Ex vivo expansion of cord blood hematopoietic stem/progenitor with support of microencapsulated mesenchymal stem cells in rotating wall vessel
Liu Y., Liu TQ., Fan XB., Liu MY., Zhao GF., Bao CY., Ma XH., Cui ZF.
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.