Primary cell culture can be applied to (1) molecular biology; (2) cell biology; (3) genetics; (4) immunology; (5) oncology; (6) virology and other fields.
Operation method
Primary cell culture of murine embryo
Principle
Primary cell culture, refers to cells, tissues or organs obtained directly from the animal body and cultured in vitro until the first passaging. In this type of culture, the desired tissues (or organs) are first removed from the animal's body by aseptic operation, digested by trypsin, dispersed into individual free cells, and cultured under artificial conditions for continuous growth and reproduction.
Materials and Instruments
Pregnant female rats Move 1. Material collection: Pregnant female rats were taken, and after execution by cervical dislocation, the whole body was immersed in a beaker containing pure alcohol for 1 minute. Remove the female rat in a stainless steel surgical tray and open the abdominal cavity with sterile surgical scissors, remove the mouse embryo and put it into a sterile petri dish. Note that the removal can be done outside the sterile operating table. 2. Wipe the periphery of the culture dish with 70% alcohol, then transfer the dish to an ultra-clean bench and wash the embryos several times with Hanks' solution containing double antibodies to remove blood stains. The head, tail, limbs and internal organs were removed with sterile surgical instruments, and the remaining tissues were cut with scissors and washed several times with Hanks' solution containing double antibiotic. Finally, it was washed with cell culture solution containing serum. 3. Cut the tissue pieces into 1 mm3 or so in the culture medium, use a sterile rubber-tipped pipette to suck up the tissue pieces, pay attention to sucking in the front of the special cell long pipette slender part, such as sucking too high, can make the tissue pieces adhere to the wall of the tube and can not be blown out. Tissue block should be evenly attached to the bottom of the cell culture flask, pay attention to the space between the tissue block, not too dense. 4. Turn over the culture flask so that the cell culture side of the adhered tissue block is facing up, and add 3--5ml of cell culture solution to the bottom of the culture flask, do not add too much in order to avoid contamination caused by leakage of culture solution. After screwing the cap on the bottle, write the culture time on the side of the bottle with an oil-based pen. The cell culture was placed in a CO2 incubator at 37℃, CO2 concentration of 5% and complete humidity to facilitate the attachment of the tissue mass. 5. 2-3 hours later, the visible tissue nuggets are slightly dry and firmly adhered to the wall of the bottle, and then slowly turn over the bottle, so that the cell culture solution slowly soak the tissue nuggets and continue to cultivate. During the operation, the action must be gentle, so as not to adhering to the bottle wall of the tissue block off, affecting the adherence culture. Then place the culture bottle in 37℃ incubator for 2-3 days. Caveat 1. the ratio of culture fluid and culture: a certain concentration of culture can only support the growth of a certain number of cells, and cannot blindly increase the concentration of cells per unit volume. 2. pH: the initial culture pH should be 7. 4， and should not be lower than 7.0 during the culture process. 3. space inside the culture flask: the ratio of the volume of the culture liquid to the space on the liquid surface inside the culture flask is generally 1:10. For more product details, please visit Aladdin Scientific website.
Culture fluid Serum Balanced salt solution Antibiotics HEPES buffer
Ultra-clean bench Filter CO2 incubator Electrically heated drying oven Petri dishes Culture flasks Drip tubes Tweezers Surgical scissors