Steps:

* Determine IC50 and IC90 dose of drug for 72h treatment.

* Treat cells with IC90 dose for 24h.

* Replace the media with fresh media containing IC50 dose and maintain it.

(The shock will kill most of the cells.)

* Some cells (less sensitive to drug) will remain alive for long time. Maintain the cells in that concentration unless it start division.

* Once confluent maintain the cells at the same dose for at least 5 generations.

* Then increase 1.5 times of the dose and repeat the same.

* Keep on increasing dose slowly for some time (till 4-5 times of IC resistance dose not come). Once cell will reach that conc, it will start expressing its resistant proteins in a stable fashion.

* Then increase the conc. twice or even thrice the previous dose, and cell will remain viable.

Note: 1) Make stock at every stable stage (after stable resistance at that dose).

2) Addition of glutamate (natural autophagy inhibitor) in the media may help some time.

3) If cell size increases (twice of the normal size) do not use that cells and start with a lower dose and with fresh stock. Some increase of cell size is expected.

I think that there's no an optimal concentration...I mean... you can start with a low concentration of drug ( a concentration that kills few cells). you can treat the survived cells with a major concentration ( you decide the range to increase the concentrations ...example: every 0,1 ug/ml )...and go on.... until you verified that the new cell line is resistant to CDDP (for example by a clonogenicity assay) and you can also verify in parallel, step by step, ERCC1 levels.

good luck

I suggest you look at this reference particularly the table in it. Godwin et al., High resistance to cisplatin in human ovarian cell lines is associate with marked increase of gutathione synthesis. PNAS April 1992 Vol 89 pp 3070-74.

They used a colon cell line as well HCT 115.

It may be helpful to do an IC50 curve and then start treating your cells with an IC5-10 and gradually escalate the dose.

This is an example, they use ovarian cancer cells though:

Yan et al.

Oncology Reports 17: 1163-1169, 2007

Biological comparison of ovarian cancer resistant cell lines

to cisplatin and Taxol by two different administrations

I suggest you that a few weeks (at least one month) cells treated with IC50 concentrations of cis-DDP. IC50 values ​​previously determined by the MTT test .

You must start by a concentration near the IC50 for cisplatin, if it is too high and you do not recover cells in a week, then get down to half IC50. You should be able to recover resistant cels then bu culturing the cells in increased concentration by intercalating periods without drug

It depends on whether you want to select your resistant cells by intermittent incremental dose or by pulse treatment. Whichever way, your first task is to establish the IC50 by dose-dependent cytotoxicity curve using CKK-8 or MTT assay. You may also need to do time-dependent cytotoxicity assay to determine the optimal duration (call it ID50) of exposure. Upon establishing the IC50, you either treat the cells in pulse at the IC50 and ID50, whilst allowing the cells to attain confluence between treatment intervals; or you treat the cells intermittently with a small dose of CDDP and gradually increase it to the IC50. Go to my profile and get my paper on isolation of drug-resistant cell lines; it could serve as a good recipe for you. All the best!

I suggest that you treat the cells with IC50 concentration of the drug and then maintain the survived cells at this concentration for one or two weeks until they show the similar growth rate as that of the parental cells. After that, you can increase the drug dosage and repeat the same procedure again and again. It will take a few months to get the resistant lines.

Thank you all. Until now i tried a concentration much higher than the IC50. I have tested it with MTT, SRB CVE assays. Unfortunately, the ERCC1 leves are decreasing. I studied and other markers, including TYMS, to find ou the cross-talking between the repair pathways.

I agree with the Benedict Odii answer. Either you proceed by progressively increasing the drug concentration starting from a very low concentration to avoid too much cell death. Or you treat your cells at a concentration similar to that encountered at the peak of plasma concentration in patients for a short time lapse (i.e. 30 min.). Then you remove the drug and let the cells to respond. Surviving cells will proliferate (hopefully) and you will treat them again and again until most of them survive the process. This last method better simulates the in vivo events and could select mechanisms of resistance which are more clinically significant than the mechanisms selected by the previous one.

I made MTT assays to calculate the IC50 and I started the culture of cells with concentrations near the IC50. After 2 passages I have very few cells, but I have live cells. Do I need to culture them for some passages without CDDP? Because I add CDDP in every passage.

Yes, you have to maintain a selection pressure with the CDDP. To help in the autocrinic process, you can add conditioned medium from proliferating untreated HCT-116 cells. This is made by centriguging medium of cells at half confluency and using it as culture medium by adding your drug. It take quite a long time to obtain resistant cells....

The real problem with making cells resistant to chemotherapy drugs is that if you add a low concentration i.e the IC-50 the cells do not really die and you end up with a population of cells that contain a mixture of sensitivities. The best thing to do is to perform a kill curve and treat the cells with a concentration which will kill 75% and 90% respectively. With Cisplatin I would change the media and wash the cells and add fresh drug every few days until the cell number remains constant. You will then have to identify clones because there are usually many mechanisms whereby cells become resistant to cisplatin; changes in ERCC/XRCCs, loss of pro-apoptotic pathways, detoxification via GST, changes in AKT activity etc, etc etc, so when you study the resultant pool you will not be able to get a clear answer. In your case you suggest that ERCC1 might be implicated. The best way is to either clone the cells in situ using ring cloning in petri dishes or do limited dilution cloning in 96 well plates (where you trypsinise your cells and plate them out at 1 cell every 3 or 4 wells of the plate and collect and then expand the colonies that grow out). Needless to say you should keep the drug in the culture media at all times as you want genuine resistance to the drug, and not some transient insensitivity which will not last more than a week. When you have isolated your clones the re-do the MTT assay and determine the IC-50/ic-90 on your clones and the parental line to determine the increase in resistance (4 fold, 8 fold 10 fold etc). When you you have done this you can screen the cells for whatever mechanisms you are most interested in, and possibly identify which ones are the most common. This is a lot of work, but worth it to generate some good quality resistant cell lines that will of use not only to you but to everyone else in the field.

good luck!

Try to select stable resistant cell lines by fluctuation analysis. See Chen G, Jaffrézou JP, Fleming WH, Durán GE, Sikic BI. Cancer Res. 1994 Sep 15;54(18):4980-7.

Steps:

* Determine IC50 and IC90 dose of drug for 72h treatment.

* Treat cells with IC90 dose for 24h.

* Replace the media with fresh media containing IC50 dose and maintain it.

(The shock will kill most of the cells.)

* Some cells (less sensitive to drug) will remain alive for long time. Maintain the cells in that concentration unless it start division.

* Once confluent maintain the cells at the same dose for at least 5 generations.

* Then increase 1.5 times of the dose and repeat the same.

* Keep on increasing dose slowly for some time (till 4-5 times of IC resistance dose not come). Once cell will reach that conc, it will start expressing its resistant proteins in a stable fashion.

* Then increase the conc. twice or even thrice the previous dose, and cell will remain viable.

Note: 1) Make stock at every stable stage (after stable resistance at that dose).

2) Addition of glutamate (natural autophagy inhibitor) in the media may help some time.

3) If cell size increases (twice of the normal size) do not use that cells and start with a lower dose and with fresh stock. Some increase of cell size is expected.

Thank you all for your suggestions.

I'm a little bit confused. The ERCC1 is a DNA excision repair protein. In cases of resistant to CDDP its expression must be increased or decreased? I think, that if a cell line is resistant, the repair mechanism are increased, so ERCC1 expression is increased in order cells can escape cell death-damage. Am I right or not?

Try add very low dose of cisplatin (below optimal dose) to the culture for long period. Then try to dilute the cell suspension and seed it in 96 multuwell plate to grow single cell. you will get different clones from your culture. few of them may adapted to become resistant to cisplatin. Repeat this procedure until you definitely get resistant cells.

You can start with vely low doses of the drug: normally 5-6 days treatment. then, increse the doses periodically. Or you can use a high doses to select the resisant clones.

I have an intermediate query, wrt cisplatin, i am presently trying to establish cisplatin resistant breast cancer cell line, which method could be a right choice to start with ?

I tried many of the above methods. You should start with Diptiman Choudhury informations..

Generally, start with a low dose and increase it passage per passage depending on the confluence and the growth rate of your cell line

Is there any other way of creating stable resistant cell line apart from high or low drug consistent exposure???

http://www.ovarianresearch.com/content/4/1/21

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054193

I'd say: 'How can I select a drug resistant cell line/strain?', the term: 'create', leads soon to concepts such as CRISPR

A Cisplatin resistant cell line was prepared by incubation with increasing concentrations of CDDP for 14 or 24 months.

Source:

PROCHAKZKA et al: MYCN EXPRESSION IN NBL AFTER CISPLATIN TREATMENT IS NOT RELATED TO COPY NUMBER

This can be done by adding to the cell culture 1/500 of the usual dose of the drug that you need to resistant to. If your cells growing well keep them on that concentration for 5 passages then start to increase the concentration gradually. However for each concentration you need  to keep you cells to grow for 5 passages. Eventually you will obtain resistant cell line but after long time.

Is there any way of creating drug resistant bacteria say M.smegmatis ?

My major problem in obtaining drug-resistant clone ( say cisplatin) is that the cells will stop grow and eventually die during the process no mater what concentration of cisplatn I start...it is so weird

i am wondering if it is important for cell confulency to begain with ic90 ,is it better to seed the cell around the confulency around70-80% or more?@ Diptiman Choudhury

Hi Dr. Liu,

You may not need to start with 70-80 % confluency. But you need to give treatment at 80-90 % confluency. Because after treatment only 5-10 % of your cell will survive. Further the rest of the cell will be in a very bad shape and will keep on dying over the next few days. Only the cells which could resist the stress will thrive and the number is pretty less.

thanks for your answer,I have another question is that your mothed for resistant chemo-drug is available for all ？I mean pharmacokinetics of different cells vary a lot，why you choose 72h IC90m @ Diptiman Choudhury