Cell Culture: “Primary Culture” General Experimental Process and 7 Operation Precautions

Primary Culture: Various tissues of the animal body are taken out of the body, treated with various enzymes, EDTA or mechanical methods, dispersed into single cells, cultured in the culture medium, so that the cells can survive, grow and multiply. This process is called Primary Culture.

The most commonly used primary culture are tissue block culture and dispersed cell culture. Tissue block culture is to directly transplant the shredded tissue block on the wall of the culture bottle and add culture medium for culture. Dispersed cell culture is the use of mechanical or chemical methods to disperse the cells. To isolate the most active free cells from the tissues of the fetus or newborn, the classic method is to digest the conjugate between the cells with proteolytic enzymes (such as trypsin and collagenase), or use a metal ion chelator (such as EDTA) ) Remove the Ca2+ on which the cells adhere to each other, and then slightly shake it mechanically to make it a single cell.

Equipment required for Primary Culture Experiment

Laminar Flow Clean Bench, culture bottle, CO2 Incubator, penicillin bottle, small glass funnel, petri dish, pipette, pipette, gauze, surgical instrument, hemocytometer, low-speed centrifuge, water bath (or incubator)

Reagents needed for the experiment: culture medium (containing 20% calf serum), 0.25% pancreatin, Hank’s solution, iodine
Sample needed for experiment: animal tissue block

Primary digestion culture method

1. Place the sterilized culture supplies on the clean surface of the Clean Bench, and sterilize with ultraviolet light for 20 minutes;
2. Wash your hands before starting work, wipe your hands to the elbow with 75% alcohol, turn on the infrared sterilizer or alcohol lamp, and install the straw cap;
3. Rinse the tissue 2-3 times with Hank’s solution (put the tissue block in a beaker) to remove blood stains (if the tissue is suspected to be contaminated, first put it in a mixture containing penicillin for 30-60 minutes);
4. Cut the tissue into pieces (2~3 mm in size) with ophthalmic scissors, add 30-50 times more trypsin solution than the total amount of the tissue pieces, and pour them into the conical flask together, ligate the mouth of the bottle or plug it with a rubber stopper ；
5. Use a constant temperature water bath or place in a 37°C constant temperature box for digestion. Shake every 20 minutes during digestion. The digestion time depends on the size of the tissue mass and the hardness of the tissue;
6. When the digestive juice becomes turbid during the digestion process, a pipette can be used to suck out a little digestive juice and observe under the microscope. If the tissue has been dispersed into cell clusters or single cells, immediately terminate the digestion, and then filter out the tissues that have not been fully digested through a stainless steel sieve. Block, centrifuge the digestion solution at low speed (500~1000 RPM/min) for 5 minutes, aspirate the supernatant, and add an appropriate amount of culture medium containing serum.
7. Count with a counting plate. If the cell density of the cell suspension is too high, add culture fluid to adjust, and then divide it into culture flasks. For most cells, the pH is required to be in the range of 7.2 to 7.4, and the culture medium is reddish. If the color is yellow, it means that the liquid has become acidic and can be adjusted with NaHCO3.
8. Placed in the CO2 Incubator, the temperature is 36.5℃, the bottle mouth needs to be blocked with gauze cotton stopper or screw cap, and a new stopper needs to be replaced every time the liquid is changed.

Precautions for aseptic operation

1. Wash your hands before operation. After entering the Laminar Flow Clean Bench, wipe your hands with 75% alcohol or 0.2% bromogeramine, and wipe the mouth of the reagents and other bottles;
2. Ignite the infrared sterilizer or alcohol lamp, and operate near the flame. Heat-resistant items should be burnt on the flame frequently. The burning time of metal instruments should not be too long. The utensils that have absorbed the nutrient solution should not be burnt again, so as not to burn and form carbon membrane;
3. The operation should be accurate and agile, but not too fast to prevent air flow and increase the chance of pollution;
4. Do not touch the working part of the sterilized utensils with your hands, and the supplies on the work surface should be arranged reasonably;
5. After the bottle is opened, try to keep the 45℃ inclined position.
6. The straws for absorbing the solution cannot be mixed.
7. Tissue blocks, trypsin, culture medium and other items that are easily damaged by ultraviolet rays, it is best to bring them into the operation field during culture; if they are placed in advance, they need to be covered with paper to avoid the effects of radiation;