From my experience you will need to use reduce matrigel., If not what you describe is what happens. Or cells in culture to long and then will not grow in vivo.

Check your mycoplasma status. Mycoplasma infections can cause tumors to regresses and have abnormal growth, which may be more apparent in immunocompetent mice vs. the incompetent mice.

They have been tested for mycoplasma recently and are clear.

Do you mean reduced growth factor matrigel? Could I dilute my existing matrigel?

Firstly , always check cells for Mycolplasma, before harvesting for injection.

IF clean, harvest cells (I used 2 large flasks per mouse) and resuspend in 50:50 HC Matrigel. (no need to dilute matrigel, just make sure you place Matrigel, on ice, in the fridge, at least two days prior to use, to ensure that the gel is liquid. Keep all tips, pippettes, vials etc on ice and/or kept in the fridge, prior to using)

Ensure all aseptic techniques are carried out throughout this process.

Inject 100ul of suspension (50:50 cells/matrigel) subcutaneously on the flank of the mouse (ensuring to lift the skin and inject, as to prevent attached tumours forming)

In my experience, tumours start to develop, within 2 weeks of injection and grow very well, thereafter.

Hope this helps :)

Sorry spotted an error, it is 1 large flask per 2 mice, NOT 2 per mouse, as stated above.

:)

Thank you Karen, unfortunately this is what I do already.

I have a successful model using HT29 cells which acts as you describe above, but in the animals injected with OE19 cells tumours either fail to form or start to shrink after 1/2 weeks of slow growth.

Although we did test our line for mycoplasma prior to injection, it has not been the standard practice here to check cells prior to every injection. Do you find OE19 cells particularly sensitive to mycoplasma infections, or is this just part of your standard practice?

We are testing the line in some other strains of immuno-supressed mice in case it is due to some remaining immune response. I've injected cells in the range from 2 million to 20 million; not sure how many cells you are getting in your large flask but I assume it is in that range....

I also checked the viability of the leftover cells after injection and the cells are definitely alive and able to grow robustly in vitro.

Many thanks to everyone who has answered so far. Hopefully switching the strain will help....

:)

Hi Laura

We did OE19 experiments in SCID mice and they grew fine. Maybe a change in strain will work, NuNu mice?

I routinely check all my cell lines prior to injection. Mycolplasma does affect the uptake and growth of cells/tumours in vivo.Always safer to check to ensure cell lines are free from mycoplasma contamination. Best to try to inject cells with a passage less than 20. Hope this helps.

:)

Still no joy with the different mouse strains. They seem to maintain just under 250mm3 with matrigel but don't seem to grow once they are in vivo. My passage number is higher than 20 but not massively so. I intend to search for a lower passage in our banks/ order some in (I was told they arrived with a passage no of 29ish so I assume you just mean less than 20 passages after receipt).

We have a delay between counting and implantation where they are kept on ice (in media) when I walk between the 2 buildings, so I'm also going to try to do every step possible in the animal unit even though cell viability is fine afterwards and has been checked.

Can anyone think of anything else that could help/ cause the cells to arrest in vivo?

Many thanks for all advice so far.

Hi Is there a specific reason you need to use OE19? We have also worked with OE21 and the tumours grow extremely well. Would it be possible for you to use this cell line?