15 replies to this topic

[jakatta70]

Can anyone tell me an acceptable final % of methanol to expose Caco-2 or THP-1 cells to?

[bob1]

certainly no more than 10%, at this concentration mnay yeasts and bacteria start to die.  I would suspect that 1-3% would probably be OK.

[jakatta70]

bob1, on Jun 4 2009, 01:13 AM, said:

certainly no more than 10%, at this concentration mnay yeasts and bacteria start to die.  I would suspect that 1-3% would probably be OK.

Hi Bob,

Could you tell me if 5% ethanol is an acceptable final concentration to use in cell culture? Will this be cytotoxic? Im using Caco-2 cells. I decided against methanol usage and have instead opted for ethanol.

[bob1]

What you need to do is test it for your system.  Presumably you are looking at the effects of some drug on the cell, so you need to look at the effect of the carrier (ethanol) on the cells as well, to make sure that the effects you are seeing are due to the drug not the carrier.

Start at a low percent (maybe 0.1%) and work up from there, studying how your genes, morhpology etc are affected.

[Stephan]

I have used 5% ethanol before as a positive control for apoptosis.  Becareful for the length of time of exposure.  more than an hour at 5% starts causing massive apoptosis.

[jakatta70]

Stephan, on Aug 28 2009, 10:56 AM, said:

I have used 5% ethanol before as a positive control for apoptosis.  Becareful for the length of time of exposure.  more than an hour at 5% starts causing massive apoptosis.

Wow! May I ask what cells you were using? Im using Caco-2 intestinal cell line.

[miBunny]

For ethanol, you do not want to go above 0.1% final concentration in cell culture.  I would apply the same number to methanol.

[Stephan]

jakatta70, on Aug 28 2009, 01:02 PM, said:

Wow! May I ask what cells you were using? Im using Caco-2 intestinal cell line.

I am using MCF-7 breast cancer cells.  I often work with CaCo-2 and HT29 as well.  Although I haven't done a proper experiment run with the 5% ethanol on the CaCo-2, maybe I should?

The CaCo-2 cell lines are alot more resilient than the MCF-7 line.  My CaCo-2 cells become confluent overnight from frozen and need to be sub-cultured every day!  I wouldn't be too worried about 5% being too high.  

I used the 5% ethanol because I didn't have any camptothecin etc and I did have a reference for the 5% ethanol.  Maybe I'll do a bit of scratching to find it.

[jakatta70]

Stephan, on Aug 31 2009, 09:17 AM, said:

jakatta70, on Aug 28 2009, 01:02 PM, said:

Wow! May I ask what cells you were using? Im using Caco-2 intestinal cell line.

I am using MCF-7 breast cancer cells.  I often work with CaCo-2 and HT29 as well.  Although I haven't done a proper experiment run with the 5% ethanol on the CaCo-2, maybe I should?

The CaCo-2 cell lines are alot more resilient than the MCF-7 line.  My CaCo-2 cells become confluent overnight from frozen and need to be sub-cultured every day!  I wouldn't be too worried about 5% being too high.  

I used the 5% ethanol because I didn't have any camptothecin etc and I did have a reference for the 5% ethanol.  Maybe I'll do a bit of scratching to find it.

Hi Stephen,

Just to let you know that I used 5% ethanol in my cell culture of Caco-2 and it certainly did cause massive cell death after an hour! So Im now taking bob1's advice and assessing the toxic effects of both methano, and ethanol on my cells via morphology, MMP-9 release via zymography and also LDH assay. My project is a nightmare at the minute trying to find a suitable carrier for these drugs. Some drugs are very polar and will onyl dissolve in 3% DMSO. Problem is at this percentage the drug comes out of solution when incubated at 37C which ends up killing my cells as well.

I was told that a student that did the experiment before me used a T75 flask and not 12 well plates and found no toxicity. However when I do the maths, the volume and number of cells and final percentage of carrier is the same between the big flask and the small wells! The joys of research!

[Stephan]

very interesting!  Not sure to the reason between the T75's and the 12 wells, although it may be due to the exponential nature of apoptosis and so fewer cells in the 12 wells die at a higher percentage rate?

I'm no expert in drug delivery but do you HAVE to incubate at 37C?  if the cells are only out of culture for a short while perhaps leaving them at 20 -25C would resist the drug leaving solution?

Keep me updated as to what you find.

[DRT]

jakatta70, on Sep 3 2009, 09:29 AM, said:

My project is a nightmare at the minute trying to find a suitable carrier for these drugs. Some drugs are very polar and will onyl dissolve in 3% DMSO. Problem is at this percentage the drug comes out of solution when incubated at 37C which ends up killing my cells as well.

Have you tried either of these?: increase the serum content (or dissolve the compound directly into serum); try one of the cyclodextrin family of carriers (this drastically alters the delivery kinetics but at least you end up delivering something).

[jakatta70]

DRT, on Sep 4 2009, 12:46 AM, said:

jakatta70, on Sep 3 2009, 09:29 AM, said:

My project is a nightmare at the minute trying to find a suitable carrier for these drugs. Some drugs are very polar and will onyl dissolve in 3% DMSO. Problem is at this percentage the drug comes out of solution when incubated at 37C which ends up killing my cells as well.

Have you tried either of these?: increase the serum content (or dissolve the compound directly into serum); try one of the cyclodextrin family of carriers (this drastically alters the delivery kinetics but at least you end up delivering something).

Hi,

Dissolving the drug into the serum is an interesting idea. I will certainly try that. Could you tell me though why you think this might keep the drug in solution? Also if the polar drugs only dissolve in DMSO would you not expect the drug to be insoluble in serum? It is insoluble in DMEM media.

[DRT]

jakatta70, on Sep 5 2009, 01:31 AM, said:

Dissolving the drug into the serum is an interesting idea. I will certainly try that. Could you tell me though why you think this might keep the drug in solution?

If it weren’t for the carrier proteins in serum, my drugs would never get to where they are needed

[jakatta70]

DRT, on Sep 8 2009, 12:40 AM, said:

jakatta70, on Sep 5 2009, 01:31 AM, said:

Dissolving the drug into the serum is an interesting idea. I will certainly try that. Could you tell me though why you think this might keep the drug in solution?

If it weren’t for the carrier proteins in serum, my drugs would never get to where they are needed

Hi DRT,

I took your advice and tried dissolving some of my highly polar compounds into FCS. Unfortunately none would dissolve even after I applied 50-60C heat and/or vortexing for an hour. So I decided to try a 1:1 ratio of 100% DMSO + FCS. This produced an intense exothermic reaction which precipitated the protein content of FCS and turned the solution a dark cream colour. So I cant use this in my in vitro assays.

So what Im going to do is make a concentrated solution of my drugs in 100% DMSO and add x microlitres to my cell assay so that the final concentration of DMSO the cells are exposed to will be less than or equal to 0.5%. At this percent I havent been finding toxicity when using an LDH assay. Fingers crossed!

[DRT]

Good luck, sounds like it should work. I like your 'try it out and see' attitude. Are we allowed to know a bit more detail about your compounds?