Rabbit intervertebral disc cell culture can be used to (1) obtain rabbit intervertebral disc cells, (2) for research on disc-related topics, and (3) for research on rabbit models.
Operation method
chunking
Principle
The in vitro culture system of rabbit intervertebral disc cells was firstly established, in which pure oxygen was given to the first group, and ozone was given to the other three groups at different concentrations: 30 ug/ml, 60 ug/ml and 80 ug/ml, and then the viable cell rate of the intervertebral disc cells was calculated by using the Taipan blue cell rejection assay after 15 min and 30 min.
Materials and Instruments
Rabbit Move I. Experimental steps Results IVD cells are chondrocyte-like cells, described as: monolayer cells with irregular shape, which can be triangular, polygonal, pike-shaped and irregular. Different from fibroblasts. As shown in the figure below: Figure 1: Tissue block apposition method Figure 2: Transmissible cell morphology Figure 3: Pre-passage state of cells 95% fusion Caveat The intervertebral disc is located between two adjacent vertebrae, and the sampling process is cumbersome and should be done quickly, within half an hour after the rabbit's death; otherwise, the tissue is less tolerant to hypoxia, resulting in failure of the culture. For more product details, please visit Aladdin Scientific website.
Culture medium DMEM Hanks' buffer Fetal bovine serum Insulin Transferrin Selenite Penicillin Streptomycin
Petri dishes Culture flasks Flasks Test tubes Ophthalmic shears, ophthalmic forceps CO2 Incubator pH meter Constant temperature water baths
(2) Shearing: On an ultraclean table, further separate the surrounding tissue and rinse it several times with Hanks' solution until the rinse is clear. Ophthalmic scissors were used to trim the tissue into small pieces of 1~2 cm3 in size , and the tissue was rinsed with Hanks' solution. Add a small amount of culture medium containing serum. (The tissue block should not be too small, otherwise it is not easy to crawl out a sufficient number of cells).(3) Inoculation: Use a pipette to suck small tissue blocks into the bottom of the culture flask, arrange evenly, a 25 cm2 culture flask can be placed 10-15 small tissue blocks, turn the flask, add a small amount of culture medium. 4~6 hours later, after the tissue blocks are sufficiently adhered to the wall, then turn it back (the action must be gentle, in order to avoid the tissue blocks that have just been adhered to the wall to detach from the bottom of the flask).(4) Incubate the cells in 5% CO2 incubator and change the solution every 3 days. When 95% of the cells are fused, 0.25% trypsin was used to passivate the cells into separate flasks.2. Enzyme digestion(1) The previous steps are the same as the tissue block method 1 and 2.
(2) The tissue block should be further sheared, and it is better not to add serum to the culture medium at this time.
(3) After completion, add the digestive solution and transfer to a 10 ml centrifuge tube for digestion in an incubator.
(4) Every 20 minutes, blow with a pipette to see if the liquid becomes turbid, and take a small amount of liquid and observe it under a phase contrast inverted microscope.
(5) Count the number of viable cells by 0.4% Formosan staining.
(6) The inoculum density was in the order of 105 . Incubate in 5% CO2 incubator and change the liquid every 3 days.
(7) When the cells were 95% fused, 0.25% trypsin was used to passivate the cells. 
