What medium do you use? Any other good tip for me (coated wells, etc.)?
PS: We are trying to establish primary cell lines from metastases.
You might want to contact the guys from Oncodesign, France who had a paper on establishing cell lines from primary tumors in Clinical Cancer Research recently (Julien et al., CCR, 2012).
We used the HT-29 colorectal adenocarcinoma cell line from CLS:
http://www.cell-lines-service.de/content/e3969/e4364/e4421/index_ger.html
The cells need standard CM: DMEM:Ham's F12 medium (1:1 mixture) supplemented with 2 mM L-glutamine and 10% fetal bovine serum (they grow easily in a monolayer)
I had no problems with colon cells till now. I used HT-29, CaCo2 and colo205. All with RPMI1640+10%FCS+1%P/S. Growing good and fine in normal cell culture flasks. Nothing special!
You might want to contact the guys from Oncodesign, France who had a paper on establishing cell lines from primary tumors in Clinical Cancer Research recently (Julien et al., CCR, 2012).
I think RPMI1640+10%FCS+1%P/S is the correct one. In the past I used HCT-116 and I used RPMI medium
I normally use this cell line and i growth them in DMEM Hi glucose, with 10% of Fetal bovin serum, 1% of pen-strep, 1% of L-glutamine. I use normal wells from Corning.
I think all of the media mentioned above could work and really you could use whatever supplements and antibiotics you find most effective. I've had plenty of success growing tumor cells in plain old RPMI supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin, but I've only worked with established cell lines. To isolate cells from a primary or metastasis you would probably have to put the tumor into a cell dissociator like Miltenyi Biotec's gentleMACS dissociator, which I've used to obtain tumor infiltrating lymphocytes from solid primary tumors (along with a cell isolation kit). I suppose you could use that to create a single cell suspension, count viable tumor cells, titrate down to, say 1 cell/10-50 uL, and put a single cell into, say 10 flasks with 5 mL R10 media each to see how it works. But, again, I've never done this, so it's only hypothetical, and the tumor cells might not survive. However, if you want a completely valid cell line from a tumor metastasis, you'll want to check for latent viral infection, like CMV, adenoviruses, &c., once you have a line of cells established. I'm sure you could find a paper to reference with a quick lit review, but this type of validation would most likely involve PCR. For that matter, you may want to sequence or probe for key mutations like BRAF V600E &c. and I know there is a good amount of literature on that topic. Hope this helps and good luck!
Dear collegue
if You start with human biopsie, the preparation of primary culture will depend on the delay between biopsie harvest and the lab.. The biopsie has to be maintained on ice in MEM or DMEM medium containing glucose 25 mM with antibiotics . In the lab, after a rapid dissection and mecanic dissociation in sterile condition, Half of the niopsie can directly be cultivated in DMEM-25 mM Glucose containing 10 % (v/v) SVF 2 mM glutamine 1% non essential amino acids 50 µM gentamycine and let on 1°%CO2 atmosphere for three days . The second part of the initial dissociated biopsie can be mixed with trypsine/EDAT solution or collagenase IV for 15 min maximum ( discard the medium, wash with PBS the pellet, centrifuge, discard PBS and add tryspine/EDTA or collagenase IV). Collect solely cell suspension., add complete medium , centrifuge again.. Resuspend cells in medium and start to cultivate the primary culture at 1 million cell /ml in complete medium
An alternative method consist to graft human biopsie in nude mice. Tumour will develop (4 to 8 weeks) Human fibroblast will be substituted by mouse fibroblast which can easier be removed by long Tryspine/EDTA incubation (15 min) during tumour dissociation.
Finally FACS or magnetic beads disposal can be used. If so, You may need one selective CD to make a selective separation.
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