This experiment was used for primary culture of osteoblasts.
Operation method
Osteoblast primary culture experiment
Principle
When the implant block culture method is used, incubate with a small amount of culture solution so that the bone tissue block adheres to the culture flask. Observe the cell growth after the bone tissue block floats up. In the monolayer culture method of cell isolation, cancellous bone was cut into 2~5 mm size and digested with collagenase and trypsin. The suspended cells were inoculated into culture flasks and incubated with F12 culture medium.
Materials and Instruments
Bone specimens Move I. Ex vivo block culture Caveat 1、The experimental materials should be fresh, and after separating the materials from the living body, they should be kept at low temperature and the cell separation experiment should be carried out as soon as possible. 2， Aseptic operation. The culture solution used in the operation can be added 5 times the content of penicillin and streptomycin in the usual cell culture solution. 3、When separating cells by enzyme method, pay attention to the concentration of enzyme solution and control the digestion time.When separating the cells by the block method, pay attention to the movement to be gentle, do not hurt the cell tissues, and try to flatten the edge of the tissue block in favor of cell freeing. 4， the choice of culture medium. Different cells have different nutritional requirements in the culture medium, choose according to the characteristics of the separated cells. For more product details, please visit Aladdin Scientific website.
Hams F12 culture medium Trypsin solution for cell passaging Digestive solution for isolation of osteoblasts
Petri dishes Culture flasks Scalpels Surgical forceps
1. Obtain bone specimens from the operating room.
2. Wash the bone tissue several times in sterile saline at room temperature.
3. If the bone tissue cannot be used in time, place the bone specimens in Ham's F12 (with 12 % FBS, 2.3 mmol/L Mg2+, 100 U /ml penicillin and 100 ug/ml streptomycin sulfate) culture medium. Bone specimens can be stored overnight at 4°C.
4 . The following morning, bone tissue is washed with Tyrode's solution containing 100 U/ml penicillin and 100 ug/ml streptomycin sulfate.
5. Bone tissue is placed in a Petri dish, and the trabeculae are removed with a scalpel and surgical forceps, and then the trabeculae are transferred to another Petri dish.
6. 10 ml of Tymde's solution is added to flood the excised trabeculae. Wash the trabeculae several times with Tyrode's liquid until blood and fat cells are removed.
7. For implant block culture, place 2 ml of complete culture solution into a 25 cm2 culture flask and pre-incubate for 20 min to equilibrate the culture solution with gas in the incubator. Adjust the pH with CO2, 4.5% NaHCO3 or HCl as needed.
8. Cut the bone trabeculae into 1-3 mm pieces.
9. Pipette off the pre-incubation medium from the culture flask and add 2.5 ml of fresh Ham's F12 culture medium.
10. Place 25-40 bone trabeculae into the culture flasks.
11. Place the flasks in an upright position, and slide the implant blocks along the bottom of the flasks with the inoculation ring to distribute them evenly. 12. The implant blocks were evenly distributed.
12. Place the culture flask in an upright position for 15 min at 37°C.
13. Slowly return the culture flask to its normal transverse position. Plants should adhere to the bottom wall of the culture flask.
14. Place the culture flask horizontally at 37℃ for 5~7 d. Thereafter, check the cell growth and change the culture medium. In order to prevent detachment of the explants, the flasks should be slowly stood upright before moving them from the incubator to an ultra-clean bench or microscope.
15. Maintain the culture by changing the culture medium twice a week.
16. When the cells grow to form a monolayer, the explants are removed, and the cells are removed from the flasks by using a trypsin (dissolve 125 mg of trypsin (type XI, Sigma) in 50 ml of Ca2+andMg2+ free Tyrode solution). Tyrode solution free of Ca2+ and Mg2+. The pH was adjusted to 7.0 and filtered to remove bacteria. Cells were digested using Pyrex tubes in 5 ml and 10 ml portions and stored at -20°C. Cells were isolated by centrifugation and then inoculated into culture flasks or petri dishes.
Monolayer culture of isolated cells
1. Repeatedly rinse cancellous bone with Tymde's solution to wash away fat and blood cells. Under aseptic conditions, remove the bone trabeculae with a scalpel and surgical forceps.
2. After more bone trabeculae are removed, wash them 3 times with Tyrode's liquid to wash away the leftover blood and fat cells. Cut the trabeculae into 2~5 mm pieces.
3. Wash the trabeculae with F12 culture solution containing FCS.
4. Put the tissue pieces into a sterile vial with a magnetic stirring bar, and then add 4 ml of digestive solution (the digestive solution for separating osteoblasts: ① Liquid A: containing 8.0 g NaCl, 0.2 g KCl, and 0.05 g NaH2PO4H2O in 100 ml of ultrapure water. Liquid B (collagenase and trypsin mixture): 137 mg of collagenase (type I, Worthington Biochemicals) and 50 mg of trypsin (type III, Sigma) were dissolved into 10 ml of liquid A. The pH was adjusted to 7.2, and then the liquid was prepared with 100 ml of ultrapure water. Adjust the pH to 7.2, and then make up 100 ml with ultrapure water (filtered and sterilized, then dispensed into 10 ml and stored at -20°C). The added digestive solution should cover the tissue mass.
5. Stir the liquid containing the tissue mass for 45 min at room temperature.
6. Discard the cell suspension as it is likely to contain fibroblasts.
7. Add 4 ml of digestive solution and stir for 30 min at room temperature.
8. Collect the digestive solution and centrifuge it at room temperature (580 g for 2 min).
9. Aspirate the supernatant off the liquid, then add 4 ml of FCS with 20% FCS to the digestive solution. ml of Ham F12 culture medium containing 20 % FCS and count the cells.
10. After centrifugation of the cell suspension (580 g, 10 min), the cells were mixed with 4 ml of complete culture medium. Use this cell suspension as inoculum.
11. Pre-incubate 75 cm2 flasks with 8 ml complete F12 culture solution for 20 min in order to equilibrate the culture solution with the gas in the incubator.
12. Aspirate off the pre-incubation solution and add 2 ml complete F12 culture solution.
13. Add 4 ml of cell suspension and inoculate with a density of 6,000 to 10,000 cells/cm2.
14. Add 6 ml of F12 culture solution. 6 ml F12 culture medium to make the total amount 12 ml.
15. During the isolation of cells, add 4 ml digestion solution to the remaining bone tissue block and repeat the digestion for 30 min .
Collect the isolated cells. If necessary, repeat the digestion several times. When there is a lot of bone tissue, the digestion time can be extended to 1~3 h . Count the cells after each digestion and inoculate the isolated cells into culture flasks.
Cell Transfection
1. Remove the implanted tissue block.
2. Aspirate off the culture medium and wash the cells with D-PBSA (0.2 ml/cm2 ).
3. Add trypsin solution (0.1 ml/cm2 ) to the culture flasks, and incubate at 37℃ until the cells detach from the wall of the flasks and the cells separate from each other. Observe the detachment and separation of cells with a microscope. Incubation for 10 min is usually sufficient.
4. Transfer the detached cells into a centrifuge tube and add an equal amount of Ham F12 culture medium containing 20% FCS.
5. Centrifuge (600 g, 5 min).
6 . Aspirate the supernatant and gently mix the cells with complete culture medium and blow repeatedly.
7 . Keep two copies of the cells for determining cell density and one copy for DNA detection.
8. Inoculate the remaining cells into culture flasks or dishes previously equilibrated with culture medium. Cells can be walled within 24 h.