Generation of clinical-grade red blood cells from human umbilical cord blood mononuclear cells
A xeno-free method for ex vivo generation of red blood cells (RBCs) is attempted in order to replicate for large-scale production and clinical applications. An efficient milieu was formulated using injectable drugs substituting the animal-derived components in the culture medium. Unfractionated mononuclear cells isolated from human umbilical cord blood were used hypothesizing that the heterogeneous cell population could effectively contribute to erythroid cell generation. The strategy adopted includes a combination of erythropoietin and other injectable drugs under low oxygen levels, which resulted in an increase in the number of mature RBCs produced in vitro. The novelty in this study is the addition of supplements to the medium in a stage-specific manner for the differentiation of unfractionated umbilical cord blood mononuclear cells (MNCs) into erythropoietic lineage. The erythropoietic lineage was well established by day 21, wherein the mean cell count of RBCs was found to be 21.36 ± 0.9 × 108 and further confirmed by an upregulated expression of CD235a+ specific to RBCs. The rationale was to have a simple method to produce erythroid cells from umbilical cord blood isolates in vitro by mitigating the effects of multiple erythroid-activating agents and batch to batch variability.
KeywordsErythropoiesis Umbilical cord blood Red blood cells Biosafety Xeno-free production
Umbilical cord blood
The authors would like to acknowledge Dr.Asra, Dr. Chandra and Mr. Fida Hussain for their kind help.
Source of funding
A part of this work is funded by the Department of Biotechnology (DBT), Govt. of India (Ref. No. BT/PR 5729/PID/6/675/2012).
Compliance with ethical standards
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Conflict of interest
The authors declare that they have no conflict of interest.
- Garritsen HSP, Brune T, Louwen F et al (2003) Autologous red cells derived from cord blood: collection, preparation, storage and quality controls with optimal additive storage medium (Sag-mannitol). Transfus Med 13:303–310. https://doi.org/10.1046/j.1365-3148.2003.00457.x CrossRefPubMedGoogle Scholar
- Glen KE, Workman VL, Ahmed F et al (2013) Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture. Cytotherapy 15:1106–1117. https://doi.org/10.1016/j.jcyt.2013.04.008 CrossRefPubMedGoogle Scholar
- Haiden N, Klebermass K, Cardona F et al (2006) A randomized, controlled trial of the effects of adding vitamin B12 and folate to erythropoietin for the treatment of anemia of prematurity. Pediatrics 118Google Scholar
- Indumathi S, Harikrishnan R, Rajkumar JS, Dhanasekaran M (2015) Immunophenotypic comparison of heterogenous non-sorted versus sorted mononuclear cells from human umbilical cord blood: a novel cell enrichment approach. Cytotechnology 67:107–114. https://doi.org/10.1007/s10616-013-9663-2 CrossRefPubMedGoogle Scholar
- Sawada K, Krantz SB, Dessypris EN et al (1989) Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development. J Clin Invest 83:1701–1709. https://doi.org/10.1172/JCI114070 CrossRefPubMedPubMedCentralGoogle Scholar
- van den Akker E, Satchwell TJ, Pellegrin S et al (2010) The majority of the in vitro erythroid expansion potential resides in CD34(−) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples. Haematologica 95:1594–1598. https://doi.org/10.3324/haematol.2009.019828 CrossRefPubMedPubMedCentralGoogle Scholar
- Vlaski M, Lafarge X, Chevaleyre J et al (2009) Low oxygen concentration as a general physiologic regulator of erythropoiesis beyond the EPO-related downstream tuning and a tool for the optimization of red blood cell production ex vivo. Exp Hematol 37:573–584. https://doi.org/10.1016/j.exphem.2009.01.007 CrossRefPubMedGoogle Scholar
- von Lindern M, Zauner W, Mellitzer G et al (1999) The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro. Blood 94:550–559Google Scholar