Hi Everyone, we have been trying to embed cells in alginate that we will mechanically stimulate and then want to extract RNA and protein downstream to look at gene expression. We have been having a horrible time working with the alginate, and if anyone can help that would be greatly appreciated! This is what we are doing, and our issues:
We have tried several types of alginate (Sigma, Pronova etc. all with different G/M ratios).
1). Our first issue was with sterilization. Autoclaveing interferes with mechanical properties, filter sterilizing through a syringe takes one of the strong guys (and several filters) in our lab to do it -- We can do it, but what a pain-- not sure if there is anything easier.
2). We typically seed 4-6 x 10^6 cells/ml. Once polymerized, and after a few days of culture, they cells do not seem to be evenly distributed, they tend to aggregate along the edges.
3). I think our mold design isn't great. We fabricated plastic molds, that look really great, but it is hard to get a completely flat, smooth plug. I have seen some papers where they have used dialysis tubing to make alginate plugs, has anyone tried this?
4). Biggest issue is the dissolution step. After "stimulating" our cells, we want to extract RNA. Ideally we would like to "suspend" the cells just after stimulation to eliminate the chance that gene expression will change (or return to normal) during the time when the alginate is dissolving. Using EDTA or sodium citrate takes 35min (minimum) for the alginate to completely dissolve. I was wondering if maybe adding RNA later to the solution would work to stop cellular functions and preserve the RNA while the alginate dissolves, but I don't know if this would work. -- Anyone have suggestions? -- We also see a lot of general cell loss (we get fewer cells out of the alginate than we put in (what the heck!) -- even counting dead cells they still aren't all there... sigh.
I guess the last thing is that we have been trying to optamize for months now, trying to make alginate plugs that remain intact following compression. THey either crack, or get smooshed. I've read a ton of papers on the mechanical properties of different alginates, and I think this is just a matter of us finding the right combination between type and source of alginate, culture time and mechanical stimulation protocols.
If you can help let me know!
Thanks, Shelley
Hi Shelley,
You can contact with Dr. Orive from Basque Country University ([email protected]). Him group are working wiith alginate capsules. They are a strong a really group in this field.
Carlos
Yes, I was thinking about trying that. Do you add RNAlater or anything? What kind of yields do you get from doing this? Our downstream application is going to be microarrays, so I need to get good clean RNA. I was thinking of trying this, and then further putting the RNA through a column (like RNEasy) to further purify it....
Do you have specifics on your protocol? I think it would be a good thing to try!!
Yes, I wasn't able to get the paper from home, I can try at the University but if you have a PDF of the protocol would be great! Thanks!
Praba, I know that alginate does not really support cell adhesion, so I don't think you are going to really get much spreading of your cells in the alginate unless you are going to add something else like RGD.
As I said, I am also seeing our cells concentrating to the outside edges of our plugs. I am assuming that given our plug size (13mm diameter x 3mm high), there are diffusion issues, and therefore cells in the center are possibly dying. Most people who work with alginate use beads, that alleviate that problem.
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