I'm practicing the bone marrow procedure on mice, and the problem is the engraftment rate is poor.
I collect BM cells from long bones of mice and make a suspension fluid directly, which means I don't use sorted KSL cells.
I prepare 5x10^5 BM cells per recipient, and transplant it by lateral tail vain injection. Almost all the injections are successful.
The other researchers used to do it by retro-orbital sinus injection, of which engraftment rate was acceptable.
I read an article that says "the engraftment rate doesn't differ between those two procedures, though heterogenous hematopoietic reconstitution is more likely to occur in those who received transplant by tail vein injection, which suggests engraftment failure.
I also read that as much as 1-5x10^6 BM cells per recipient is needed to achieve total hematopoietic reconstitution.
Do I need more BM cells to transplant ? Do you think it would improve engraftment rate ?
Thank you.
If you're using total bone marrow cells, then you probably need more cells (does your count include red cells?). Another factor may also be the level of cytoablation you're using. If you're doing allogeneic transplants then you need high cytoablation and cell numbers.
Marcus O. Muench
Thank you very much for your comment.
As you mentioned, I'm doing allogenic transplant, but within the same mouse line which is C57BL/6.
I use 9.5Gy of total body gamma-ray irradiation as cytoablation procedure, which I believe ensures mice to die.
I'll try with more numbers of cells via tail vein injection.
By the way, my cell count doesn't include red cells.
Best regards,
Daisuke
When you do BM transplantation, the idea is that all haematopoietic cells in the recipient are wiped out by irradiation and any mice that do not receive donor BM should die within a couple weeks. Therefore, in all radiation experiments, you need to have a control group that receives the radiation but not recipient bone marrow. We have tried both lateral tail vein and retro-orbital injections and when done properly, tail vein works better. Retro-orbital injections are also not allowed by certain institutional review boards. We typically transplant 10 million bone marrow cells into the recipient mice (no separation, just processed to remove RBCs). You should be able get close to 100 million BM cells and this should be enough to transplant a group of 10 mice. Increasing the number of BM cells might help you get better results.
Julie Joseph
Thank you for your suggestion.
You transplant 10 million BM cells per mouse. That's 20 times as many as I use. That sounds like it'll improve the success rate of my transplant to increase the cell number.
You mentioned that retro-orbital injections might be taken as ethically wrong thing to do. That's what I'm afraid of. My data can be required revision because of the way to transplant, right?
I'll increase the number of cells to transplant, and stick to tail vein injection.
Thank you.
Daisuke Koga, editors and reviewers do not have that power. The techniques you use are up to you. What I meant is that your University and its animal care committee might not be ok with it. But it depends on the institution. If done incorrectly, retro orbital injections can blind mice. Really bad ones can go into the brain and could be fatal. Incorrect tail vein injections can cause the tail to go necrotic and fall off. So overall, it could be less risky. Regardless of whatever mode of administration you choose, make sure you are trained and are comfortable doing it.
Julie Joseph
Thank you so much again!
I'm kind of relieved to hear that editors and reviewers can't make me redo the experiment again with the procedure they prefer.
I'll think about which way of transplant to choose. Actually, I'm a little afraid of holding mice for anesthetic procedure, so I feel more comfortable with tail vein injection.
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