I would like to perform Western blotting analysis of 3D-cultured cancer cells in collagen gel. In the case of Matrigel, Cell Recovery Solution (Corning) is very useful, but I do not know exactly how to isolate cells from the gel. I hope for the kind advice!
Have you considered collagenase digesting your gel, prior to the lysis protocol? Or simply collect your 3D cultures and proceed through standard homogenization protocols as you would use for animal tissue (sonication, rotor stator, ...)? good luck!
Are these 3D cultured cancer cells aggregates (spheroids) or as single cells embedded in Collagen?
I recommend a collagenase/dispase solution (http://lifescience.roche.com/shop/products/collagenase-dispase14301). You can incubate briefly (10-15min), pipet up and down to break up the gel completely, and add to an EDTA/EGTA-containing solution to quench the reaction before proceeding.
Collagenase/dispase will work. Heating, stearing, pipetting will help. adjust concentrations of the enzyme mix. Avoid trypsin due to cell damage. Collagenase will disolve the tissue, but dispase is important for single-cell suspension
The HyStem hydrogel technology uses HA and gelatin to make a gel. This gel can be dissolved through a non-enzymatic process when the gel was made with an alternative crosslinker PEGSSDA. See the link for more details.
Go J Yoshida
Hello Professor. Have you been successful to retrieve cell from collagen matrix? I am culturing tumor spheroids in rat tail collagen matrix. I want to do single cell western blot. I am trying to develop the process of collecting spheroids and prepare single cell suspension. But still confused.
Hello, I'm also working on human collagen I coated plate for T cell culture, and I want to collect cells for FACS. Is there any method to get single cells? Thank you very much.
Hi, one good alternative 3D cell culture matrix is nanofibrillar cellulose (NFC, GrowDex). It is animal-free and well-defined hydrogel that supports the 3D cell growth. One benefit in using GrowDex is also the possibility to isolate the cells from the matrix without affecting the cells - cells will retain their morphology, surface proteins and functionality.
Pleas take a look of this paper where they cultivated and isolated pluripotent stem cells from GrowDex.Article The Use of Nanofibrillar Cellulose Hydrogel As a Flexible Th...
You can find more information here GrowDex® Hydrogel Range | Animal-free 3D cell culture | UPM Biomedicals
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