Hello, I added thiol RGD peptide into PEGNB polymer and after UV-photopolymerization, MSCs are seeded onto 2D hydrogel. However, the cells can not attach to the RGD-modified surface and there is no migrating at all after 24 hours. Instead, the cells form aggregates and remain alive. Has anyone done this before?
Thanks for any reply!
Thank you.
I am also wondering if the thiol ene RGD is properly conjugated, but it seems to be quite tricky because I can not find any dye that can simply stain this peptide. I have to do modifications or radio label it.
I think you are right that the gel is probably too thick so most of the RGD is buried inside instead of displayed on the surface. I will try to polymerize thinner gel 'sheet' to see if that helps.
As John said, the RGD needs to be immobilised to the polymer, otherwise the cells will just bond to free RGD and aggregate and the integrin receptors will be occupied and unable to bind even non-specifically. After a few days in culture, the MSCs, which are adhesion dependent, will apoptose.
You should use an analytical technique – perhaps NMR – to verify that the RGD is bound to the polymer before you polymerise the gel.
Thank you for your answer, Nick!
Just like you said, MSCs in my RGD-modified model always form aggregates while in my control model with no RGD at all, the cells just stay at the bottom, remain unattached, but no aggregates are formed the whole time. So the aggregates is probably due to the binding of free RGD in the medium, which means most of the RGD is either buried inside the hydrogel or it is not conjugated into PEG.
Also, I made modifications based on John's suggestion. I tried to polymerize PEG hydrogel with larger surface area and smaller thickness, some MSCs ended up attaching to the surface and migrating!
That's good that you improved it, but ultimately, in order to have full control over your system and be able to claim that it is a defined system, you need to verify that the RGD is bound to the PEG and purify the functionalised PEG to remove any unbound RGD. Any unbound RGD will bind to cells and if this varies from sample to sample, your results will be all over the place.
You are absolutely right, I am thinking about an easier way to remove any unbound RGD in my model, which is polymerized in 48 well plate.
Thank you so much for your suggestions!
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