What is a Cell Culture?

Cell culture refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH, and certain nutritional conditions, etc.) in vitro to enable it to survive, grow, reproduce and maintain its main structure and function. Cell culture is also called cell cloning technology. The formal term in biology is cell culture technology. Regardless of the entire biological engineering technology or one of the biological cloning technologies, cell culture is an indispensable process. Cell culture itself is a large-scale cloning of cells. Cell culture technology can transform a single cell into a simple single cell or rarely differentiated multi-cells. This is an essential part of cloning technology, and cell culture itself is the cloning of cells. Cell culture technology is an important and commonly used technique in cell biology research methods. Cell culture can be used to obtain a large number of cells, and to study cell signal transduction, cell anabolic metabolism, cell growth and proliferation. ^[1]

Animal cell culture

Facilities : ultra-clean bench, constant temperature incubator, inverted microscope, liquid nitrogen storage tank, electric blast drying oven, freezer, electronic balance, constant temperature water bath, centrifuge, pressure steam sterilizer.

equipment:

First, glass equipment

I. Cell Recovery

After the frozen cells were removed from the liquid nitrogen, they were continuously shaken in a 37 ° C water bath to promote their melting. Transfer to a 15ml centrifuge tube, add 10ml of pre-warmed DMEM complete medium, gently blow, centrifuge, 2000rpm × 2min, discard the supernatant. 10 ml of DMEM medium was added for washing, and the supernatant was discarded. Add 10 ml of DMEM complete medium, gently pipette, inoculate in a 10 cm dish, and culture in a cell incubator containing 5% CO ₂ .

Cell Passaging

When the cell density reached 80% ~ 90%. Remove medium and wash twice with 10ml PBS. Add 3ml trypsin containing 0.25% EDTA and place in a cell culture incubator for 3min. Add 1 ml of DMEM complete medium to terminate the trypsinization and transfer to a 15 ml centrifuge tube. Add 10ml PBS to wash the cell culture plate, transfer to a 15ml centrifuge tube, 2000rpm × 2min, discard the supernatant. Add another 10ml PBS (autoclaved and stored at 40C), mix well, pipette 10 microliters for counting, inoculate at 1 × 10 ⁵ per plate, and continue to culture in a cell incubator containing 5% CO ₂ .

Cryopreservation of cells

When the cell density reached 80% to 90%, remove the medium and wash twice with 10ml PBS. Add 3 ml of trypsin containing 0.25% EDTA and place in a cell incubator for 3 min. Add 1 ml of DMEM complete medium to terminate the trypsinization and transfer to a 15 ml centrifuge tube. Add 10ml PBS to wash the cell culture plate, transfer to a 15ml centrifuge tube, 2000rpm × 2min, discard the supernatant. Add 1ml of cryopreservation solution (90% serum, 10% DMSO), put it in a cryopreservation box (isopropyl alcohol in the box to ensure the speed of temperature reduction), immediately put it in a 80 ° C refrigerator overnight, It can be stored in liquid nitrogen for at least two years, or three months without liquid nitrogen.

Preparation of cryopreservation solution: 70% complete medium + 20% FBS + 10% DMSO. DMSO should be added slowly and shake while dropping. ^[5]

(1) When you start to cultivate a certain cell for the first time, be sure to search on WWW. ATCC. ORG with the name of the cell, you can get detailed information about the cell, including the culture medium, serum, additives, The usual digestion time, passage time, etc. For specific cells (such as primary cultured cells), you need to consult the relevant literature for more accurate culture methods.

(2) When entering the cell to start cell culture, you must strictly follow the steps below:

Make sure that all the solutions and consumables for cell operation have been sterilized and tested without problems. Do not use uncertain solutions and consumables. Unless special circumstances, do not borrow other people's solutions.

Make sure the cuffs of the clothes have been rolled up or the cuffs of the white coat have been tightened.

Determine the amount of alcohol in the alcohol lamp, and replenish it if necessary.

Make sure that all the solutions and consumables you need are within reach. In order to easily open the caps with one hand, all caps can be unscrewed before the experiment.

Try not to pour the solution directly, unless the bottle mouth is not burned out. If the pouring fails and the solution sticks to the bottle mouth, use a 75% alcohol sprayed paper towel to carefully clean around the bottle mouth (do not touch the bottle mouth) and simply burn on the flame.

If you are not sure that the consumables used are clean during operation, you must replace them in time.

Pack 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 contamination

Experimental supplies prevent contamination. The reagents, consumables, and equipment used for cell culture must be thoroughly cleaned and disinfected. The various solutions must be sterilized carefully and used only after the sterility test is negative. The operating room and the remaining sterile equipment should be cleaned, disinfected and sterilized regularly.

Prevent pollution during operation.

Clothing that is prone to static electricity or dust absorption must be changed to a white coat before entering the cell.

Before starting the experiment, make sure that there are no problems with the gloves you wear. As long as you have touched the items outside the biological safety cabinet, you must disinfect the gloves in time.

Close the door after entering the cell culture room, sit down and walk as little as possible to avoid affecting the curtain of the biological safety cabinet. Wipe your hands and bottle caps with a 75% alcohol cotton ball before starting work. Strictly check the equipment, solutions and cells used in advance, do not bring contaminated or unsterilized items into the sterile room, and do not use it casually, so as to avoid large-scale pollution.

The action should be light during cell operation. The bottle mouth must be opened in a sterile area around the flame, and the bottle mouth should be placed around the flame and simply turned to burn. Be careful not to let the flame burn the plastic bottle mouth.

During experiment operation, the partition of the biological safety cabinet should be kept as low as possible to minimize conversation. Never sneeze or cough towards the work area to avoid unnecessary pollution.

The bottle cap should be placed upside down to avoid contamination of the bottle cap due to mishandling.

Do not pass over the mouth of the open container to avoid contamination of cells by unknown objects falling from the clothes.

Pay attention to replacing Pasteur pipettes, pipette tips and pipettes in the experimental operation. Disposal of non-clean or non-determinable clean items must be discarded. After the experiment, you should clean up in time, keep the laboratory clean and tidy, and finally clean the table with 75% alcohol.

(4) Prevent cell cross-contamination

When performing a variety of cell culture operations, the equipment used must be strictly distinguished, and it is best to mark it for easy identification. The operations are performed in sequence, only one cell is processed at a time, and t-kun disorder is prone to occur when multiple cells and multiple operations are performed together.

Do not touch the mouth of the reagent bottle with the pipette tip and pipette with the cells adhered during the liquid exchange or passaging operation, so as not to bring the cells into the medium to contaminate other cells.

Once all cells are purchased, introduced from other places, or established by themselves, they must be kept frozen in time. Once contamination occurs, the cells can be revived and culture continued. · ^[6]