Hello. I am performing an experiment that involves decellularizing pieces of lung and heart tissue (porcine) in a petri dish (about 150-250 um thick). I am finding that the further along in the decellularization process (washes with various detergents over 4 days), the ECM gets stickier and starts sticking to my metal forceps as well as plastic pipette tips. As I am interested in ECM integrity and want to image these pieces of tissue, I was wondering if the research community had any advice/tips/tools that would allow me to physically pick up my ECM pieces without having it stick to my tools. Thank you!
The pieces of tissue that start out cellularized are washed first with 1% SDS for a few 1-hr cycles, then left overnight. The following day the same is repeated with 1% Triton, and at this stage the tissue slices are still relatively easy to pick up and don't stick too much to the metal forceps. The third day is multiple rinses w/ PBS and water, and then 1 hr with 1M NaCl. All of these washes are at room temperature at 30 oscillations per minute so really slow. The final wash is DNase w/ MgCl for an hr at 37C. Then a few more rinses and a final 30 min wash with PBS with antibiotics, rinsed, and then left in DMEM-F12 media with 20% fetal bovine serum overnight at RT. It's after I add the media that the stickiness becomes the biggest issue. I do plan to cellularize the tissue afterwards so I use media that I grow my cells in (primary human bone marrow mesenchymal progenitor cells) .
Hi Alison!!
I have compared different methods of decellularization for mice intestine. The evaluated tissue got really stickier when I used some methods, such as NaCl. In order to hand these tissues, I used plastic spatulas (cell spatules), metalic smooth tip pincers (for not dilacerate the tissues), petri dishes and PBS. To image the tissues, I always left them wet. If they got dried, it is very difficult to hand them.
Best regards
Ana Celeste
It seems that SDS charges the scaffold significantly and NaCl also contributes to this, and this may be a reason of that sticking. May be you can use something to neutralize the charge?
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