I agree with Francis - the best way is to use a "realistic" concentration of drug based on clinical measurements (if available) and wait for resistant clones to grow out. This is tedious - depending on the combination of cell line and drug you are using it can take months - but from my own experience and from talking to other people who have done this, cell lines generated by selecting with a "lethal" dose may develop different mechanisms of resistance compared to those selected via escalating doses. I would add that some cell lines are so sensitive to certain drugs that you will have no choice but to drop the dose below the max. plasma concentration. Doing a basic GI50 type of analysis (using a cell viability/proliferation assay) will help you find the right concentration of drug, as others have suggested. Good luck.

There is plethora of drugs what kind or which type was not mentioned in your query, best suggestion is to obtain clinical samples from drug resistant cancers like Breast and Prostate and maintain them in vitro. I am not sure about the other cancers as I work mostly in this field. You can over express Hedgehog signaling genes like GLI TFs to make BCa and PCa cell lines drug resistant.

We developed drug resistant ovarian cancer cells by starting with very low doses and stepwise increase of concentration...works well and needed round about 8 to 10 weeks...resistance was checked by IC50 compared to sensitive "wild" type cell line and cells kept under permanent pressure by adding high drug concentration in the culture media

Generally the drug resistance mainly caused by MDR proteins (P-glycoprotein-Pgp) in cancer cells. If you want to produce cell line which is drug resistant then try to enhance the expression of Pgp, and there are plenty of literature available on the overexpression of Pgp.

It depends which drug you are interested in. Pgp is one of the mechanisms of drug resistance but a lot of drugs are not substrates for Pgp. An alternative way of generating resistant cells is to culture them in the presnce of the drug at a concentration in the range found in vitro in the patient plasma, for a duration corresponding to the half life of the drug in vitro. Then, you wash the cells and further culture them for a long time. Most of them will die but hopefully some will survive, proliferate and will be resistant. You need to know the pharmacokinetics of your drug to perform this generation of resistant cell but you will simulate what happens in vivo and thus you will obtain a more reliant model than by starting at a low concentration and increasing it step by step. If possible, try both methods.

I agree with Francis - the best way is to use a "realistic" concentration of drug based on clinical measurements (if available) and wait for resistant clones to grow out. This is tedious - depending on the combination of cell line and drug you are using it can take months - but from my own experience and from talking to other people who have done this, cell lines generated by selecting with a "lethal" dose may develop different mechanisms of resistance compared to those selected via escalating doses. I would add that some cell lines are so sensitive to certain drugs that you will have no choice but to drop the dose below the max. plasma concentration. Doing a basic GI50 type of analysis (using a cell viability/proliferation assay) will help you find the right concentration of drug, as others have suggested. Good luck.

I would ask if you want to create cells that are of de novo resistance (follow Francis's and Harvey's suggestions) or acquired resistance (follow Sebastian's, Rohan's, and Karen's suggestions). Good luck.

I suggest that you culture desired cells with the drug at IC-5% concentration for 3 days and gradually increase the conc. by IC-5% every changing media (3 days) till reaching to IC45%.

to confirm cells resistance to drug, measure the MRD proteins specified for your cell line.

The question is first of all: do you want to generate clonal populations of resistant cells, which has the advantage that all cells should have (in theory at least) acquired the same resistance mechanism. Or do you want to generate a mix of cells from your original cell line, which is likely heterogenous in the first place unless you have subcloned it (Heterogeneity is not a bad thing, but important to keep in mind for some of your later experiments and comparisons! Which differences are really caused by any given resistance gene?).

I am not sure what your aim is but if you want to identify resistance mechanisms then you need to generate individual resistant clones.

We have used actually 5x the IC50 for treatment of cells (BRAF inhibitor and/or MEK inhibitor).

The cells are seeded at a sensible density in plates (so you can later pick resistant clones, there are also nifty flasks on which you can remove one side to pick colonies) and treated regularly with 5x IC50 (or the drug conc you estimate as most appropriate).

We replenished the media including fresh drug every 3-4 days. Dependent on the growth rate of your cells you will end up with individual cells forming growing colonies, IF YOU CAN GENERATE RESISTANCE after ~2-8 weeks. Make sure you don't have your colonies growing into each other (your initial seeding is the key here!). You pick your colonies when they consist of at least 20-50 cells. If you do it earlier you will loose to many colonies as you will not be able to transfer all cells.

There are several picking methods if you need info let me know.

You can help your picked colonies and in fact your generation of clones along if you use condition media if dealing with low cell numbers. Condition media should be collected from happily growing parental cells (that do not show media exhaustion). This media needs to be sterile filtered (22um) and can then be used straight or 1:2 with fresh media. Add normal amount of drug to this. Once your colonies grow (and not all will!) you can switch back to normal media, but keep drug in.

I would suggest the over expression of PGP which might be easy to, initially . Then use the IC50 cloning selection that Mahmoud Singer suggests

Hello,

I agree with the answer of Harvey and Francis. To begin your study,

I recommand also the article from Lesuffleur et al (Lesuffleur T, Barbat A, Dussaulx E, Zweibaum A. Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells.Cancer Res. 1990 Oct 1;50(19):6334-43, free in pub Med).

Good luck

I agree with Frances. I have been growing Metformin resistant MCF7 cell lines. Started with a low dose (dose used in Tissue Culture, based on clinical dose, not MTD ) and escalated as and when cell lines were confluent. I always kept a 'buddy' flask, alongside the increasing dose flask, to maintain the cell line.

Do cell death curve for the cell line. Take the IC50 concentration of the drug and treat the cells in flask with the drug for one or two days. Remove the media and fresh media only. After confluent split and again add the drug like that 3 or 4 splits increase the concentration repeat this for while then test with cell death curve check for change in IC50. Other way is to start with low concentration of drug and treat the cells. I think takes long time. I like the first way with high drug concentration because when you treat the cells only resistant cells live then you can grow them further.

Hi Lukas, my recommendation for colonies relates to the ability to identify resistance mechanisms. If you have several of these mechanisms within your cell line, you should (ideally!) find these represented in your different colonies. The colony strategy makes it far easier to cleanly identify mechanisms because every cell of a given clonal population should have the same mechanism.

Now, what Jason really wants to do in detail he has not told u,s so he may just want to create "pools" of resistant cells for his purpose, which is much easier as it does not involve small cell numbers, condition media and so on....(much faster also!)

Thanks for all your answers. They are extremely helpful.

Now, I can't really say what drug I'm using but it is on a leukemic cell line. The aim is to generate a resistant line after escalating doses of drug.

I wonder what kind of problems would such a methodology result in? Compared to say, throwing in high doses of a drug right at the start and sieving out cells that survived.

You should be patient to get resistant cell lines with stepwise drug increments. We have developed several sub lines from MCF-7. Please check our papers.

Vincristine

Aout colon cancer drug resistance you can refer to our paper I herein enclosed

good work

It seems that there are a few different ways to generate resistant lines. But i am wondering whether or not each procedure may initiate different resistance mechanisms (e.g. p-glycoproteins, Bcl-2 increase..). If so, everbody should explore the mechanism which is supposed to be responsible for the resistance developed.

also you can stably trasnfect them with some genes such as GSTP

I agree with Francis that at high conc. cell may die. However, escalating dose patiently or even at same dose, selecting the survivors (let it may be 5-10%) is best approach, that is how in body also cell become resistant.

Hello Jason,

Start from LD50 dose of an anticancer drug(select marketed drug of same target) and then a stepwise increment of drug after every passage will lead to generate a drug-resistant cancer cell lines.

Thanks,

milind

If you are working with different Cancer cell lines. Transfect each cell line with a unique DNA barcode with varying lengths (at least 20bp difference just to avoid sequencing step) using Lipofectamine or other TA (8hours and trypsinize). Culture test cell lines together, after 48hrs of treatment with low to high concentration of drugs perform FACS (Dead and live cells) and PCR on live cells. This gives you the cell lines that are resistant to drug at various concentrations. This is will be useful if you are working on different cell lines on a holistic basis. However, with this fast track appraoach you can get your solution with in a week. I hope this approach suits your needs. Good luck Chan.

Hi I am currently developing a BT20 and a specific drug, resistant cell line. Again it is based on a dosing regime and escalating, until you can see an effect in the growth of the treated line and maintain that concentration until it grows at the same rate as the UT. It is a long, steady progress, so ensure that the cells passage is low, before you start.

Jason Y.C.,

By coincidence, I noticed this article in my RSS news feed backlog yesterday. Useful to you?

http://journal.frontiersin.org/Journal/10.3389/fonc.2014.00040/abstract

Why do you want to incraese resistance?

How about trying to decrease it..Those APC channels remove drugs...what is the interest to create such resistance?

It is better to produce the drug having polyfunctionality

I think best approach is to use escalating doses over a period of time. Start with the low dose first and than increase the dose gradually over a period of time. This way you will get the resistant tumor cell line simulating the resistant primary tumor with few variations.

Take a example of solid tumor, where drug interaction at peripheral is much higher, getting diluted at the same time or getting access toward inside after peripheral tumor cell death. So best way is to incubate at low dose for 2- 3 weeks where cell will acquired a genetic alteration to resist cell death, then could escalate for observing cell phenotype ( acquired resistance ?).

Maybe some siRNA is helpful to do this

 I agree with Francis Belloc. In the past I used a breast cancer cell line, MCF-7, resistant to medroxyprogesterone acetate.The MPA-resistant sub-line was generated in the ‘‘Laboratorio di Oncoematologia Sperimentale della Divisione di Oncologia Medica della Fondazione S. Maugeri di Pavia". MCF-7 cells were grown in medium containing increasing concentrations of MPA (from 0.025 to 12.5 μM) for 20 passages (MCF-7/MPA). The sub-line exhibited a reduced PR content compared with the parental cell line and showed cross-resistance to doxorubicin. Moreover, it expressed p170 glycoprotein (Gibelli et al., 1994). The MCF-7/MPA cells were maintained continuously in the presence of 12.5 μM MPA. Obviously the concentration of compound used to induce resistance depend on the type of compound used.

In case it's of any use, I've managed to generate a daunorubicin (DNR) resistant cell line by exposing an early passage of the cell line to increasing doses of DNR, as has been mentioned before. I started with a 1/1000th of the therapeutic dose for 4 days with 3 days in the absence of DNR, and each week increased the dose 10-fold and continued this until the cell line could not cope with the concentration of DNR. I found that, on the days without DNR, it was important to replace the medium every day to remove dead cells and give the live cells a good chance of survival before the next exposure to DNR.

Dr. Jacques: i think he need them as a model for MDR to begin to apply his own drug to test it as an inhibitor for this proteins, as i do.

How can I distinguish the cells are not devided any more from they are death(just the death rate equal the birth rate) when the cell number is not change in a short term, such as the first two days after drug assisant?

Hello,

I aggree with the answer of Mr Belloc Begin the resistance protocole with pharmacological dose of the drug. Change cells with medium and drug for a long period ( greater than  3 weeks).  You will find in the dishes or flasks subclones containing resistant cells. You have  to trypsinate  and cultivate the cells in the presence of drug until cells reached a similar growing curve as the parental cell population.  You obtain  therefore a resistant cell population, which however needs to be characterised in terms of genotype and protein cell expression.

Best regards

Can i split the cells with trypsinization before increasing the dose of drug during the establishment of drug resistant cell line??  

Dear Jason,

I think a proper answer depends on many factors, including the cancer cell type you are investigating. About colon cancer cells you may refer to the following study: N.A. Dallas, L. Xia, F. Fan, M.J. Gray, P. Gaur, G. van Buren II, S. 692

Samuel, M.P. Kim, S.J. Lim, L.M. Ellis, Chemoresistant colorectal 693

cancer cells, the cancer stem cell phenotype, and increased 694

sensitivity to insulin-like growth factor-I receptor inhibition, 695

Cancer Res. 69 (2009) 1951–1957.  Additionally, I attach herein a paper from my lab in which we produced resistant colon cancer cells with increasing sub-lethal doses of 5-Fluorouracil.

Best,

Mariano

different cancer cell type needs different methods to induce，and whether successful or not depends on many other factor. If you want to learn more, creative bioassay is a good choice.http://www.creative-bioarray.com/Services/drug-resistant-cell-model-generation.htm

what if you ganna to establish murine derived cell line，you will still using a concentration reffered to the vitro of patient plasama? how about started with IC50?@ Francis Belloc

Starting with IC50 would be too high. Start very low as several people have correctly suggested and gradually increase drug concentration.