Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three‐dimensional culture system

Abstract

One of the key factors critical for a successful human cord blood transplantation in treating patients with hematopoietic disorders is the number of hematopoietic stem/progenitor cells derived from human cord blood. Here, we report an alginate three-dimensional (3D) culture system for the expansion of CD34+ cells in cord blood mononuclear cells (CBMCs). Cord blood mononuclear cells were isolated from human cord blood and encapsulated in 3D alginate beads. The cells were grown with different concentrations of cytokines. At day 0, 3, 6, 9, and 12, respectively, the percentage of CD34+ cells was quantified by flow cytometry. Colony-forming cell assay was performed to determine the potential of hematopoietic reconstruction of the amplified cells under the 3D culture system. After culturing for 12 days, the CBMCs encapsulated in the 3D alginate beads were amplified 5.89 ± 0.72 fold, CD34+ cells increased from 2.60 ± 0.52% to 13.27 ± 2.65%, and the colony-forming assay showed that the colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) increased from 363.34 ± 34.47/10⁵ cells to 3423.33 ± 645.14/10⁵ cells (P < 0.001). In comparison, the conventional two-dimensional (2D) culture system showed that the CBMCs, CD34+ cells and the CFU-G/GM were 0.68 ± 0.16 fold, 0.45 ± 0.17%, and 532.92 ± 82.97/10⁵ cells, respectively. This study demonstrates a new and efficient method to amplify the CD34+ human cord blood hematopoietic stem/progenitor cells in a 3D alginate culture system ex vivo for extended periods while retaining the hematopoietic reconstruction capacity.