I'm looking into how you could do siRNA transfection of cells once they are cultured as spheroids in a 3D matrix (collagen). Has anyone tried this before?
Would you use the transfection reagent mix on top of the collagen mix containing the spheroids? Or would the siRNA not be able to penetrate the collagen gel?
Should you then have to embed the siRNA into the collagen gel and if so, how would you do that (concentration and volume-wise)?
Hey ya Julie, give me a call. I am currently working on 3D transfection with Matrigel at the moment with the magnefect-nano. I may have some ideas that may help you!
Aims xx
What cells are you using?
Would it be an option to transfect the cells before formation of the spheroids? Maybe using stable expression of the siRNA as shrNA, coupled to a fluorescent protein for detection of transfected cells?
@Elad, thank you for the information. Hope to find a cheaper option.
@Stephan, I'm using pancreatic cancer cell lines. The idea is that I would like to do the knockdown once the cells have formed a spheroid to make sure I don't disrupt cell-cell adhesion by the knockdown. However, I might have to go for transfection before spheroid formation and hope for the best ...
@Aimee, thanks for the call. Keep me updated.
@Mark: is a good suggestion and will keep this in mind. However this would take quite a bit of time to get going and I would like to have some preliminary data asap.
Hey Julie, I was just thinking, has Laura tried anything like this with her work in Cambridge - I am sure she has done some 3D work. Analysis of my transfected cells takes place tomorrow - I shall keep you informed.
In the meantime, I hope you find something that will give you some preliminary data soon!
You could try something that Theresa Reineke is working on ( http://www.chem.umn.edu/news/news.lasso?serial=433 ), I've seen her recent results presented at the ASGCT conference in May and it looks like she has some active transport for example into the heart from the outer side. Very impressive results. So that might work for your scaffold system maybe too.
I'm trying to do pretty much the same experiment using mammary epithelial acini and I would be great if you could share your protocol if you find one that works nicely. At the moment I'm optimising 3D FectIn and DreamFect gold from OZ biosciences. I can see some transfected cells, but the efficiency is quite low.
Best regards,
Elena
Dear Julie,
If you are still working on siRNA transfection in spheroid 3D cell culture, I would propose you si3D-Fect and si3DFectIN. These two reagents are dedicated to siRNA transfection in 3D (http://www.ozbiosciences.com/content/16-3D-transfection-3Dscaffolds-3Dhydrogels).
Basically the idea is to first preapre what we call "Gene Activated Matrices" - GAM - composed by either scaffolds (solid 3D matrices) or hydrogels (gel matrices) containing complexes of nucleic acid and transfection reagents:
- 1. Complexes of nucleic acid and transfection reagent (3D-Fect, 3D-FectIN, si3D-Fect or si3D-FectIN) are formed during a 20 minutes incubation step at RT
- 2. The complexes are mixed with gel or added onto the 3D scaffold. Gel is let to polymerize at 37°C for 30 min to 1 H, and 3D scaffolds are put under agitation for 1H.
in this way, complexes will be embedded into the gel or will interact with the surface of the 3D scaffold.
- 3. cells are added onto the gel or the scaffold. They are transfected while invading the hydrogel or colonizing the 3D scaffold.
for hydrogels, we propose 3D-FectIN and si3D-FectIN for DNA and siRNA respectively,
for 3D-Scaffolds, we propose 3D-Fect and si3D-Fect for DNA and siRNA respectively.
It is important to note that the goal of this reagent is to prepare GAM; complexes are formed and added onto/into the 3D support before the addition of cells.
@Elena,
Dear Elena,
I hope you are doing well since ou last mail exchange, and I am really glad to know that you can see transfected cells (even if, as you say efficiency is low) ; this means that the system works and only need some optimization.
would you need any advice or would you like to try si3D-FectIN, please do not hesitate to contact me.
Good luck with your experiments,
Cedric
http://www.ozbiosciences.com/3d-transfection/63-si3d-fect-transfection-reagent-silencing-3d-scaffolds.html
http://www.ozbiosciences.com/3d-transfection/64-si-3dfectin-transfection-reagent-silencing-hydrogels.html
http://www.ozbiosciences.com/content/16-3D-transfection-3Dscaffolds-3Dhydrogels
Is there a way to perform the siRNA knockdown AFTER the ells have been growing in Matrigel a while? In my experiments I want my spheroids to form (about 7 days) before siRNA knockdown. I can't find any transfection reagents for this purpose.
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