I am having difficulty understanding how I can replicate a study's success. Could you take a look at this old study (patent really)? They have done what I hope to, which is achieve a volume of around 2ml of cells at about 5x10e7. http://www.faqs.org/patents/app/20120039856
It says in one of the later paragraphs that 2 ml of suspended cells were injected into a patients scalp to help with some sort of hair loss study. In previous paragraphs, it says that they used the isolation kit from miltenyi and only used 100uL of microbeads/blocking reagent. They also used cord blood, which to my understanding has less cd133 cells than bone marrow. I know they cultured the cells for about a month or so to get the 5x10e7 cell number, but I have a few questions.
If you have 2 vials of 2ml microbeads (and 2 vials of 2ml Fcr blocking reagent) can you achieve the same or greater volume and cell count as the study? I am pretty sure you can since they only used 100ul of microbeads/FcR blocking reagent versus 4ml of microbeads/FcR blocking reagent. Granted they did culture them for a long time, but if you used that much more reagents, you could surely get the same if not more volume/cell numbers right?
If so, I would like to know how much bone marrow will be needed?
I just don't understand how they were able to get two milliliters of cells. Will the cell number 5x10e7 usually equate to about 2ml in volume? I guess the big question here is how can I get the same (or greater eg 5ml's of cells) volume and cell number as the study without culturing for so long?
I have 2 kits and 2 vials of blocking reagent. Will that be enough?
Hi Ellie,
as I pointed out before in the other discussion at:
https://www.researchgate.net/post/CD133_cells_Is_it_possible_to_get_2-5_milliliters_of_cells_from_the_miltenyibiotec_kit
The volume of cell suspension after MACS-Separation is not a good parameter to compare because it just depends on the volume of MACS-Buffer you use for elution of the MACS column. You can have 2 or 20 ml, but the only important value to consider is the total cell number of positively selected cells.
I just had a brief look at the study and doubt that MACS for CD133 and cultivation with "SCF (Stem Cell Factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3) and IL-6 (interleukin-6)" is a good method to isolate or propagate MSCs. You will end up with a heterogeneous mix of hematopoietic progenitors, I guess, but not with MSCs and not with stem cells. Of course, this does not mean that the method they describe may not work for treatment of baldness. The identitiy of the stem cell type before and after culture in this patent is quite unclear to me (at least at first glance). If you want to isolate and expand MSCs, you will find well established protocols in Medline.
To come back to your question: Just estimating very roughly: For the isolation of approx. 1x10^6 CD133+ cells out of approx. 1x10^8 mononuclear cells from bone marrow, you will need 50 µl MACS antibody. Thus, you will need around 2,5 ml antibody to isolate around 5x10^7 CD133+ cells. Of course, the amount required will vary dependent of the percentage of CD133+ cells in your bone marrow sample.
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