I am facing a problem with plasmid DNA transfection in the 3D hydrogel system.
I am using a two-component hydrogel system, where you mix the Comp A and B at a definite ratio it forms a hydrogel. So what we do is, we mix the mesenchymal stem cells in comp B which is hyaluronic acid, and the plasmid DNA complex prepared using Mirus trans-IT x2 transfection reagent is mixed with comp A which is chitosan. In this way, we make a 3D hydrogel with both MSCs and pDNA complex encapsulated in a 3D scaffold.
After encapsulation, it should ideally release the pDNA complex slowly and it should be able to transfect the MSCs in the gel system and produce the GFP signal.
Anyone working on the same area can help me understand what would be the reason that I don't see any cells getting transfected with GFP pDNA?
Any experts in this field? working with chitosan ?
hi Ashis Kumar
I don't work with chitosan but works with mesenchymal stem cells and transfection kinds of stuff. Seeing your post, thought to discuss, as you said transfection is not happening. I'm thinking that does your components are compromising the transfection efficiency, due to which the transfection is not taking place.
First transfecting the MSC with pDNA and then encapsulating can't be an option?
Regards
Saurabh
hi Saurabh Mandal
Thanks for the response.
We are using the hydrogel system for sustained delivery of pDNA. That is the reason we are encapsulating it in the hydrogel. It's very true that the hydrogel is affecting the transfection since it is a polycationic polymer
In your experience, Which transfection system works better for MSC?
Without a hydrogel system, most of the standard transfection reagents work well with MSC. We have used Lipofectamine 2000, Lipofectamine 3000, ABM DNAfectin, and Polyplus JETprime. These all have worked well for plasmid transfection.
Hi,
Wont u allow the cells to grow in the hydrogel system? If u do transfection after that then only it ll show the expression of ur interest isnt it? That's how we do transfection in 2D na? (We culture cells on 2D cell culture plate and add lipofectamine or any other carrier which makes complex with the gene of our interest)
Balavigneswaran Karthikeyan
Yes, it's true that we add the transfection complex after plating the cells in 2D conditions. but in 3D, we encapsulate the cells and the transfection complex together with hydrogel components. This is mainly for achieving sustained delivery of our target to the cells.
R u encapsulating pDNA with some carriers or the carrier itself chitosan or the gel u use?
If u r using hydrogel itself as a carrier I dont think the complexation would occur. because the amt of polymer u use to encapsulate pDNA will be huge. You can try making complex (pDNA+lipofectamine /viral / catioinci peptide) then load into the hydrogel system, where u have MSCs or other cells encapsulated.
Hi Balavigneswaran Karthikeyan
We prepared pDNA complex with different liposomes, and then we encapsulated in chitosan hydrogel. the problem here is the liposome pDNA is not getting released from the hydrogel system for it to enter into the cell. there is some electrostatic interaction between the chitosan and liposomes that is not allowing it to release into the medium.
Ashis Kumar
Have you not tried putting the transfecting agent, the cells in one component (HA in this case), and then making the gel?
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