Hey Jonathan,

I do 3D cultures regularly in Matrigel and unfortunately Dispase is the way to go. Expensive, but worth it. It just dissolves the Matrigel, the architecture stays intact. I usually incubate my spheres for about 30-45 minutes with Dispase in the medium 1:1. I found it most beneficial and cheapest if I take everything of the plates first, spin to reduce volume and then add Dispase. In your case I would avoid that. Aspirate most of the medium without touching the Matrigel and then add Dispase in equal volume. Your cells should be free of the matrix in max 45 minutes. Hope that helps.

Hi, collagenase works well with collagen I (as expected) but not so well with matrigel. You could lift the gel out and transfer into a tube then add collagenase containing 0.5% FBS to protect the cells. Gentle shaking should disrupt the collagen and cells can be harvested by centrifugation. For Matrigel you mat need to use a different digesting agent such as dispase or the Cell Recovery Solution from BD Biosciences. Hope this helps!

Thanks Sunil! I am trying to keep the intact architecture of the cells and I have used collagenase/dispase with mechanical force to isolate single cells. Do you know if the collagenase and dispase will disrupt the architecture? I am thinking it might and by adding these agents perhaps some of the signaling pathways might change in response to the enzyme having to be warm. Usually I can get the matrigel to dissolve in ice cold PBS but obviously collagen is trickier because its made up of a soluble and insoluble fraction.

Have any idea about how long should the incubation with the collagenase be at 37 degrees?

I normally incubate fairly thick collagen gels in collagenase for 30-60mins at 37C which results in comprehensive digestion. I agree with you in that the architecture is likely to be disrupted as the tension within the gel helps to maintain the structure. As signalling pathways are likely to be dependent upon structure, you probably will see changes. Not sure how you get around that one!

Hi Jonathan, you may have your good reasons why you use matrigel or collagen for your 3D cultures. But as you discussed already, the use of proteinases like collagenase etc. have probably unwanted effects on the cells. You may not want to switch to other culture protocols now but perhaps this may still be of interest to you: Our company Cellendes offers a new defined hydrogel that consists of dextran polymers crosslinked with PEG (polyethylenglycol). This gel can be dissolved by the addition of dextranase which does not affect cells because it is not a protease. We incorporate the adhesion peptide RGD into this hydrogel and culture MDCK cysts in such gels with great success. The isolation of the cysts out of a 30 µl gel lasts only 30 min at 37 °C. But it also can be done at room temperature. The cysts are well polarized and the percentage of cysts in the gel is basically almost 100. If you go to our webpage www.cellendes.com and go to the Application Notes you will find a detailed description of this culture and results including a protocol. If you need more information, just let me know!

Remember, cells secrete imediately their own extracellular matrix which aggregate them witth their matrix when cultured for longer than a few days.. Digesting the collagen does not release necessarily from their own complex tight 3d matrix.

Try to add dispase to the digestion protocol.

We have tried to use collagenase before to isolate cells from 3D gels, and although it works, it will very likely disrupt intracellular signalling. If you plan on extracting protein from the cells once isolated, we have also just completely and immediately digested gels using an 8% Urea solution, and subsequently ran western blots from these protein lysates. To help get rid of the collagen, you can always put it through a high molecular weight spin column before going through with the western. Working with cells in 3D matrices is always a tricky business.

Jonathon, we used trypsin with the collagenase as per the following:

...collagen lattices were washed in PBS for 10 min at room

temperature followed by incubation with 0.05% trypsin, 0.53 mM

EDTA, in PBS (Life Technologies) for 10 min at 37°C and subsequently

digested in 3 mg/ml collagenase (type I; Sigma) in buffer (130

mM NaCl, 10 mM Ca acetate, 20 mM Hepes, pH 7.2) for 8 min at

37°C [28]. The enzymatic reaction was stopped by adding 50 ml of FBS...

Exp Cell Res. 2000 May 25;257(1):180-9.

You might also try to lower the initial concentration of collagen to the minimum required to achieve expected results. Good luck.

Hey Jonathan,

I do 3D cultures regularly in Matrigel and unfortunately Dispase is the way to go. Expensive, but worth it. It just dissolves the Matrigel, the architecture stays intact. I usually incubate my spheres for about 30-45 minutes with Dispase in the medium 1:1. I found it most beneficial and cheapest if I take everything of the plates first, spin to reduce volume and then add Dispase. In your case I would avoid that. Aspirate most of the medium without touching the Matrigel and then add Dispase in equal volume. Your cells should be free of the matrix in max 45 minutes. Hope that helps.

Hi Neil,

I take it the urea solubilizes the collagen? Have you used that protocol in any published work? I'm curious to see how it works out.

Stupid question about the HMW spin column: would you try to retain or eliminate the HMW fraction, and what kind of cutoff would be reasonable? Sigma mentions on their Type 1 collagen product info page that "fresh" collagen runs at around 120 and 220 kDa in SDS-PAGE -- I guess I'd infer that you expect some fraction of the collagen from the gels to be considerably shorter after culture and urea treatment?

Thanks!

Tim

Thanks for all these helpful suggestions. I'll be giving a few of your suggestions a try and hope for the best. Thanks everyone! Cheers!

Hi Jonathon;

I am culturing mammospheres and my medium has matrigel in it. So my mammospheres attach it. You wrote that you are using ice cold PBS to dissolve your matrigel. Is there any protocole for it or do you just put them into ice cold pBS and wait for a while? If you could help me i would appreciate.

Thanks

Hey Jonathon,

Very interesting discussion. Do you have any progress in your tests?

Hi Berrin,

If its just matrigel you can just add ice cold PBS and put them on ice for a bit (5mins). Then after that you just gently pipette up and down with ice cold PBS. This will break up the matrigel. I also recommend cutting off some of the tip of the 1000uL tip so you dont break up the sphere. Then take that suspension and place it in a 50mL conical with ice cold PBS. This adding more PBS and since its cold it also helps to remove the matrigel. Gently spin down at 4 degrees C. Wash a couple more times with Ice cold PBS. You should know have intact spheres and they should be out of the matrigel. Hope that helped. Thats what I do.

Hi E. Ainbinder,

I was unable to get the collagen to break up without breaking my spheres up. So I have switched to just isolating all the RNA and protein and going that route. Its seems like the enzymes are too either too strong and break up both the matrix and the sphere or they don't break up the matrix. It could be a concentration issue but i have done some concentration/titration experiments and I am still unsuccessful. So I can't really get the staining done that I would like too but I can at least look at the signaling pathways.

Hi Jonathon,

Look at this old manuscript:

Richards J, Larson L, Yang J, Guzman R, Tomooka Y, Osborn R, Imagawa W, Nandi S: Method for culturing mammary epithelial

cells in a rat tail collagen gel matrix. J Tissue Cult Meth 8: 31–36, 1983

They describe there a way to release live cells out of collagen.

I have no idea what kind of cells you are culturing but it is always important to choose a right Type of collagenase. Use the enzyme of high quality (Worthington), high specific activity (not less then 300 ug/mg), keep the dry enzyme in dessicator. Collagenase solution is light sensitive. Prepare 0.1-0.15% solution in your medium base (like DMEM), filter, keep it cold for not more then two weeks.

You can also try to use some Liberase (Roche). We have recently tested one of them for a sensitive procedure and the result was very good. Look at Roche's site.

If you need to do some staining, go to Nature 501, 373–379, 2013. They describe it in methods.

It will be nice to know if it was helpful.

Good luck. Elena

Thanks Elena! I'll check this out.

Jonathon,

I see this is an old discussion, but just in case you still need advice:

There are two older Fred Grinnell papers (1991, 1993) with the details about collagenasing lattices. 3mg/ml or 5 mg/ml depending on the toughness of the gel. Type 1 collagenase from Sigma (C-0130). Also pretreating with trypsin is necessary. I still do this and it works great. Not sure how it would affect embedded matrigel spheres though.

References:

PMID:1995294

PMID:8335692