I recently contacted Miltenyi and asked for an estimate on an average PGMC cell count from 50ml of bone marrow. They replied and said that in theory, there are usually 1x10e8 mononuclear cells in 50ml's of marrow.
Here I just received a different answer to the same question:
"On average you should get about a million cells per ml of blood (5x10^7 PBMCs for 50ml blood)"
So I'm just wondering which is wrong and which is right? I understand that you must count the cells after each isolation, but estimation on average is what I am dealing with here. It is to my understanding that bone marrow contains more mononuclear cells that peripheral blood right?
Hi,
Donor and operator (i.e. hematologist) variation is so high that both are reasonable estimates. Unless you know your operator's average (is determined by technique and needle preference) nobody can say exactly. I've gotten as much as 1.2 * 10^8 MNCs from only 9 ml of BM, but very rarely less than 1M per ml. If you're not interested in an exact average, but more in a reasonable minimum in the planning of future experiments then 1M/ml is a good ball-park figure.
Al is based on the isolation technique. In any case, there are always more such cells than those which eventually adhere to plastic and expand by the conventional methods.
I fully agree with Hendrik, BM being a biological sample has lot of variations wrt to the cell count, age of the donor, peripheral blood contamination and the collecting physician. I have been working with the bone marrow for almost a decade now, processed over a more than 50 samples for various reasons such as R&D, clinical trials, crypreservation of the patients BMMNCs for future use and believe me every sample that i processed behaved differently in terms of the recovery of the cells. A good sample ( good collection, no peripheral blood mixing) may even yield 3-4 million MSCs per ml. So there is nothing like wrong and right, it is all do with the sample. But yes a good and a consistent processing technique will help in good recovery of the cells.Yes, bone marrow definately contains more MNCs than the peripheral blood sample.
Hope u r successful in getting a high number of cells when u process the BM.
Best of luck!!!, revert back if any queries or clarifications
I concur with Hendrik & Prathibha - MNC yield from human bone marrow samples is highly variable, for a number of reasons. One major issue is blood contamination - relatively rare in mouse, but significant [up to 10%] in human, and that leads to high numbers of T lymphocytes in the MNC preparations. A second issue is how the marrow was harvested - were multiple plugs taken, or was 50 mL aspirated from a single tap?
Other issues are what type of MNC are you looking for, and if you plan to deplete red cells, and how you plan to do it? Ficoll-Paque leads to depletion of neutrophils and potentially some hematopoietic stem cells. Both ammonium chloride lysis and hypotonic shock [dH2O] tend to be toxic to HSC.
Mesenchymal stem cells are often associated with the spicules in the marrow, and are reduced if the marrow has been run through the nylon mesh filters found in a marrow harvest filter pack; in turn, the spicules [and MSC] can be harvested by back-flushing the filters.
As a side note to Lauren , what strain of mice were you using to isolate 10^8 BMMNC from 2 femurs? The best I could do was about 7.5 x 10^7 from BALB/c mice.
Thank you, Lauren. That makes sense, since I was just doing the syringe-flush method - a long time ago. IIRC, the bone crush technique came into use after I had stopped working with mice.
At one point as a post-doc, I tried using a collagenase-dispase incubation on bones after syringe flushing out the red marrow. I was trying to isolate bone marrow stromal precursors [probably MSC], hoping that the 'empty' bones would be enriched for those cells. That did not work out all that well, but my boss found the cells we recovered that way had lots of HSC.
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