall of the cell room, and the air shower; add water to the water bath or change the water, put the wardrobe and slippers in the cell room neatly, mop the floor, and disinfect the cell room with ultraviolet light for 30 minutes. Note: If the student on duty added enough water to the water tank in the incubator in time last week, according to experience, the incubator will not dry up in the next week, but the student on duty should still check frequently to find abnormal conditions in time. Remember to add sterile water to the incubator.
4. If there is no problem, clean the incubator every 2 months: first take out the items in the incubator and temporarily store them in the clean bench. For more sensitive cells, they can be temporarily stored in another incubator; take out the stainless steel plate in the incubator, including 4 horizontal plates and 2 vertical shelves on the left and right, and clean the steel plate and the inner wall of the incubator with alcohol cotton balls; start the incubator self-disinfection program.
Article 9 Personnel Training
1. The person who will do the cell experiment shall apply and fill in the cell room use application form.
2. The teacher in charge of the cell room shall arrange the training time according to the number of applicants. The training content includes safety training, theoretical knowledge and practical operation training.
3. After passing the training assessment, the instructor, training teacher, and the person in charge of the cell room shall sign and confirm to grant the right to use the cell room.
4. Those who fail the training shall re-participate in the training.
Article 10 Implementation and punishment of the system
1. The person in charge and management personnel of the cell room pay close attention to the use of the cell room. If they find that the operator violates the regulations, they will be reminded first, but records must be made.
2. Those who violate the rules within 3 times in a week will be warned, those who violate the rules 3-5 times will be suspended from using the cell room for 2 weeks, and those who violate the rules more than 5 times will have their access to the cell room suspended and will need to retrain before they can use the room again.
Chapter 3 Supplementary Provisions
These rules and regulations will be implemented from the date of publication, and will be interpreted and corrected by the Laboratory Management Committee of the Biomedical Engineering Team. This laboratory pursues continuous improvement. If teachers and users have good suggestions, they can communicate with the person in charge of the cell room or members of the Laboratory Management Committee. We will coordinate in a timely manner to provide harmonious and efficient experimental conditions for the team.
