Rat Mammalian Osteoblast Culture Experiment
Rat Mammalian Osteoblast Culture Experiment
Rat Mammalian Osteoblast Cell Culture Cultures are used for (1) obtaining rat mammalian osteoblasts; (2) cytological mechanism study of bone repair.
Operation method
enzymatic digestion
Principle
Osteoblasts were cultured in DMEM culture medium containing 20% fetal bovine serum from the skulls of mice born 2~3 days after birth, and collagenase digestion was used to isolate osteoblasts, and the cells were inoculated and passaged. Collagenase digestion is a reliable, simple and rapid method for primary cell isolation and culture.
Materials and Instruments
SD rat Move I. Experimental steps 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. Discussion Osteoblasts are encapsulated in hard tissues, making them difficult to handle. Osteoblasts can be cultured by bone tissue block method, enzyme digestion, periosteal tissue block method, bone marrow culture method, and thin bone slices treated with EDTA and digested with collagenase. Osteoblasts cultured in vitro maintain some of the characteristics of bone tissue cells. It has been reported in the literature that the morphology of osteoblasts cultured by different methods is different [1]. For more product details, please visit Aladdin Scientific website.
F12 Complete culture solution Trypsin Type II collagenase Alcohol
Surgical instruments Magnetic stirrer
1. Newborn SD rats within 24 hours of neck dissection were soaked in 75% ethanol for 5 min, the skull was removed, the parietal and frontal bones were separated, the cranial specimens of rat suckling mice were washed repeatedly with iced saline solution to remove fatty tissues and residual blood, and then put into another Petri dish with F12 complete medium for further washing, and then the cranial bone was cut into 2-5 mm2 pieces , and the washed bone fragments were pre-digested for 15 min with 0.25% trypsin 2 ml to remove fibrous tissue cells, and the supernatant was discarded (containing fibroblasts). The washed bone fragments were pre-digested with 0.25% trypsin 2 ml for 15 min to remove fibroblasts, and the supernatant (which mainly contained fibroblasts) was discarded.
2. Then digest with 0.1% collagenase type II 10 ml at 37°C for 20 minutes and with magnetic stirring for 20 minutes at room temperature. The digested solution is collected and centrifuged at 1200 rpm for 10 minutes at room temperature. Remove the supernatant, suspend the cells with 4 ml of F12 culture medium with 20% fetal bovine serum, inoculate the cells in 75 ml culture flasks, and replenish 8 ml of culture medium to make the volume of liquid in each flask up to 12 ml; for the precipitated portion of the cells after resting, repeat the digestion with collagenase for 20 minutes, the digestion with magnetic stirring for 15 minutes, and centrifugation for 10 minutes, and then put the obtained 3 flasks of cells in carbon dioxide incubator under 5% CO2, 95% air, and 37℃ temperature. 95% air, 37 ℃ temperature culture, 24 hours after the cells can be seen attached to the wall growth, cytoplasm began to stretch, change the fresh culture medium, and then every 48 hours to change the culture medium (Note: digestion depending on the specific circumstances, can also be digested more than 1 times).
3. Generally, primary culture can be full grown on the 7th day after inoculation. For passaging, take 1 bottle of osteoblasts with good growth and loose wall adherence, discard the culture medium, rinse with PBS for 2 times, add 0.25% trypsin 1 ml, digest for 3-5 minutes at room temperature, discard the trypsin solution, and add F12 culture medium and blow well for 8-10 minutes. The digested cells were collected, combined and counted; adjusted with F12 complete medium until the cell concentration was appropriate. Generally, 2-5 generations of osteoblasts are taken for experiments. Too many generations will result in aging or differentiation of the cells.
II. Results
As shown in the figure, osteoblasts are very similar in shape to fibroblasts, but are different in that they are much less digestible than fibroblasts, especially when they are passed on a lot and the cells are old, they are very difficult to digest and do not digest easily into single cells.
Figure 1: Osteoblasts
Figure 2: Osteoblasts 100 times
Figure 3: Mineralized nodules formed by osteoblastsIII. Methods of identification
1. Alkaline phosphatase (ALP) activity assay
2. bone glass protein (BGP) assay
3. Mineralization Nodule Test
[1] Szeto Zhenqiang. Cell Culture [M]. First Edition. Xi'an. World Book Publishing Xi'an, 2000:115.