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What are the methods for culturing vascular smooth muscle cells with fetal bovine serum?


The commonly used methods for culturing vascular smooth muscle cells with fetal bovine serum include the probe method and the enzyme dispersion method. Let's take a look at these two methods together.

1. Patch method

Method: After bleeding the animal through the carotid artery, the thoracic and abdominal aortic segments were quickly removed under aseptic operation and rinsed three times in a petri dish containing Hank's solution. The clot was washed clean, and the fibrous fat layer of the outer membrane was peeled off. Then, the blood vessels were longitudinally cut, and the inner and middle layers of the intima and endothelial cells were scraped off. The inner and middle layers of the intima were quickly torn off, cut into small strips about 1mm wide, soaked in Hank's solution containing serum, and the torn tissue blocks were planted on the wall of the culture bottle. Place in a constant temperature incubator at 37 ℃ for about 2 hours, remove the culture bottle and add 20% HyClone fetal bovine serum culture medium, then return to the incubator for static cultivation for 4 days. Four days later, cells can be seen migrating and swimming out of the tissue.

The vascular structures of different animals vary, with pigs and monkeys being easier to operate, but small animals such as rabbits and mice are difficult to separate due to their small blood vessels and thin vessel walls. Additionally, the tissue block is too thin to adhere to the culture medium, making it difficult for cells to grow. To ensure the success of cultivation, attention should be paid to:

① The number of implanted tissue blocks, such as 75cm2 long neck bottle tissue blocks, shall not be less than 30;

② Add appropriate amounts of different serum according to the species of cultured cells. Generally, bovine serum can be added. If bovine serum has poor proliferation effect, fetal bovine serum (FBS) should be added, usually at a concentration of 10% to 20%;

③ Selection of culture medium: Commonly used media include DMEM (Dulbecco's modified Eagles' medium) and M199 medium. DMEM has a better effect on cultivating smooth muscle cells in rabbits.

2. Enzyme dissociation method

After cutting along the longitudinal axis of the artery, scrape off the endothelial cells and tear off 2/3 of the inner membrane. Be careful not to mix in endothelial cells. Cut the tissue block into 1-2mm2 and place it in serum-free M199 medium with 3mg/ml collagenase at 37 ℃ for 0.5-1.5 hours. Centrifuge the supernatant, repeat the above digestion steps, and then centrifugate (9000r, 4min), collect cells, adjust the number of cells in M199 medium with 5%~10% of the same serum or HyClone fetal bovine serum, and then transfer to 30~90mm culture dish for culture. For rabbits, it is better to add high concentration of glutamine (0.2g/L). There are significant differences among researchers in the selection of culture medium, enzyme concentration, and digestion time.

3. Summary: The patch method has the advantages of obtaining high purity, large quantity, and easy operation of smooth muscle cells. However, using the patch method can easily lead to the loss of muscle filaments in the cytoplasm of smooth muscle cells and an increase in subcellular organelles. On the contrary, enzymatic dissociation method causes the loss of intercellular connections due to the action of enzymes, resulting in abundant muscle fibers inside the cells and maintaining a contracted state. Smooth muscle cells are often cultured from the thoracic and abdominal aortic segments of rabbits, pigs, mice, and monkeys. Due to the different treatment methods of block sticking and dissociation, there are differences in cell morphology and properties in the initial stage of cell culture. With the extension of cultivation time, the cultivation environment tends to be consistent, and the cell characteristics also tend to be similar.

For more experimental information, please consult our sales representative. Our company provides comprehensive experimental technology services, greatly saving the time and energy of repeated labor for scientific researchers. We have excellent technical consultation and perfect after-sales service. Purchasing ELISA reagent kits comes with free testing services and online one-on-one technical guidance to solve your worries.
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