Cell Technology Topic: Rat Lactate Osteoblast Culture Experiment

1. The newborn SD rats were sacrificed within 7 hours after the neck was sacrificed, soaked in 75% ethanol for 5 min, the skull bone was removed, the parietal bone and frontal bone were separated, and the ice-physiological saline solution was repeatedly washed to remove the adipose tissue and residual blood. Put it into another Petri dish containing F12 complete Petri solution, wash it, cut the skull into 2~5 mm ² pieces, and pre-digest the washed bone fragments with 0.25% trypsin 2 ml for 15 min to remove Fibrous tissue cells, the supernatant is discarded (mainly containing fibroblasts).

2. Then digest with 10 ml of 0.1% type II collagenase for 20 minutes at 37 ° C, and digest for 20 minutes with magnetic stirring at room temperature. After standing for a few minutes, the digest was collected and centrifuged at 1200 rpm for 10 minutes at room temperature. Remove the supernatant, suspend the cells with 4 ml of F12 medium in 20% fetal bovine serum, inoculate the cells in a 75 ml culture flask, and make 8 ml of the culture solution to make the amount of liquid per bottle reach 12 ml; Repeat digestion with collagenase for 20 minutes, magnetic stirring for 15 minutes, centrifugation for 10 minutes, and place the obtained 3 bottles of cells in a carbon dioxide incubator, culture in 5% CO ₂ , 95% air, 37 ° C, visible after 24 hours. The cells grow adherently, the cytoplasm begins to stretch, and the fresh culture medium is changed. The culture medium is changed every 48 hours (Note: Digestion depends on the specific conditions, and can be digested more than once).

3. Primary culture can be overgrown on the 7th day after general vaccination. During passage, take 1 bottle of osteoblasts with good growth and moderate adherence, discard the culture solution, wash twice with PBS, add 0.25% trypsin 1 ml, digest for 3 to 5 minutes at room temperature, and place the pancreas. Discard the protease solution and add F12 medium for 8-10 minutes. The digested cells were collected, pooled, and counted; adjusted to a suitable concentration with a F12 complete medium. Generally, 2-5 generation osteoblasts are used for experiments. There are too many algebras, and the cells age or differentiate.

Second, the results

As shown in the figure, the shape of osteoblasts is very similar to that of fibroblasts, but the difference is that it is less digestible than fibroblasts, especially when the number of passages is high. When the cells are aging, they are very difficult to digest and are not easily digested. Single cell.

Figure 1: Osteoblasts

Figure 2: Osteoblasts 100 times

 
Figure 3: Mineralized nodules formed by osteoblasts

Third, the method of identification

1. Alkaline phosphatase (ALP) activity assay

2. Bone glassy protein (BGP) detection

3. Mineralized nodule detection