Hi Nick I have culture cells in hydrogels (mainly Fibrin and collagen) and there are a few factors to consider:

a) Nutrients. Cells in 3D use a LOT more media than those in the same wells of a 2D gel. You may find you have to re-feed the cells every day or so rather than 2-3 days for cell culture. This is especially true if seeding at an initial high cell density.

b) Duration of staining: if you intend to stain live cells within the gel with eg: Calcein/Dapi etc you may need to extend staining times considerably (this is also true for fixatives) as it often takes some time for the dye/fixative to diffuse down to the bottom of the gel.

c) Embedding for sectioning: I found it pretty hard to freeze and section these gels (it was like sectioning an ice cube!) so we ended up paraffin embedding the gels (and paraffin sections are a lot harder to perform IHC on than frozen*1).

It took me quite a while to get a good protocol for wax embedding as, as the name suggests, these gels are mostly water and can contract considerably during the dehydration process (where the H20 is replaced by ethanol). My tips are to extend the time of each graded ethanol bath from what you would normally do for tissue embedding, include more gradiation steps and perform gentle shaking during each ethanol bath (I did dehydrated, infiltrated etc with the gels in a tissue cassette between two biopsy pads in Duran bottles on a shaker this worked well and the wax went in ok too). In the end we got the gels down to ~10% contraction post vs pre embedding. We also used LMP wax (MP of ~ 55-58oC as we were also using collagen gels and I found they contracted if wax > 60o - 65oC).

d) Over time (we cultured for up to 6 weeks) we found that the cells (fibroblasts) migrated towards the top of the gel (where the oxygen was better) and formed a "skin". If you intend to culture for prolonged periods you may find the same and I think this is a natural effect of this kind of (non-perfusion) culture.

e) Imaging. A confocal can easily image 1-2 mm through a hydrogel - the only thing to be aware of is that (as mentioned before) labels take longer to perfuse to the bottom and so cells at the base will be less bright - I recommend that you image (blank) areas above and below the gel as well (i.e. if gel = 1 mm image 1.2 mm above, through and out of the other end of the gel - this will allow you to measure the thickness of the gel as I think you will find that the gels rapidly contract over the first few days of culture as the cells "tug" at the matrix - this is especially true of fibroblast-like cells).

I hope this helps,

Gary

*1: This is because the rough treatment the tissue/cells get can ruin the epitopes and so some tough antigen retrieval methods are necessary. If you intend to perform IHC on wax sections let me know and I can give you a few tips on this.

Very nice response above. I could add the following:

Rinsing times are much longer when staining gels. Depending of its size, sometimes 15-30 minutes under agitation to repeat 3-4 times.

I used to block everything for 1 h with 2% BSA, 10 µL/mL fish gelatin, 10 % FBS.

If you can best is to stain your cells before embedding them in gels.

It works well with CFSE.

Of course if you don't have perfusion, gels should not be too thick as you will have an oxygen gradient problem.

Hi Nick,

An addition on how to recover cells from gels:

I don't know what kind of polysaccharide and poly(ethylene glycol) gels you are using. We sell dextran gels that make it very easy to recover cells by adding dextranase. Cells can be either alive or chemically fixed, both works.

www.cellendes.com

Thanks to everyone who has offered advice so far, especially Gary for his very comprehensive response.

I am working with gellan gum, a polysaccharide used in food with recent applications in tissue engineering. GG is digested by galactomannanase, so I have been considering using that to recover cells. The issue is that cells may respond very rapidly to having their matrix degraded, so gene expression may not be representative of the situation before digestion. Any thoughts on this?

Hi yes. If you are looking at gene expression (qPCR/dPCR) then I would fix the cells prior to removing them from the matrix (for RNA work we tend to use 95% EtOH/5% acetic acid (-20oC) for 10 minutes (although for 3D you can extend this to 20 or 30 min).

However, saying that, people routinely Trypsinise and FACS sort cells (such as single sorting) prior to analysis and don't seem to be worried about the initiation of Anoikis these cells probably undergo. 

Totally agree, there's no point in doing this kind of work if you can't be sure that gene expression hasn't changed during the collection of the cells. If you're looking at the effect of matrix properties on cell behaviour and you have to then take the matrix away from the cells, you need to know you're not changing gene expression. Thanks for the tip regarding fixing, that's really helpful.

Me again - ignore my last post. It is pointless using a precipative/dehydration fixative if you have then to re-hydrate the gel to get the cells out (RNA would degrade).

You could use 4%PFA or similar (although that's not ideal for RNA work).

Personally I would check the qPCR as follows:

a) plate X number of cells in 2D and same number in matrix (i.e. half a million)

Note: seed at density in 2D so cells are not confluent the following day

b) allow to settle for 24 hours.

c) extract RNA from 2D cells, remove cells from 3D following protocol and extract RNA from these too.

d) Compare qPCR of several reference genes to see if there are any statistical differences in RNA expression over the time taken to remove from 3D.

You could always compare a fixed (not Ethanol) too.

Sorry about that and good luck

Gary

Think about when you compare 2D with 3D cultures. Just the culture condition can change gene expression. After all, you don't know where the difference came from.

Hi Brigitte that is a good point. Indeed there will be a huge difference in gene expression between the two systems - just think about the differences in  integrin engagement!

I was just thinking about HK genes in which (hopefully) the difference will be minimal (but as you say - who knows!). That is why I think pre-fixing (or not) with 4% PFA or Formalin may be best. However PFA is probably not the best fixative for RNA hence my other suggestion.

Gary

Thank you both for your extra points.

Hi Nick,

I don't know why this didn't come earlier into my mind: I know from one of our customers that, at least with our gel, the gel can be frozen (or schock frozen) and then be mechanically broken up with a pestle in frozen condition, such that an RNA extraction becomes possible. There are micro pestles commercially available which work for small amounts in Eppendorf tubes. Perhaps this is a solution for you as well!

All the best, Brigitte

That sounds like an excellent suggestion, thanks very much. It may remove the need to enzymatically degrade the gel, which could affect gene expression quite rapidly.