Veterans Affairs

Efficient isolation and culturing of CD34+ stem cells from human peripheral blood (and other media)

Method obtains pure populations with high yield

Medical & Biotechnology

Human stem cell

Scientists at the U.S. Department of Veterans Affairs (VA) have developed a method of isolating and enriching stem cells from either bone marrow or peripheral blood which takes advantage of the non-adherent property of stem cells, as opposed to the adherent property of differentiating cells, by serially passaging the suspended cells in liquid media. The patented technology is available via patent license agreement to companies that

Common methods for obtaining human stem cells for therapeutic cell replacement include, for example, purifying cells by flow cytometry and then growing them in growth medium containing serum on a feeder layer of primate cells. The growth medium in such methods contains fetal bovine serum on the feeder layer and this makes them ill-suited for re-implanting into patients. Furthermore, double sorting CD34+ cells by flow cytometry is tedious, gives yields of low abundance, and presents sterility problems.

VA researches have addressed the above with a method to derive pure cultures of stem cells, by continuous growth in liquid culture medium in the absence of methyl cellulose, matrigel, blood clot, or other matrix. Only suspension cells are passaged by removing suspended cells and conditioned medium from stromal cells, macrophages, endothelial cells and other cells that attach to the wall of the culture flask. Suspension cells are passaged with cell-conditioned medium into fresh culture flasks containing fresh culture medium. Alternatively, the hematopoietic stem cells can be grown in defined serum-free medium.

This cell culture system yields pure populations of bone marrow stem cells, such as CD34+ or CD34− cells, in large numbers, grown in continuous cultures ,that can be expanded from microliters of cells to thousands of liters of cells. This method solves the problem of immune rejection of transplanted cells, pathogen transfer (e.g. hepatitis, HIV) from donor to host, limited availability of embryonic and fetal stem cells, and the ethical issues of human embryonic and fetal stem cells.

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