BACKGROUND: In vitro generation of red blood cells (RBCs) is an important alternative to donor RBCs. It was impossible, however, to generate a large quantity of RBCs due to necessity of supporting stromal cells or xenogeneic or human serum for in vitro culture, which had restrictions in safety, supplies, and expenses. In addition, the low viability of erythroblasts during terminal maturation in vitro required highly efficient production protocols. Here, we present a protocol for mass production of clinical-grade RBCs from cord blood (CB) CD34+ cells in stroma-free culture by using poloxamer 188 (P188), a polymer known to be cytoprotective against hydrodynamic stress. STUDY DESIGN AND METHODS: Umbilical CB CD34+ cells were cultured for 21 days in medium containing several cytokines, and roughly fibrin-eliminated CB derived plasma was added from Day 13. To enhance the final RBC production, P188 was added from Day 13 and the cell count and viability were compared with controls lacking P188. RESULTS: Erythroid expansion between Day 17 and Day 21 was significantly higher in cultures treated with P188, with a mean of 1.5 times and maximum of 3.6 times higher expansion than in controls lacking P188. The enhanced survival resulted from increased stability of the RBC membrane and decreased fragility. The erythroid cells were enucleated up to 95% and demonstrated hematologic variables similar to those of reticulocytes. CONCLUSION: This RBC production protocol is a simple stroma- and serum-free culture method ensuring enhanced viability of terminally mature erythroid cells and can be easily applicable to mass production of clinical-grade RBCs as well as erythropoiesis research.
BACKGROUND: In vitro generation of red blood cells (RBCs) is an important alternative to donor RBCs. It was impossible, however, to generate a large quantity of RBCs due to necessity of supporting stromal cells or xenogeneic or human serum for in vitro culture, which had restrictions in safety, supplies, and expenses. In addition, the low viability of erythroblasts during terminal maturation in vitro required highly efficient production protocols. Here, we present a protocol for mass production of clinical-grade RBCs from cord blood (CB) CD34+ cells in stroma-free culture by using poloxamer 188 (P188), a polymer known to be cytoprotective against hydrodynamic stress. STUDY DESIGN AND METHODS: Umbilical CB CD34+ cells were cultured for 21 days in medium containing several cytokines, and roughly fibrin-eliminated CB derived plasma was added from Day 13. To enhance the final RBC production, P188 was added from Day 13 and the cell count and viability were compared with controls lacking P188. RESULTS: Erythroid expansion between Day 17 and Day 21 was significantly higher in cultures treated with P188, with a mean of 1.5 times and maximum of 3.6 times higher expansion than in controls lacking P188. The enhanced survival resulted from increased stability of the RBC membrane and decreased fragility. The erythroid cells were enucleated up to 95% and demonstrated hematologic variables similar to those of reticulocytes. CONCLUSION: This RBC production protocol is a simple stroma- and serum-free culture method ensuring enhanced viability of terminally mature erythroid cells and can be easily applicable to mass production of clinical-grade RBCs as well as erythropoiesis research.
Authors: Sandeep N Shah; Monique P Gelderman; Emily M A Lewis; John Farrel; Francine Wood; Michael Brad Strader; Abdu I Alayash; Jaroslav G Vostal Journal: PLoS One Date: 2016-12-13 Impact factor: 3.240