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Techniques for tumor xenograft establishment

xenograft tumor inoculation cells cancer

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8 replies to this topic

#1 shamash119

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Posted 16 October 2013 - 07:09 AM

Hi all,

 

Recently I attempted a tumor innoculation using MDA-MB-468 cells combined with Matrigel , the suspension was a 1:1 volume to volume mixture, and the final suspension had  2X10^6 cells in the total 200 uL volume injected.

 

I injected the dual flanks subcutaneously , and once I penetrated into the SC I made a small pocket with the syringe, then while slowly injecting the cells I moved the needle to the left and right in a fan motion to spread the cells. 

 

Now, it has been 2 weeks since the innoculation, and you can see a very small lumps that are very spread out.

 

Is it better to inject it directly as one mass without moving the syringe needle? Also, should I have put much more cells because the growth is really small and limited?

 

What do you guys recommend, can somebody give me some tips? Was it such a small volume that was dispersed?

 

I was thinking to inject again using 200uL of the 1:1 mixture with matrigel with a total of 5X10^6 cells directly as once lump?

 

When you guys penetrate the SC and form a pocket do you inject all the cells together as a lump, or do you sway the needle left and right to deposit the cells over the entire suface to increase the surface area?

 

I have had really bad luck with this tumor induction and would really appreciate some tips from you guys!

 

Thanks again!



#2 pcrman

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Posted 24 October 2013 - 07:27 PM

Hi I think you are doing quite right. The only thing that I don't like is to move the needle in a fan motion to spread the cells. This is unnecessary. Once the needle has penetrated the skin to SC space, push the needle a short distance further in the SC space (so that the cells won't easily come out once you withdraw the needle) and inject your cells to let them form a visible lump. If you spread the cells, it is harder for them to grow into visible tumor.  

 

In you case, I think you can wait for another week to see if tumors appear.  If not, you can increase the number of cells to 3-5 million. Beware if you reinject cells to the same location you may get very nasty tumors (maybe the matrigel previously injected can potentiate cell growth and invasion.)  



#3 shamash119

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Posted 08 November 2013 - 10:17 AM

Hello I have a question

 

I am planning to induce tumors on 30 mice on the dual flanks for a total of 60 tumors. I am prepare the cell suspension and matrigel mix for 70 tumors just to have some extra on hand. After i form the cell suspension and matrigel suspension, do i load all 70 syringes at the same time in increments and store these on ice, and then inject the mice one by one?

 

I am trying to minimize the variation by doing this as a big experiment in one batch, is this the proper technique?



#4 shamash119

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Posted 08 November 2013 - 10:18 AM

I am assuming the loaded syringes will have to sit at least 1 hour or so while I am loading all the syringes, what do you think is the best way to go about doing this?



#5 shamash119

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Posted 18 November 2013 - 09:28 AM

Should the 30 mice be inoculated at the same time or seperated into 15 each and done after another so the cells are not sitting too long in syringe.



#6 pcrman

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Posted 18 November 2013 - 11:25 AM

whether to divide the mice to batches depends on how you are experienced and the time needed to finish 30 mice. For me, I can do the 60 injection in 20 min and feel there is no need to do it in two different times. I don't pre-load the syringe with cells. I use one syringe (1 cc) to take 1000 ul cells each time and inject 5 sites (200 ul each) while keep the tube containing cells on ice. After finishing the 5 sites, I use the same syringe to take another 1000 ul. I change to a new syringe if I have to inject a different cell line. 



#7 shamash119

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Posted 18 November 2013 - 11:32 AM

Thanks alot buddy! Are you using a 1:1 mixture with matrigel?


Edited by shamash119, 18 November 2013 - 11:32 AM.


#8 shamash119

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Posted 18 November 2013 - 11:32 AM

I know they have different formulations of the matrigel, are you using the high protein content or the normal version of matrigel?

 

I believe the protein content can vary from 10mg/mL to around 5mg/mL or so after reconstitution in media


Edited by shamash119, 18 November 2013 - 11:33 AM.


#9 pcrman

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Posted 18 November 2013 - 01:27 PM

We use the normal version of matrigel and mix it with cells at 1:1 ratio (volume). 







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