Cell and Tissue Research

, Volume 375, Issue 2, pp 437–449 | Cite as

Generation of clinical-grade red blood cells from human umbilical cord blood mononuclear cells

  • Suneel Rallapalli
  • Soma Guhathakurta
  • Shalini Narayan
  • Dillip Kumar Bishi
  • Venkatesh Balasubramanian
  • Purna Sai KorrapatiEmail author
Regular Article


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.


Erythropoiesis Umbilical cord blood Red blood cells Biosafety Xeno-free production 





Umbilical cord blood


Mononuclear cells



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.

Supplementary material

441_2018_2919_MOESM1_ESM.jpg (437 kb)
Supplementary Fig 1 Cell recovery and purity was determined on samples obtained from the UCB before (pre) and after volume reduction (post) using differential cell counts coupled with flow cytometric analysis of specimens stained with 7-amino-actinomycin D (7AAD) and CD45+ cells. Top panel: FACS plots of unfractionated cord blood (a, b and c). A gate was placed around the population of interest (a) and the events selected were then sent to a 7-AAD /SSC plot to discriminate the live and dead cells (b). The selected even were then sent to a CD45 + /SSC plot, where viable CD45 + cells were counted (c). Bottom panel: FACS plots of MNCs obtained after density gradient centrifugation. The same gating strategy was applied for viable CD45+ cell counting in MNCs fraction (d, e and f). (JPG 436 kb)
441_2018_2919_MOESM2_ESM.jpg (42 kb)
Supplementary Fig 2 Flow cytometry results using anti– HbF antibody. The high HbF content of cultured RBCs (~80%) with bright fluorescence, corresponding to HbF, allowed discrimination between day 21 ex vivo generated RBCs (a) and essentially HbF negative adult peripheral blood RBCs (b) . (JPG 41 kb)


  1. Boehm D, Murphy WG, Al-Rubeai M (2010) The effect of mild agitation on in vitro erythroid development. J Immunol Methods 360:20–29. CrossRefPubMedGoogle Scholar
  2. Brune T, Garritsen H, Witteler R et al (2002) Autologous placental blood transfusion for the therapy of anaemic neonates. Biol Neonate 81:236–243. CrossRefPubMedGoogle Scholar
  3. Chandrashekar S, Kantharaj A (2014) Legal and ethical issues in safe blood transfusion. Indian J Anaesth 58:558–564. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Cheung JOP, Casals-Pascual C, Roberts DJ, Watt SM (2007) A small-scale serum-free liquid cell culture model of erythropoiesis to assess the effects of exogenous factors. J Immunol Methods 319:104–117. CrossRefPubMedGoogle Scholar
  5. Christensen RD, Jopling J, Henry E, Wiedmeier SE (2008) The erythrocyte indices of neonates, defined using data from over 12,000 patients in a multihospital health care system. J Perinatol 28:24–28. CrossRefPubMedGoogle Scholar
  6. Constantino BT (2011) The red cell histogram and the dimorphic red cell population. Lab Med 42:300–308. CrossRefGoogle Scholar
  7. Crauste F, Pujo-Menjouet L, Génieys S et al (2008) Adding self-renewal in committed erythroid progenitors improves the biological relevance of a mathematical model of erythropoiesis. J Theor Biol 250:322–338. CrossRefPubMedGoogle Scholar
  8. de Lima M, McMannis J, Gee A et al (2008) Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial. Bone Marrow Transplant 41:771–778. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Drake JR, Fitch CD (1980) Status of vitamin E as an erythropoietic factor. Am J Clin Nutr 33:2386–2393CrossRefPubMedGoogle Scholar
  10. Duran JM, Taghavi S, George JC (2012) The need for standardized protocols for future clinical trials of cell therapy. Transl Res 160:399–410. CrossRefPubMedGoogle Scholar
  11. England SJ, McGrath KE, Frame JM, Palis J (2011) Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus. Blood 117:2708–2717. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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. CrossRefPubMedGoogle Scholar
  13. Giarratana M-C, Kobari L, Lapillonne H et al (2005) Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol 23:69–74. CrossRefPubMedGoogle Scholar
  14. Giarratana M-C, Rouard H, Dumont A et al (2011) Proof of principle for transfusion of in vitro-generated red blood cells. Blood 118:5071–5079. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Giarratana MC, Marie T, Darghouth D, Douay L (2013) Biological validation of bio-engineered red blood cell productions. Blood Cells Mol Dis 50:69–79. CrossRefPubMedGoogle Scholar
  16. 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. CrossRefPubMedGoogle Scholar
  17. 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
  18. Hiroyama T, Miharada K, Kurita R, Nakamura Y (2011) Plasticity of cells and ex vivo production of red blood cells. Stem Cells Int 2011:195780. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Icardi A, Paoletti E, De Nicola L et al (2013) Renal anaemia and EPO hyporesponsiveness associated with vitamin D deficiency: the potential role of inflammation. Nephrol Dial Transplant 28:1672–1679CrossRefPubMedGoogle Scholar
  20. 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. CrossRefPubMedGoogle Scholar
  21. Jin H, Kim H-S, Kim S, Kim HO (2014) Erythropoietic potential of CD34+ hematopoietic stem cells from human cord blood and G-CSF-mobilized peripheral blood. Biomed Res Int 2014:435215. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Khodabux CM, von Lindern JS, van Hilten JA et al (2008) A clinical study on the feasibility of autologous cord blood transfusion for anemia of prematurity. Transfusion 48:1634–1643. CrossRefPubMedGoogle Scholar
  23. Kögler G, Sensken S, Airey JA et al (2004) A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 200:123–135. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lahoti V, Murphy W, Al-Rubeai M (2012) Mathematical approach for the optimal expansion of erythroid progenitors in monolayer culture. J Biotechnol 161:308–319. CrossRefPubMedGoogle Scholar
  25. Lapillonne H, Kobari L, Mazurier C et al (2010) Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica 95:1651–1659. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Leberbauer C, Boulmé F, Unfried G et al (2005) Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood 105:85–94. CrossRefPubMedGoogle Scholar
  27. Lim M, Panoskaltsis N, Ye H, Mantalaris A (2011) Optimization of in vitro erythropoiesis from CD34+ cord blood cells using design of experiments (DOE). Biochem Eng J 55:154–161. CrossRefGoogle Scholar
  28. Lu S-J, Feng Q, Park JS et al (2008) Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood 112:4475–4484. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Moore DC, Carter DL, Bhandal AK, Studzinski GP (1991) Inhibition by 1,25 dihydroxyvitamin D3 of chemically induced erythroid differentiation of K562 leukemia cells. Blood 77:1452–1461PubMedGoogle Scholar
  30. Mountford JC, Turner M (2011) In vitro production of red blood cells. Transfus Apher Sci 45:85–89. CrossRefPubMedGoogle Scholar
  31. Neildez-Nguyen TMA, Wajcman H, Marden MC et al (2002) Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotechnol 20:467–472. CrossRefPubMedGoogle Scholar
  32. Pranke P, Hendrikx J, Alespeiti G et al (2006) Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount-BD and ISHAGE protocols. Braz J Med Biol Res 39:901–906. CrossRefPubMedGoogle Scholar
  33. Ramesh B, Guhathakurta S (2013) Large-scale in-vitro expansion of RBCs from hematopoietic stem cells. Artif Cells Nanomed Biotechnol 41:42–51. CrossRefPubMedGoogle Scholar
  34. Rosen CJ, Ackert-Bicknell C, Rodriguez JP, Pino AM (2009) Marrow fat and the bone microenvironment: developmental, functional, and pathological implications. Crit Rev Eukaryot Gene Expr 19:109–124. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Rubinstein P, Dobrila L, Rosenfield RE et al (1995) Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 92:10119–10122. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Saraiva BCA, Soares MCC, Dos Santos LC et al (2014) Iron deficiency and anemia are associated with low retinol levels in children aged 1 to 5 years. J Pediatr 90:593–599. CrossRefGoogle Scholar
  37. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Stocchi V, Cucchiarini L, Magnani M, Fornaini G (1987) Adenine and pyridine nucleotides in the erythrocyte of different mammalian species. Biochem Int 14:1043–1053PubMedGoogle Scholar
  39. Taguchi T, Suita S, Nakamura M et al (2003) The efficacy of autologous cord-blood transfusions in neonatal surgical patients. J Pediatr Surg 38:604–607. CrossRefPubMedGoogle Scholar
  40. Timmins NE, Nielsen LK (2011) Manufactured RBC - rivers of blood, or an oasis in the desert? Biotechnol Adv 29:661–666. CrossRefPubMedGoogle Scholar
  41. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 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. CrossRefPubMedGoogle Scholar
  43. 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
  44. Wang J, Kimura T, Asada R et al (2003) SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection. Blood 101:2924–2931CrossRefPubMedGoogle Scholar
  45. Xi J, Li Y, Wang R et al (2013) In vitro large scale production of human mature red blood cells from hematopoietic stem cells by coculturing with human fetal liver stromal cells. Biomed Res Int 2013:807863. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Xie X, Li Y, Pei X (2014) From stem cells to red blood cells: how far away from the clinical application? Sci China Life Sci 57:581–585. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Suneel Rallapalli
    • 1
  • Soma Guhathakurta
    • 2
  • Shalini Narayan
    • 2
  • Dillip Kumar Bishi
    • 2
  • Venkatesh Balasubramanian
    • 2
  • Purna Sai Korrapati
    • 1
    Email author
  1. 1.Biological Material Laboratory, CSIR-Central Leather Research InstituteChennaiIndia
  2. 2.Department of Engineering Design, IIT MADRASChennaiIndia

Personalised recommendations