Rat vascular endothelial cell culture can be used for (1) studies on blood fluidity, prevention of thrombosis, and regulation of vascular tone; (2) studies on selective permeability as well as immune regulation.
Operation method
chunking
Principle
The patch method is suitable for endothelial cell culture in low endothelial cell yields and in vessels with small tubular diameters, and it is a simpler and more economical method than enzymatic digestion.
Materials and Instruments
Male Wistar rats Move 1. Dislocate the cervical vertebrae to execute the rats, soak the whole rats in 75% ethanol to sterilize for about 1 min. under aseptic condition, cut open the thoracic cavity layer by layer, isolate and take out the aorta, and put it into a petri dish containing sterile D-Hanks' liquid. 2. 2. Use ophthalmic scissors and forceps to remove as much fat and fibrous tissue as possible from the vessel periphery. Then, rinse the outer surface of the blood vessel and the coagulated blood in the lumen of the vessel several times with D-Hanks' solution containing antibiotics, and then put it into another sterile Petri dish. 3. The vessel was cut longitudinally with ophthalmic scissors and spread flat in a Petri dish. Use a very sharp scalpel to cut them into 1 mm3 sized arterial implants (or cut them with scissors, according to personal preference), and then plant them into culture flasks or petri dishes (pre-set the flasks or petri dishes with 1% gelatin at 37℃ overnight, and then transfer them into a CO2 incubator for 2h before using. The flasks or dishes can be rinsed with culture medium containing 10% calf serum). Place the endothelial surface of the arterial block in contact with the bottom of the bottle, and plant at a density of about 1 block/cm2. 4. 4. Allow to stand in the incubator for 2 h (dry wall). Then, add 0.5 ml of culture medium containing 25% fetal bovine serum, the amount of which should slightly cover the intimal surface of the arterial implant without causing the implant to float. 24 h later, replace the culture medium with 2-3 ml of culture medium. 5. At about 72 h, the implant should be placed in contact with the bottom of the bottle. 5. After 72 hours, when the endothelial cells were fully grown and the fibroblasts were just about to grow, remove the arterial implant, scrape off the fibroblasts and change the culture medium once. After that, the culture medium was changed once every 2 days, and each time, 1/2~1/3 of the volume of the medium was changed. Continue to culture for 8-10 d, the cells will be fused into a monolayer and can be passed on. Caveat 1. Maintain all tissue cells in sterile conditions from the time of sampling. Cell counting may be performed in a sterile environment. 2. In the ultra-clean table, tissue cells, culture solutions, etc. should not be exposed for too long to avoid evaporation of solutions. 3. where steps are operated outside the ultra-clean bench, each vessel needs to be covered with a lid or rubber stopper to prevent bacteria from falling in. 4. Wash your hands before operation, and wipe your hands with 75% alcohol or 0.2% Neosporin after entering the ultra-clean bench. The mouth of the reagent bottle should also be wiped. 5. Light the alcohol lamp, operate near the flame, heat-resistant items should always be burned on the flame. Metal instruments should not be cauterized for too long to avoid annealing and should be cooled before clamping the tissue. Appliances that have absorbed nutrient solution should not be cauterized again to avoid charring and forming a carbon film. 6. the operation should be precise and agile, but not too fast to prevent air flow, increasing the chance of contamination. 7. the working part of the sterilized utensils should not be touched by hand, and the arrangement of supplies on the workbench should be well laid out. 8. bottles should be kept in a 45° slanting position as far as possible after opening. 9. Pipettes, etc., for sucking up solutions should not be mixed. Common Problems I. Experimental discussion Plants are suitable for the culture of endothelial cells with low endothelial cell yield and small vessel diameter, and the method is simpler and more economical than the enzymatic digestion method. However, the disadvantages make it easy to mix with fibroblasts and smooth muscle cells, the former's adherence time is close to that of endothelium, and the latter's adherence is slower than that of endothelium, and it will be inhibited by heparin. For more product details, please visit Aladdin Scientific website.
DMEM Fetal bovine serum Endothelial growth factor Sodium heparin Penicillin Gelatin Trypsin PBS D-Hanks' solution
Surgical Instruments Ophthalmic Scissors Ophthalmic Forceps Petri Dish Scalpel Culture Bottle CO2 Incubator
1. Remove the arterial block at the appropriate time, i.e., when the endothelial cells have already climbed out and become incipient (roughly at about 72 h, according to the conditions of the culture medium in this paper, because of the presence of growth factors, the endothelium is easy to climb out and proliferate), remove the arterial block and scrape off the fibroblasts.
2. The purity of endothelial cells can be improved by using heparin-containing culture medium (adding 100 U/ml of heparin to the culture medium);
3. a high concentration of serum and 50 µg/ml ECGF can sufficiently promote the proliferation of endothelial cells, and endothelial cells become dominant. In addition, the dry walling time of arterial explants should not exceed 2 h, and the addition of culture medium is especially critical, too little will prevent the endothelium from contacting the culture medium, while too much will cause the arterial explants to float, resulting in failure of walling. Therefore, the addition of culture fluid after dry walling and the addition of a small amount of highly nutritious culture fluid (0.5-1 ml) can avoid these problems.