hello dear,

use collagenase 1 in 2-3mg/ml concentration and no need to use trypsin at digestion step. Flush the chord 4-5 times with PBS to remove any remanants of blood. Then cut it inti small slices of few cm diameter and then digest them after proper washing with PBS properly and incubate as per your incubation protocol. Once you get the digested extract, to remove the blood cells and any non specific components you can centrifuge it at 800g for 10 minutes. Discard the supernatant and then seed the pellet by resuspending in media+FBS. It will remove endothelial contamination. Hope it will be helpful..

Hello Tatenda,

another option that worked quite well is to remove the umbilical arteries and the vein, because these are the only sources of endothelium within the cord.

All you need is a strong anatomical forceps in each of your hands and a good grip:

- place a 5-10 cm section of the cord into a large Petri dish

- grasp one end of the cord with one forceps and pull away a piece of tissue with the other one.The outer portion of the cord tissue has an amazing tensile strength, so with a bit of practise you will manage to rip off long strips of cord tissue, which you may directly transfer into your collagenase. 

- as soon as you have exposed the vessels, pull off the two arteries and then the vein. This is hard work, as 'Wharton's jelly' is much tougher than any other jelly ;-).

- the remaining tissue is ready for digestion - I agree with Ajay that collagenase alone will do nicely.

If you retrieved fine strips of cord tissue (< 1mm in diameter), you may also try explant culture, which worked well in DMEM/F12 (1:1) with 5 % of human serum. Just resist to move your culture vessel during the first 3-4 days of culture.

Finally, you should know that cord tissue MSCs are more polygonal and less spindle-shaped than bone marrow MSC.

Hope this will be of help,

Andreas