I plan to grow isolated circulating tumor cells (CTCs) on a Matrigel-coated well plate.
I have learned that there are 2 types of Matrigel-coated well plates and that one is thick and the other is thin. However, I am very new to this technique so I did experiments on both thick and thin coated plates.
Matrigel acts as an extracellular matrix and thus results in different growth appearances.
For thick coating, 100% Matrigel cells will form as spheroid and for thin coating, 2% will be in normal growth in culture media (please see attached images).
My questions are:
- how much Matrigel is considered "thick" and "thin"?
- what type of Matrigel would be good for growing isolated CTCs (thick or thin)?
In the future I'd like to work on harvesting cells on Matrigel. Does anyone have any useful tips? (And perhaps also for trypsinization?)
I am not an expert in CTCs, but have indeed been working on growing cells in and on Matrigel. The type of culture you want to perform depends on what experiments you are planning.
Say for instance you want these cells to be growing in a more physiologically relevant context, you may want to grow them embedded in 100% Matrigel. So the way to do this would be to re-suspend the cells in 100% Matrigel, and seed them into a suitable dish. Once the Matrigel gels at 37 for 10 minutes, you can add complete growth medium. This will allow the ceclls to grow in a 3D matrix similar to in vivo conditions.
Another way to do what I just suggested is to do a coat and an overlay of Matrigel. In this case, you will have to do a thing coat of 100% Matrigel on you dish. Resuspend the cells in growth medium supplemented with Matrigel to a final concentration of 2%. You can then overlay this cell suspension on the gelled Matrigel coat that you gave the dish..
Both of these methods above will give you 3D structures similar to what you have shown with a thick coat. As far as harvesting cells is concerned, you will have to place the dish on ice for 20 minutes and then centrifuge the spheroids that you have grown. Discard the supernatant and then you will have the spheroids. You can either trypsinize these in the tube or stain, etc...
The other option is to merely coat the dish as you have done in the 2% Matrigel coated plate. This only gives the cells ECM ques from the bottom. This is probably why you are seeing a growth resembling a 2D epithelium.
I also believe that BD has a dispase or something similar, to harvest cells from Matrigel.
I am not an expert in CTCs, but have indeed been working on growing cells in and on Matrigel. The type of culture you want to perform depends on what experiments you are planning.
Say for instance you want these cells to be growing in a more physiologically relevant context, you may want to grow them embedded in 100% Matrigel. So the way to do this would be to re-suspend the cells in 100% Matrigel, and seed them into a suitable dish. Once the Matrigel gels at 37 for 10 minutes, you can add complete growth medium. This will allow the ceclls to grow in a 3D matrix similar to in vivo conditions.
Another way to do what I just suggested is to do a coat and an overlay of Matrigel. In this case, you will have to do a thing coat of 100% Matrigel on you dish. Resuspend the cells in growth medium supplemented with Matrigel to a final concentration of 2%. You can then overlay this cell suspension on the gelled Matrigel coat that you gave the dish..
Both of these methods above will give you 3D structures similar to what you have shown with a thick coat. As far as harvesting cells is concerned, you will have to place the dish on ice for 20 minutes and then centrifuge the spheroids that you have grown. Discard the supernatant and then you will have the spheroids. You can either trypsinize these in the tube or stain, etc...
The other option is to merely coat the dish as you have done in the 2% Matrigel coated plate. This only gives the cells ECM ques from the bottom. This is probably why you are seeing a growth resembling a 2D epithelium.
I also believe that BD has a dispase or something similar, to harvest cells from Matrigel.
The answer provided by pavan is very interesting and you should reall yconside to grow your cells in a 3D environment. I would like to add another aspect. Normal Matrigel contains quite a number of growth factors in varying amounts. If you want to perform further studies on your cells, you may want to look at the growth factor deprived version of Matrigel, where the amount of growth factors is not zero but at least reduced. Then you can add your own factors rather than culturing your cells already in a colorful mixture of factors.
We used to use around 150ul of Matrigel, just enough to cover the bottom of the insert, and then dried it for around 1 hour at 37 degrees C. Cells were seeded onto this in a minimal amount of media.
Can we make Matrigel coated well plate as stock?
I have an idea to make 50% Matrigel coated well plate and keep it at 4 C. But not sure how long can we keep it, is it has expired date?
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