Hello all,
I want to re-isolate MCF7 cells from xenograft masses and grow them in vitro (just a wee or two).
In different protocols I red that people use always colagenases but sometimes also DNAse and /or hyaluronidase. But depending on what and why ?
I found here http://www.worthington-biochem.com/cls/default.html
that collagenases have divers levels of proteolytic and tryptic activities and to not harm you membrane proteins to much you might choose type IV.
But why should I add DNAse ?!
and when do I use which type pf hyaluronidase ?
Does anyone have experience with kits to eliminate fibroblasts ? And do I have to pay attention to other contaminating cells ?
Thanks for any explanation/help !
The "kitchen sink" approach to using a cocktail of different enzymes to isolate cells reflects the state of the art where enzymes could damage cells leading to increased cell clumping that could be reduced by adding DNAse to the collagenase enzyme mixture. Hyaluronidase was also used in cell isolation in early applications of enzymes but if you do a google scholar search with proximity limits searching for hyaluronidase to an adjacent term "cell isolation", then you find that this represents only about 10% of the hits when expressed as a percentage of a similar search where collagenase was substituted for hyaluronidase. This approximate percentage did not change from 1995-2005 and 2006 to 2016. Hyaluronidase is used for applications where you know there are high amounts of hyaluronic acid.
A wide range of collagenase-protease enzyme mixtures with known enzyme compositions are sold by VitaCyte as DE Collagenase enzyme products (www.vitacyte.com). In contrast to traditional collagenase products, these equivalently priced products have known enzyme composition and contain > 85% bacterial enzymes. The range of collagenase to protease ratios is much wider, enabling use of lower protease activities then found in type IV collagenase, potentially minimizing damage to membrane surface markers.
Thanks Robert!
Do you have also personal experinece and could tell me whats a good yield?
I tried on the weekend now on 1 big mass 2 divers protocols +/-a disgregation step with Gentle MACS.
Overall my outcome was similiar but not so high (2.5-3x10^6cells for a 5x5x3-4mm piece) .
But growing the cells then I saw that the first protocol seemed to not stress the cells to much. MCR7 cells form quicker islands like usually.
Prot a) Liberase Collagenase and Trypsin in a later step
Prot B) Collagenase (Type I-A) and Hyaluronidase also Trypsin in a later step.
Both incl DNAse
Anyway I see fibroblast like cells in both cultures (even though I was told Cholera toxin should inhibit their growth).
So now I would try to add a stepp to get rid of the fibroblast.
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