Rat lung fibroblast primary culture experiment
Rat lung fibroblast primary culture experiment
Rat lung fibroblast cell culture can be (1) used for cell preservation, (2) used for molecular biology research, and (3) used for gene therapy research.
Operation method
trypsin digestion
Principle
Lung fibroblasts from rats were removed from the organism, treated with trypsin, chelating agent (commonly used EDTA), dispersed into single cells, and placed in the appropriate growth medium culture medium to allow the cells to survive, grow and multiply.
Materials and Instruments
Wistar suckling mouse Move I. Preparation of experimental materials Caveat 1. Pay attention to the aseptic operation. 2. During the operation, the action should be gentle, let the liquid slowly cover the small pieces of tissue, it is strictly prohibited to move too fast, resulting in the impulse generated by the liquid to make the pasted pieces of tissue float up and cause failure of primary culture.Lung fibroblasts can secrete a variety of cytokines and other substances, and have close interactions with other cells in the lung, thus playing an important role in the development of bronchial asthma, chronic obstructive pulmonary disease, pulmonary fibrosis and other diseases. In vitro culture of lung fibroblasts is an important means and tool for the study of lung diseases such as bronchial asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. Common Problems Pulmonary fibroblasts secrete a variety of cytokines and other substances and have close interactions with other cells in the lungs, thus playing an important role in the development of diseases such as bronchial asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. In vitro culture of lung fibroblasts is an important means and tool for the study of lung diseases such as bronchial asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. For more product details, please visit Aladdin Scientific website.
PBS Bovine serum Penicillin Streptomycin EDTA Trypsin Iodine Alcohol
Sheep Balls Ophthalmic Straight Scissors Ophthalmic Curved Scissors Ophthalmic Straight Tweezers Ophthalmic Curved Tweezers Glass Petri Dish Plastic Culture Bottle
1. AnimalOne Wistar suckling mouse 1-4 days after birth.
2. Reagents
PBS, culture medium (Hyclone's low sugar DMEM, 10% neonatal bovine serum containing Hyclone, 100 U/ml penicillin, 1% gelatin PBS, 0.25% trypsin (containing EDTA, GIBCO), iodine and alcohol wool balls.
3. Instruments2 ophthalmic straight scissors, 2 ophthalmic curved scissors, 2 ophthalmic straight forceps, 2 ophthalmic curved forceps, 3 sets of glass petri dishes, 25 cm2 plastic culture bottles (Costar).
Specific operation
1. Gelatin-coated culture bottles overnight (prepare 2-3), remove the gelatin, rinse the bottles with 2 ml of culture solution once, and place them in the ultra-clean table.
2. Lung dissection: remove the suckling mouse after soaking in alcohol, transfer to a glass petri dish on the ultra-clean table, disinfect the chest skin with iodine, and then de-iodize it with an alcohol cotton ball. The skin on the back of the neck was pinched with the left hand to fully expose the chest, and the right hand used ophthalmic straight scissors to cut the skin, fully tear it, and then sterilized it with alcohol cotton balls, and then used another pair of ophthalmic curved scissors to cut the ribs upward along the left lower edge of the sternal stem, and then cut the sternum horizontally in the middle of the incision. The lung was removed with ophthalmic forceps and placed in a glass dish containing PBS (containing 200 U/ml penicillin) and rinsed to remove blood.
3. The lungs were divided into lobes with ophthalmic scissors, the surrounding blood clots and fibrous tissue were removed with forceps, the bronchi and blood vessels at the hilum were cut with ophthalmic scissors, and the lungs were rinsed once more with PBS containing double antibiotic.
4. Use ophthalmic curved scissors to cut the lung tissue into 1 mm3 size, add PBS containing dual antibody, blow open the lung tissue block, and let it stand for 15 min before replacing it with new PBS.
5. Using a 200 ul micro-sampler (preferably the special one in the ultra-clean bench, and wipe the handle of the gun well with an alcohol wool ball when ready to use) take one 200 ul spiking gun tip, cut off the tip, and bend it with a flame on an alcohol lamp. Use the tip of the gun to suck the tissue block inoculation in the culture bottle, each bottle of about 20-25 blocks, each small block spacing of about 0.5 cm. After placing the tissue blocks, gently turn the culture bottle over so that the bottom of the bottle is facing upward, add about 2 ml of culture solution to the bottle, cover the bottle, place the bottle at an angle in a warm box, and after 2-4 h of dry walling, turn the bottle over slowly and lay it flat, and continue to incubate it statically. Pay attention to the above operation process to move gently, let the liquid slowly cover the small pieces of tissue, it is strictly prohibited to move too fast to cause the impulse generated by the liquid to make the pasted tissue pieces float up and cause the failure of primary culture. 48 h after the replacement of the liquid, replacement of 2-3 ml can be.
6. 72 h after the paste block wall, microscope can see a large number of fibroblasts climbed out, remove the tissue block, continue to culture for 2-3 days, to be full of cells, can be passed on. Note: Because of the low serum concentration, endothelial cells can climb out a small amount, but will die soon.
2.7 Passage with 0.25% trypsin routine digestion, 1:2 passaging, passaging, the use of differential apposition method of purification once, that is, wait for the cell wall 1.0 -1.5 h (i.e., the vast majority of the fibroblasts have been affixed to the wall), discard the unaffixed cells and the culture medium, and replace with a new culture medium.Fibroblasts have a long shuttle shape and often grow in a swirling pattern. See figure below:
