Ex vivo expansion of cord blood hematopoietic stem/progenitor with support of microencapsulated mesenchymal stem cells in rotating wall vessel

Coculture of cord blood mononuclear cells (CB MNCs) and alginate-chitosan-alginate (ACA) microcapsules containing rabbit bone marrow (BM) mesenchymal stem cells (MSCs) was carried out in a rotating wall vessel (RWV) bioreactor for 7 days to construct a novel dynamic coculture system. The medium was supplemented with conventional doses of purified recombinant human cytokines (SCF 50 ng·mL-1, FL 50 ng·mL-1, TPO 50 ng·mL-1 and IL-3 25 ng·mL-1) without any serum. The total nucleated cell density, pH and osmolality of the culture medium in the bioreactor were measured every 24 hours. Flow cytometric assay for CD34+ cells and methylcellulose colony assays (colony-forming units in culture, CFU-Cs) were carried out at Oh, 72 h and 168 h, respectively. Results show that the pH of the medium in the bioreactor maintains between 7.2-7.4 and the osmolality is between 280-310 mmOsmol·kg-1, both of which are in the proper ranges for ex vivo expansion of hematopoietic stem cells (HSCs) and progenitors during the dynamic coculture. And at the end of 168 h culture, the total nucleated cell number is multiplied by 107.05±6.08 fold, CD34+ cells 26.52·1.5 fold, and CFU-Cs 19.2±3.18 fold. This novel dynamic microencapsulated coculture system supports the large-scale ex vivo expansion of HSCs and progenitors. In this system, the stromal cells inhibit the HSCs differentiation, the microcapsules protect cells from immunreaction and the RWV provides a better suspension culture environment with low shear force for cells and microcapsules.