Thank you so much! 2021-11-20 17:40:24

I would suggest not to pre-digest the aorta before removing the adventitia. By doing that, you reduce the elasticity of the basal membrane. This elasticity is what protects the cells from the mechanical stretching. In this protocol we show how, when the aorta is not predigested, the adventitia can be removed just by pulling it out. Once that you have the aorta clean (see figure 3) you can start the digestion. I hope that this small change in your protocol would increase that number of cells that you obtain. If you want to keep troubleshooting do not hesitate to contact me. 2021-11-17 00:12:40

Thank you for your reply, I didn't think I would get a reply so soon! I usually pre-digest the aorta for 10-15 min after removing the surrounding fat and tissue to make the adventia loose for removing. But during the removal process, the aorta can rupture easily and breaks into small segments. Other precedent literatures say ruptures could cause mechanical stress for the vsmcs. My whole process of dissection, pre-digestion and adventia removal of 3 mice is up to 1 hour. After digesting overnight, and leaving the dish for 3days , only a small amount of cells can adhere to the bottom of the dish. 20%FBS+DMEM was changed every 3 days. The cells didn't proliferate and never reached confluency even after 30 days. Another problem is that after centrifuging the digested cells for 200gx 5min, only a tiny amount of cells is visible at the bottom of the tube, a lot of cells are left in the supernatant. I was wondering if I can increase the centrifuge force or prolong the time to obtain more cells. Thank you! My e-mail is wagyt85@mail2.sysu.edu.cn . You can reply to this message via e-mail if replying through this website is too inconvenient. Best wishes. 2021-11-11 19:38:09

I just sent and email. 2021-11-11 01:29:41

I am very glad to see your message. We are alumni. We can communicate about it. sxlijunping@163.com 2021-11-10 05:28:13

The separation of the adventitia, when done properly, does not generate too much damage. You have to consider that the media is a very elastic tissue that absorbs most of the screeching. How do you detect dead cells? How do you know that the dead cells that you observe in the incubation are VSMCs? Do you perform any kind of staining to determine the nature of these fragments? This digestion step is mainly going to dissolve the collagen fibers within the media so that VSMCs can migrate out of the aorta ring. Therefore, a lot of debris is observed but this is mainly collagen fibers. 2021-11-10 00:33:52