There is no universal protease that works with Matrigel as it comprises several proteins. BD cell recovery solution is relatively efficient - keep the cells on ice (low temperature aids in de-polimerizing Matrigel facilitating cell recovery) and use an orbital shaker for a few hours. Spinning the plate at 4 oC and 1000 RCF may help if you transfer the remaining gel with the cells to a cell strainer. For spheroids, the next step would be to use buffered saline without Calcium and Magnesium or EDTA chelator to disrupt Cadherins and reduce cell-cell adhesion, followed by trypsinization if needed. Note that chelating divalent ions does not necessarily reduce cell-matrix adhesion due to different integrins present. Following recovery, isolated cells are often sticky. While my culture system is different, what worked was a BD cell recovery solution on ice for 2 hours, followed by a 0.25% Trypsin – 1 mM EDTA. If the gel is not fully de-polymerized with the cell recovery solution, trypsinization may be done at 4 oC and Trypsin applied longer than it would be at 37 oC. At 4 oC Trypsin is also gentler to the cells. Good luck!

Jelena, thank you for your comments. We have been successful using Cell Recovery Solution at 4oC for 1 hour to dissolve the Matrigel, followed by 3 washes of the pelleted cells with PBS to make sure no interfering substances are carried through into the RNA isolation and cDNA synthesis steps. The qPCR results for housekeeping genes in our enteroid system are now very reproducible.

I'm doing the normal mechanical way and it works fairly well with qPCR. No RNA degradation and housekeeping along with Icam1 genes after stimulation give the expected results.

Hi,

we use Dispase digestion for 1h at 37 C and it worked out very well for qRT-PCR. Even the amount of RNA was very sufficient as well as the ratios for RNA purity.

We use matrigel in a lower concentration and it was almost completely dissolved after this 1 h incubation. Even further cell culturing is not a problem.

Dear Sabine 

I want to know that collaginase IAS will work in digestion of matrigel ?

I have another question, how much cell recovery solution do you put in your tube with the Matrigel?

Héloïse, to answer your question, my lab's standard procedure is to add 500 microliters of Cell Recovery Solution to 50 microliters of Matrigel containing enteroids. We allow 1 hour at 4 oC for the Matrigel to dissolve, and then wash the pelleted cells 3 times with 1 ml of PBS before extracting RNA using a Qiagen RNeasy Mini Kit.

Thank you Ifor.

I'd like to use that because I have a problem with my 3D model, it need to concentrate my organoids and the only way to do that is to remove the remaining matrigel to only have fresh one. There is also cell debris in the matrigel that I would like to remove. 

Is it sure that the cell recovery solution will not damage my organoids and just help to remove the matrigel? 

The method I described is designed to recover organoid cells for preparation of RNA to be used in qPCR analysis. It could also be used to obtain organoid cells for flow cytometry (with fewer washes needed). If your goal is to concentrate the recovered organoids, remove debris, and then put the organoids back into culture in Matrigel, you may also be able to use Cell Recovery Solution for this. But you should consult the manufacturer's instructions for advice on whether Cell Recovery Solution has any adverse effects on cell viability. Results may vary depending on the specific type of organoids you are working with. For subculturing organoids in Matrigel, most labs just incubate the Matrigel containing cells with added media on ice to dissolve the Matrigel and allow the cells to be pelleted and washed.

 Thank you Ifor for sharing your protocol. 

I have a question:

When you incubate your plate at 4 oC, do you use an orbital shaker or any other means of gentle mechanical shaking? I am also interested recovering enteroid cells for qPCR analysis.

Bryan, the incubations of enteroids embedded in Matrigel with Cell Recovery Solution are done in microcentrifuge tubes. Gentle shaking of the tubes for 1 hour is performed by attaching them to a flat shaking platform in a 4oC cold room.

Hi Pedro Saavedra, would you be able to describe your mechanical method in more detail or send me the protocol,

Thank you.

Hi everybody,

In case I want to do flow cytometry after the disolve the Matrigel, which option will be the best one, the cell recovery solution or the dispase??

Which would be the mechanical protocol?

It is important for me to be able to do cytometry after disolve the matrigel, I hope you can give me a good advice.

Thank you very much in advance.

Luis, the best answer to your question may depend on the specific markers you are interested in detecting by flow cytometry. Dispase contains proteases that may cleave some markers off the cells. While I think Cell Recovery Solution is probably a better bet as a precursor to running flow analysis, I recommend doing a pilot experiment trying both methods in parallel to optimize the method for your markers of interest. Also, some mechanical agitation by a few rounds of up and down pipetting is needed with both Cell Recovery Solution and Dispase after the incubation to dissolve the Matrigel. I do not recommend recovery of cells from organoid cultures for either qPCR or flow applications with a method that relies on just mechanical dissociation of the Matrigel droplet and the cells found within.

Hello Nikitha,

I recently did flow analysis of cells that were cultured in matrigel, depending on which recovery solution you have it will be the protocol you should use. For me it turn out to be not so complicated, I tested the Cell Recovery solution from Corning and also the Dispase from corning, and I was able to perform my staining and analysis using both. But I distinguished a slightly better cell recovery with the dispase.

I used the protocol that comes with the data sheet of the product, the only thing you have to calculate is the amount of Dispase you need to use based on the size of the wells where you plate your matrigel and also the amount of matrigel. You wash the medium, add the enzyme and then incubate at 37°C until the matrigel is completely disolved (takes about 30 min to 1 hour in my case for matrigel plated in 24 well plates) but you should check every 15 min after the first  half hour in order to know  how much time it takes for your conditions.

sometimes you will not see a complete dissolution of the matrigel, but those little pieces diperse by gently pipete up and down the cell suspension.

you stop the action of the dispase with with 5mM EDTA in PBS, spin the cells at low speed and wash a couple of more times, then you can continue to do the staining.

I should say that in my case I analyzed tumor cell lines, and I didn´t notice any difference regarding the detection of the surface markers after using both reagents, but If you are planning to analyze other cells like for example  PBMCs then I will suggest to test both to make sure your detection is not affected by the enzymatic action of the dispase. As a reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956959/

In case that you consider the Corning cell recovery solution then the protocol that comes in the data sheet works perfectly, and this is more gentyl with the cells, main differences is that you incubate at 4°C on a rocking table, the matrigel dissolves in around 30 min (again you should monitor the process to optimize times according to your experiment) and you don´t need to stop any enzymatic reaction.

I hope that helps, if you have any question I will be glad to help you.

@Luis: Is there any other inhibitor available to be used for Dispase?

Hi everyone,

I am using enteroids to analyze the effects of some metabolites (that I add in the media) on enteroid gene expression. In this case, I am wondering that if I incubate the enteroids embedded in Matrigel with Cell Recovery Solution for 1 hour, the gene expression will change and I won't be able to see the effects of the metabolites, right? Would you have any other tip to do qRT-PCR in this case?

Thank you in advance!

Yes Alice, that's what I did. Now, I will start doing the PCRs this week. It is very good to know that you had good results. Good look in your future experiments and thank you for sharing this information.

Hi Luis Felipe Olguin Contreras this is a great discussion.

I would like to know once the matrigel has been dissolved by dispase/cell recovery solution, how do you perform IF on the cell organoids. Ideally, I want to culture my organoids in a matrigel dome and then image them directly, but I have read some sources stating that there could be a lot of background fluorescence after fixing the matrigel. Thus, if I remove the matrigel and am left with the organoids for staining, how exactly do I get them to stick on an imaging surface and go through the rigorous staining procedure of IF (primary, incubate, wash etc...)

Hello everybody and thanks for the interesting discussion,

Answering to Anthony L Eiliazadeh 's last question, you might want to consider switching from solid-ECM organoid cultures to suspension-like organoids cultures, as described here:

Article High-throughput automated organoid culture via stem-cell agg...

Here, organoids are cultured in microwells in presence of low (or no) Matrigel. The organoid culture process is streamlined thanks to a side pipetting port, which allows media and buffer exchange without disrupting the organoids. Moreover, the immunostaining procedure (from PFA fixation to antibody incubation) is performed directly on the plate, and organoids can be imaged within the microwells.

The plates are commercially available as Gri3D at https://sunbioscience.ch/products/

Let me know if you have any questions. Cheers and best of luck!

Hi Ifor,

If you don't want to stress the cells with ice cold culture media, there is a possiblity to use another 3D culture matrix, such as plant cellulose -derived GrowDex. Cells can be isolated from GrowDex with simple and safe method with GrowDase enzyme treatment. The enzyme does not affect the cells at all, retaining e.g. pluripotency of iSPCs and ESCs. Also cell surface proteins are intact.

Please take a look of this article where they used GrowDex to 3D culture of iPSCs and ESCs: Article The Use of Nanofibrillar Cellulose Hydrogel As a Flexible Th...