The purification of CD34⁺ cells from human cord blood: comparison of separation techniques and cytokine requirements for optimal growth of clonogenic progenitors

Abstract

This study was performed to assess the methods which gave maximal recovery of purified CD34/45+ cells from a cord blood specimen and optimal growth of progenitors cultured from the purified cells. Cord blood samples were separated using Percoll gradients (either one (1.080) or two successive (1.080 and 1.068) gradient(s)) and commercially available devices for CD34+ cell isolation (affinity columns as manufactured by CellPro Inc. or immunomagnetic separation procedure as devised by Baxter Inc.). "CellPro' or "Baxter' techniques gave similar results in terms of nucleated, CD34/45+ and progenitor cell concentration; however, the yield of CD34/45+ cells in the CD34+ enriched fraction was significantly higher when using the "CellPro' technique. We also found significantly higher numbers of CD34/45+ cells in the CD34+ enriched final fraction when using only one, 1.080, Percoll density gradient in the first separation step. Using one density separation step followed by the "CellPro' technique, we obtained an average of 3 x 10(6) purified CD34/45+ cells from samples containing 8.5 x 10(8) nucleated cells. Granulomonocytic progenitors (CFU-GM) and mixed progenitors (CFU-GEMM) cells from light-density and purified CD34/45+ cell fractions were evaluated. We found that 20-30% of the light-density cells and the purified CD34/45+ cells, yielded a granulomonocytic colony in serum free medium in the presence of interleukins 3 and 6, erythropoietin, granulomonocytic and granulocytic colony-stimulating factors and stem cell factor. The addition of tumour-necrosis factor alpha to the cocktail significantly improved the growth of CFU-GEMM allowing 10% of the purified CD34/45+ cells to yield a mixed colony, which confirms the role of this cytokine on CD34+ cells from cord blood. This study provides an improved method for recovery of CD34/45+ purified cells and their colony formation. These methods may serve as a basis for studies on CD34/45+ cell amplification and gene transfer.

Multicentre European study comparing selection techniques for the isolation of CD34+ cells

“…Only five experiments were performed with the Isolex 50 magnetic beads where the purities fell within the reported range. 31,32 All centres using the MiniMACS columns obtained good yields in agreement with the findings of other investigators. 12,[28][29][30]33,34 Though yields from Dynabeads and the Ceprate columns varied widely, those of the Ceprate columns were within the range reported in other studies.…”

Isolation and Characterization of Human Clonogenic Osteoblast Progenitors Immunoselected from Fetal Bone Marrow Stroma Using STRO-1 Monoclonal Antibody

“…However, a distinct advantage of MACS is that it ensures high percentage viablity after cell sorting as it reduces the duration that the cells are manipulated. (46)(47)(48)(49) This was critical because the sorted cells needed to be cultured subsequentially. In addition to these isolation steps, the immunoselected cells were immortalized and then cloned.…”

Comparison of CD34+ bone marrow cells purified by immunomagnetic and immunoadsorption cell separation techniques

“…Until now, all teams who compared different selection techniques used different samples for each technique. 4,5 Thus, in the present study, the same bone marrow sample was used simultaneously for both CD34 + cell separation methods while, in addition, the expansion capacity of the selected cells was investigated in liquid cultures. …”