I know Dispase is an option to recover cells from matrigel, but it is a bit too expensive.
I do have my doubts how many cells will still be in the plug at the time of harvest. We do have the experience that cells in an ECM (or Matrigel or Collagen) plug can and do leave this plug(see our recent paper in PLOS one about neural stem cell transplantation).
In case there will still be a plug left and you can remove it, the extracellular matrix can easily be digested by collagenase. Collagen is the most abundant and stable component in these gels. That works quite well. We could extract cells like that from ECM plugs and get RNA from these (Ceyhan et al 2008).
The consequences of tissue remodeling in the implantation site (contributed by the xenograft and the host) may require enzymatic dissociation by the time you harvest the tissue.
I used trypsin for 30min /37C with tritruation once every 5min for my ic xenografts. Add DNAse inhibitor to the trypsin and block it with soybean trypsin inhibitor after you are done. Centrifigue and put the pellet on ornithine/laminin coated plates or for sphere formation in suspension.
If you need, subcutaneous Matrigel implants can also be removed and inspected under confocal microscope or used for RNA extraction and gene expression analysis. In addition, in order to assess functional microvasculature formation by your cells, the high molecular weight fluorescent tracer FITC-dextran can be injected through animal tail lateral vein before animal sacrifice. For further details, I attach one of our papers regarding this topic.
Personally, I think that cooling the Matrigel will take too long until the gel turns to liquid again, which will consequently have an effect on your cell survival and growth afterwards. I think that enzymes are the best option to keep your cells viable.
I completely agree with Petra if you just want to isolate and analyse embedded cells...
I do have my doubts how many cells will still be in the plug at the time of harvest. We do have the experience that cells in an ECM (or Matrigel or Collagen) plug can and do leave this plug(see our recent paper in PLOS one about neural stem cell transplantation).
In case there will still be a plug left and you can remove it, the extracellular matrix can easily be digested by collagenase. Collagen is the most abundant and stable component in these gels. That works quite well. We could extract cells like that from ECM plugs and get RNA from these (Ceyhan et al 2008).
thank you for all replies.
I wanted to add couple of more details. the matrigel is growth factor reduced and i plan to explant the matrigel plug 24-72 hrs after implantation with goal of evaluating GFP expression in the cells. (the GFP expression will be induced in vivo).
From the above replies I gather mere cooling is time consuming and insufficient, but I wanted to make sure you knew my goal, timeline and matrigel type and if cooling would be an option at all knowing the conditions ? Alternatively, it is perhaps easier (?) to preserve the matrigel plug for anti-GFP antibody staining on paraformaldehyde fixed, paraffin embedded slices. Any comments or anyone have a specific protocol for the latter ?
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