Experimental principle:
The various tissues of the animal body are taken out from the body, treated with various enzymes (common trypsin), chelating agent (usually EDTA) or mechanical methods, dispersed into single cells, cultured in a suitable medium to allow the cells to survive. , growth and reproduction, this process is called primary culture.
After the cells grow into a dense monolayer, the cells are substantially saturated. In order for the cells to continue to grow and the number of cells to be enlarged, passage (re-culture) is necessary. Subculture is also a way to preserve cell types. It is also a necessary process for culturing cells for various experiments. Suspension cells can be dispensed directly, while adherent cells need to be digested before they can be divided.
laboratory apparatus
Incubator (adjusted to 37 ° C), culture flask, penicillin bottle, small glass funnel, plate, pipette, pipette, gauze, surgical instruments, blood cell counting plate, centrifuge, water bath (37 ° C)
Experimental reagents:
1640 medium or DMEM medium, fetal bovine serum, 0.25% trypsin, Hank's solution, iodine, 75% alcohol
Experimental steps:
First, the primary cell culture step
1. Preparation: Take all kinds of disinfected culture products on the clean bench and UV disinfection for 20 minutes. Wash your hands and wash your hands to the elbows with 75% alcohol before starting work.
2. Layout: Ignite the alcohol lamp and install the straw cap.
3. Dispose of the tissue: place the tissue block in a beaker, rinse with Hanks solution 2-3 times to remove blood stains; if the tissue is suspected to be contaminated, it can be placed in a mixture containing streptomycin for 30-60 minutes.
4. Shearing: Cut the tissue into pieces of 2-3 mm size with an ophthalmic scissors to facilitate digestion. Add more than 30-50 times more trypsin solution than the total mass of the tissue block, then pour it into the Erlenmeyer flask and ligature the bottle mouth or stopper with a rubber stopper.
5, digestion: or use a constant temperature water bath, or placed in a 37 ° C incubator can be digested, every 20 minutes in the digest should be shaken, such as using an electromagnetic thermostat mixer to digest better. The digestion time depends on the size of the tissue block and the hardness of the tissue.
6. Separation: When the digestive juice is turbid during the digestion process, a little digestive juice can be sucked out by the pipette and observed under the microscope. If the tissue has been dispersed into cell clusters or single cells, the digestion is immediately terminated, and then filtered through a suitable stainless steel sieve. Fully digested tissue blocks. Centrifuge the digestive juice at low speed (500-1000 rpm) for 5 minutes, aspirate the supernatant, and add an appropriate amount of serum-containing medium.
7. Counting: Counting with a counting plate. If the cell density of the cell suspension is too large, adjust the culture medium and add it to the culture bottle. For most cells, the pH requirement is in the range of 7.2-7.4, and the culture medium is reddish. If the color is yellowish, the liquid is sour and can be adjusted with NaHCO3.
8. Culture: Place it in a 37 ° C incubator. If it is cultured in a CO2 incubator, the bottle mouth should be blocked with gauze tampon or screw cap. The gauze plug is easy to mold, and each time you change the liquid, you need to change the plug.
Second, the original organization block culture method
1. Shearing: Place the small pieces of tissue in a small beaker or penicillin vial, rinse with Hanks solution two or three times to remove the surface blood stains, absorb the Hanks solution, and cut the 1 mm block repeatedly with an ophthalmic scissors.
2, at the right time: use the elbow pipette to suck a few small pieces, put them in the culture bottle, use the pipette elbow to place the small pieces of tissue on the bottom of the culture bottle. The distance between the small pieces is 0.5cm, and the bottom of each 25ml culture bottle can be used. At the mercy of 20-30 blocks.
3. Gently flip the culture bottle, and the bottom of the bottle is up. Pay attention to the flow of the small pieces when you turn the bottle. Plug the stopper and place it in a 37 °C incubator for 2 hours (do not exceed 4 hours) to make the small pieces dry. .
4. Culture: remove the culture flask from the micro-cartridge, open the stopper, hold the culture flask at 46 degrees, gently inject the culture solution into the bottom of the bottle, and then slowly turn the culture bottle over, let the culture solution slowly cover. A small piece of tissue attached to the bottle. Static culture in a thermostat. After the number of cells swimming out of the tissue block increases. Add the culture solution.
Third, the steps of adherent cell passage:
1. Aspirate the old culture solution in the culture flask.
2. Add a small amount of trypsin and EDTA mixture to the bottle to limit the bottom of the bottle.
3, in the temperature box for 2-5 minutes, when the cytoplasm retraction is found, the cell gap is increased, and the digestion is terminated immediately.
4. Aspirate the digestive juice, inject a few milliliters of Hanks solution into the bottle, gently rotate the culture flask, and wash away the residual digestive juice. Note that when the cells are washed with Hanks liquid, the action should be light, so as not to wash away the loose cells, such as digesting with trypsin solution, after aspirating the trypsin solution, it can be directly added to the culture solution without washing with Hanks liquid.
5. Drain the nutrient solution with a pipette and gently blow the bottle wall cells to separate them from the bottle wall to form a cell suspension.
6. After the counting plate is counted, the cell suspension is divided into aliquots and placed in several culture flasks, and cultured in an incubator.
Fourth, suspension cell passage
1. Aspirate the cell culture medium, place it in a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.
2. Aspirate the supernatant, add an appropriate amount of fresh medium, mix well, transfer to a new culture flask according to the dilution ratio, and culture under normal culture conditions.
Precautions
1. Wash hands before operation. After entering the clean bench, use 75% alcohol or 0.2% Xinjieer to wipe the test. The reagent bottle should also be wiped.
2. Ignite the alcohol lamp and operate it near the flame. The heat-resistant articles should always be burned on the flame. The burning time of the metal equipment should not be too long, so as to avoid annealing, and the tissue can be picked up after cooling. The utensils that have absorbed the nutrient solution can no longer be cauterized. In order to avoid charring to form a carbon film.
3, the operation should be accurate and agile, but not too fast, in order to prevent air flow, increase pollution opportunities.
4, can not touch the working part of the disinfected utensils by hand, the layout of the countertops should be reasonable.
5, after the bottle is opened, try to keep the 45 °C oblique position.
6. The pipette of the suction solution cannot be mixed.
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