I am currently a student looking to get into the field of biotechnology upon graduation and am currently interning at a privately owned biotechnology facility.
At said facility, there is an upcoming project that I require assistance with. We (the other interns and I) have been daunted with the task of isolating CD34+ cells from human peripheral blood. I had hoped that I could get some help with this as I have never dealt with this specific type of cell before and would really like to land this position. The rules mandate that we use a specific isolation kit (I will list the link to it below) and that the amount of CD34+ cells in the end equates to 5ml. My questions concerning this project and the link to the kit I must use are below.
The kit that we will be using is here : http://www.stemcell.com/en/Products/All-Products/EasySep-Human-CD34-Positive-Selection-Kit.aspx
Questions:
1.) My first question regarding the kit is the amount of milliliters the kit provided will yield as it is. I assume of course that in order to obtain 5ml of CD34+ cells, there will be a considerable amount of culturing involved. I would just like to know how many ml of cells the kit will yield as it is. Or if anyone here has used the kit provided and has experience with it.
2.) How to culture them? I have read many different answers and publications that all say something different. I would like to know what is the best medium for culturing this type of cell, as well as how long and what temperature they will grow best at. Basically as the project mandates an end result of 5ml, I am asking how to culture them and how long it will take to culture them to 5ml.
That is basically it for my questions. I have a background in cell biology and am studying biotech, specifically cancer cells with no experience with CD34+ cells. So I appreciate any help anyone can offer.
Hi Ellie,
I think you are worried a bit since you are a fresher and understandably so. But trust me, the isolation of CD34+ cells using this stem cell kit as as easy as it says. Regarding your first question, the 5mL volume is required in the isolation step because some magnetic separators accommodate 5ml polystyrene tubes. But the final volume of the isoated CD34+ cells can be flexible (from 500uL-1mL generally). Rather that the volume (which is flexible) how much was the cell-yield is more important. secondly, for culture, serum-free medium is better to expand them rather than serum containing medium. Xvivo medium, strempro, stemspan etc are good serum-free medium that gives robust expansion of CD34+ cells in culture. Having said so, the culture method may definitely vary with company needs/lab conditions.
I hope this information may be of some help. Good luck with your venture!
On a second thought I was wondering whether you mentioned 5 million cells? If so you may need minimum of 5x108 mononuclear cells to start with.
You mention that you want to isolate CD34+ cells from peripheral blood. Keep in mind that in order to get a good amount of CD34+ in the peripheral blood a healthy individual would have needed to have been mobilized with G-CSF. Otherwise, there are not many CD34+ cells in the blood. Alternative sources, such as bone marrow and umbilical cord blood contain much more CD34+ cells and might more suitable.
In our experiments with umbilical cord blood we get 0.1%-1% of the mononuclear cells that are CD34+ cells. We usually do a Ficoll density gradient to get rid of the erythrocytes and then use the interphase layer for the CD34+ selection, using about 10^8 mononuclear cells to get 0.5-1 x 10^6 CD34+ cells. Take care to check the purity of the isolated cells by flowcytometry.
Culturing human HSC is a big topic of interest: many people try and there are a few good protocols available. Note that the HSC is a notoriously difficult cell to culture, because expanding the cells usually results in loss of the repopulating capacity. So: more cells does not necessarily mean more functional HSC, so the functionality of the cells should be tested preferably by transplantation into NOD/SCID or NSG xenotransplant models. A few interesting protocols: Biotano Science 2010, Goessling Cell Stem Cell 2011, Zhang C.C. Blood 2005, Drake PLoS One 2011.
I think as a starting point to sort out this problem we need to know the volume of peripheral blood you are starting with, this will give an expectation of how many CD34+ you may isolate and will allow estimation of how many cells you may expect after expansion. Also the culture conditions depend to some extent on the cells you want from the expansion eg neutrophils, rbc and techniques you may use for further analysis eg flow cytometry, molecular biology.
if you can provide the information above I can give you some advice.
regards
kay
Dear Ellie,
as previous people said, just follow the instruction of the kit, which will provide you also with information on how many ml of blood you need to start your extraction to obtain a decent yeld. Is this a limiting point? Are you short of samples (in terms of ml available)? As others said, knowing this will help giving you a more targeted answer. Anyway I know very well StemCellTech products: they are totally reliable.
About culture media to expand your cells, there are many commercial media you could use: I would use the Stempro from the same company providing the kit.
Also, a little hint: as you are working for a private company, maybe you are performing SOPs based experiments which have been pre-developed. Ask some further directions to your supervisors and be careful not to disclose the details of the procedures around, even in here...I know it is sad, I've been trough it, but you have to be careful.
All the best with your new position
Federica
Hello. Do you get good expansion of CD34+ cells cultured in commercially available media from stemcell/miltenyi?
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