We are trying to establish primary cell culture from ovarian tumors or ascites. At the beginning, most epithelial-like cells grow in cluster, forming a gland-like structure, which seems to be embraced in a membrane. These cells seem not to be able to expand over this membrane border. Instead, they grow "up" into medium, forming again cell clusters which hang over the gland structure. This is a very short period of time that cannot lead to enough cell numbers, so that we can analyze them.
After a certain period of time, the cells stop growing, or there will only be cell debris shedding over the gland. The cells inside the gland start to have more "dark" particles and seem to fuse to each other, making a huge structure with many nuclei. It seems that they are undergoing apoptosis.
Would someone give me tips, how to bring the cells into proliferation, maybe at the early stage, so that we can get enough cells to analyze?
There is a problem to cultivate ovarian cells in vitro, as factors controlling the proliferation of these cells are at present unknown. However, some published results indicate better results when FGF and EGF are added
because of the low number of cells, I still can not analyze all of them. But a couple of ones which have p53 mutations in their primary tumors have been confirmed to have the same mutation. So, I can be quite sure that these are ovarian cancer cells.
You can try either of two things. 1) Culture the cells with a variety of cytokines/hormones. One approach is to add the ascites fluid from where the tumor was obtained. Use the clarified ascites fluid at about 10% with another 10% of serum. The ascites fluid may contain the appropriate cytokines/hormones that may promote growth. Rather than buying a number of cytokines, you can utilize supernatants from a mixed lymphocyte response (MLR) harvested on day 2 of culture. There are a plethora of cytokines produced. Again, use it at about 10%. 2) We had a similar problem with a rat ovarian tumor cell line. We had to keep the cell numbers low otherwise, the cells grew as a clump for a while and then stopped, Keeping the cell numbers very low may help. 3) Make sure there are no T cells or NK cells in your initial cultures since they can have a very negative effect by killing the tumor cells from the initial seed from the ascites fluid.
the use of AREG may help cells to proliferate with and higher serum concentrations (10% / 5% )
It is not easy to cultivate primary cell especially ovarian cancer cell.
In my case, I do sphere assay using human ascites of ovarian cancer cell.
It needs 21 days to see some sphere using MEBM-based sphere media.
So you should check the thawing media and look specific media for culture.
I do not know how to cultivate primary human ovarian cell line.
you can use this strategy: go to ATCC, search for ovary, you will get 8 cell lines organ: ovary. Look at the references (at the bottom of the record) on how they isolated and established each cell line.
Find here a paper on the isolation of normal and tumoral cell lines from ovary
Good luck!
Hi,
I'm not an expert of ovarian cancer cell culture, but I worked some years in an ovarian tumor banking lab. After asking them about your question here is their comments: It's hard to get an answer without knowing all your protocol! But, they suggest to check which kind of flask or petri you are using. You should use flask specially designed for primary cell culture because they have a special coating. Secondly, are you supplementing your media with hormones? Your cells probably needs it. Thirdly, the way you isolate your cells before putting them in culture is important. You could try to simply put the tissue in the flask and wait for cells to spread on it, or try to dissociate it with enzymes like Dispase (Roche). Finally, at what density are you cultivating your cells?
I wish you good luck, but as far as I know, it is hard to keep primary culture of ovarian cancer cells longer than 10 passages.
Sincerely,
Jean-Philippe Babeu
I agree with Andreas Moor. An attempt at sphere cultures (basically cells grown in suspension or non-adherent cells) would be prudent at this point. They do not need to adhere in order to grow. Unfortunately you do have to supplement with several expensive growth factors or you could buy the media (again very expensive)
.
Primary cancer cell cultures do not all necessarily grow immediately as attached monolayers. they may need to be grown as spheroids or unattached 3-dimensional cultures in soft agar - the literature should contain many examples of this type of culture.. Also tumours established initially as xenografts in immune suppressed mice, may often subsequently go more easily into tissue culture.
Dear Dan Cacsire
It seems that some cells have this kind of nature. The BT-474 breast cancer cell line also forms clusters (gland structures). One of the reasons that make this kind of behavior is that these cells have low growth therefore cells support each other by gathering to each other and by their secreted growth factors.
So if you want to grow these kinds of cells you should know how to behave. You mentioned cells inside the gland start to have more "dark" particles and seem to fuse to each other, that looks like volcano, the darkness means that the cells that are in the inner layers are dying (for out of food, oxygen …). So before seeing the darkness you should passage the cells. Even if you still have a lot of space in the flask. During harvesting be sure that you separated the cells from each other by pipette (check the cells under the microscope). If you saw dark point in the gland (dead cells) less time centrifuge is required, because the alive cells deposit faster than the dead cells so you can separate the dead cells.
During passaging it is very important that you add enough cells in the culture flask. It would be useful to use small culture flask at the beginning.
If you want to exchange (yellow) culture medium, for these slow growth cells, do not empty all the medium, leave 20-25% for them (for normal grow cells we usually leave 15-20%).
If the growth is very slow you may increase the serum (FBS) to 15%.
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