Neutrophils produced in bioreactor
Neutrophils produced in bioreactor

Project keywords

Cell and Tissue Engineering, Systems Biology, Systems Biotechnology, Manufacturing, Health, Blood, Cancer, Stem Cells

Project summary

Millions of people are saved every year through the transfusion of blood products. Supported by the generosity of donors, transfussion of blood products demonstrate that inexpensive, generic cell therapies are possible. However, some blood cells are difficult to obtain from donors and this project develops bioprocesses suitable for routine clinical scale manufacture of blood cells from haematopoietic progenitor cells (HPC).

Ex vivo manufacture of neutrophils

Many anti-cancer drugs are so toxic that the body's blood generating tissues are severely damaged. When this happens, the number of infection fighting neutrophils (a type of white blood cell) can become so low that life-threatening fungal and bacterial infections occur. This condition is known as neutropaenia, and is a major cause of chemotherapy associated morbidity and mortality. By manufacturing neutrophils ex vivo, and giving these to chemotherapy patients, it may be possible to prevent neutropaenia and associated infections.

Dose-intensive chemotherapy results in an obligatory period of neutropaenia (less than 0.1 x 109 neutrophils/L of whole blood), during which patients are at high risk of infection. While transfusion support with donor neutrophils appears to be an obvious solution to the problem (analogous to red blood cell and platelet transfusions for anaemia and thrombocytopaenia), obtaining sufficiently large numbers of neutrophils for efficacious dosing is challenging. The limited number of neutrophils that can be obtained from a single donor, combined with an inability to store neutrophil collections, results in a single dose/donation situation at best. Neutrophil transfusion is practised in only a few specialist centres around the world, and only in a therapeutic setting. Ideally, neutrophils should be provided as infection prophylaxis to all patients undergoing chemotherapy associated with severe neutropaenia.

Several research groups have trialed the administration of ex vivo expanded haematopoietic progenitor cells in an attempt to accelerate recovery of endogenous neutrophil production. While clinical benefit was obtained, this approach does not address the imbalance between supply and demand. Precursor cells retain the ability to differentiate into a variety blood cell types, some of which are linked to immunological rejection and graft-versus-host disease (GVHD). As such these products can only be used in an autologous setting, or for recipients that are immunologically matched and receiving immunosuppressive treatments. Thus, a single HPC donation can provide only a single dose of transfusable precursor cells.

In a combination of the two approaches described above (transfusion of donor derived neutrophils and ex vivo expansion), we have developed a bioprocess for ex vivo production of a mature neutrophil product. A readily available supply of neutrophils could enable routine administration as infection prophylaxis in chemotherapy patients. Routine production of neutrophils for clinical use depends on the availability of cultivation system capable of operating at 10’s to 100’s of litres. By screening multiple culture parameters, we developed and optimized a process allowing us to conduct multi litre neutrophil cultures in Wave bioreactors. We are able to obtain cell yields equivalent to as many 20 – 40 doses of neutrophils from a single HPC donation. The process itself is amenable to routine production of a therapeutic product and has been demonstrated at volumes up to 10 L. This is the first ever demonstration of clinical scale manufacture of neutrophils from HPC. The process is currently undergoing pre-clinical evaluation at the Centre for Commercialization of Regenerative Medicine (CCRM) in Toronto.

High efficiency generation of RBC from HPC

Increasing demand for transfusable red blood cells is paralleled by increasingly stringent donor exclusion criteria and prevalence of blood borne disease, placing pressures on global blood supply. Logistical considerations and donor distribution further hinder supply, with only 45% of whole blood donations collected in regions accounting for 80% of world population. It has been proposed for some time that ex vivo manufactured RBC could provide a secure and safe alternative to donor RBC. While in principle this is true, the numbers of cells that are required to have any significant impact are extremely large. A number of processes have been developed for the generation of RBC from umbilical cord blood derived HPC, but the methods used yield only several transfusion units per donation at best, and are not well suited to routine manufacture. In order to have any meaningful impact on global blood supply, it would be necessary to generate several hundreds of units of RBC from a single umbilical cord blood donation.

We are developing high efficiency culture methods for production of very large numbers of RBC from umbilical blood derived HPC. We have developed a culture process capable of generating over 200 RBC units/donation. In conjunction with the development of advanced bioprocess technologies, these methods could enable large scale manufacture of RBC. While it will be many years before we see ex vivo manufactured RBC replacing routine donor blood transfusion used in the clinic, development of a high yield process may find application in the supply of rare blood types.


Schwaber JL, Brunck ME, Levesque JP, Nielsen LK (2016) Filling the void: allogeneic myeloid cells for transplantation. Current Opinion in Hematology 23: 72-7. PMID: 26554894

Clark MB, Mercer TR, Bussotti G, Leonardi T, Haynes KR, Crawford J, Brunck ME, Cao KAL, Thomas GP, Chen WY, Taft RJ, Nielsen LK, Enright AJ, Mattick JS, Dinger ME (2015) Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing. Nature Methods 12: 339-342. PMID: 25751143

Mercer TR, Clark MB, Andersen SB, Brunck ME, Haerty W, Crawford J, Taft RJ, Nielsen LK, Dinger ME, Mattick JS (2015) Genome-wide discovery of human splicing branchpoints. Genome Res 25: 290-303. PMID: 25561518

Brunck MEG, Andersen SB, Timmins NE, Osborne GW, Nielsen LK (2014) Absolute counting of neutrophils in whole blood using flow cytometry. Cytometry 85: 1057-64. PMID: 24995861.

Brunck MEG, Nielsen LK (2014) Next generation cell therapies to prevent infections in neutropenic patients. Stem cells translational medicine 3: 541-548. PMID: 24598780

Peatey CL, Watson JA, Trenholme KR, Brown CL, Nielsen LK, Guenther M, Timmins NE, Watson GS, Gardiner DL (2013) Enhanced gametocyte formation in erythrocyte progenitor cells: a site specific adaptation by Plasmodium falciparum. J Infect Dis 208: 1170-1174. PMID: 23847056

Doran M, Aird I, Marturana F, Timmins NE, Atkinson K, Nielsen LK (2012) Bioreactor for Blood Production. Cell Transplantation 21: 1235-1244. PMID: 22405378

Timmins NE, Nielsen LK (2012) Large scale manufacture of blood cells. In N. Jenkins, N Barron, PM Alves (Ed.) Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), Dublin, Ireland, June 7-10, 2009 (pp. 557-571) Dordrecht, Netherlands: Springer.

Timmins NE, Athanasas S, G?nther M, Buntine P, Nielsen LK (2011) Ultra high yield manufacture of red blood cells from hematopoietic stem cells. Tissue Engineering, Part C 17: 1131-7. PMID: 21736478.

Timmins NE, Nielsen LK (2011) Manufactured RBC - rivers of blood, or an oasis in the desert? Biotechnology Advances 29: 661-6. PMID: 21609758.

Marturana F, Timmins NE, Nielsen LK (2011) Short term exposure of umbilical cord blood CD34+ cells to GM-CSF early in culture improves ex vivo expansion of neutrophils. Cytotherapy 13: 366-77. PMID: 20860426.

Osiecki M, Ghanavi P, Atkinson K, Nielsen LK, Doran M (2010) The Ascorbic Acid Paradox. Biochemical and Biophysical Research Communications 400: 466-470. PMID: 20732307

Doran MR, Markway B, Clark A, Athanasas-Platsis S, Brooke G, Atkinson K, Nielsen LK, Cooper-White JJ (2010) Membrane Bioreactors Enhance Microenvironmental Conditioning and Tissue Development. Tissue Engineering Part C: Methods. 16:407-415. PMID: 19622005.

Timmins NE, Palfreyman E, Marturana F, Dietmair S, Luikenga S, Lopez G, Fung YL, Minchinton R, Nielsen LK (2009) Clinical Scale Ex vivo Manufacture of Neutrophils from Hematopoietic Progenitor Cells. Biotechnol Bioeng 104:832-40. PMID: 19591208.

Timmins NE, Nielsen LK (2009) Blood cell manufacture: current methods and future challenges. Trends Biotechnol 27:415-422. PMID: 19500866.

Doran MR, Markway BD, Aird IA, Rowlands AS, George PA, Nielsen LK, Cooper-White JJ (2009) Surface-bound stem cell factor and the promotion of hematopoietic cell expansion. Biomaterials 30:4047-52. PMID: 19481255.

Hines M, Nielsen LK, Cooper-White J (2008) The hematopoietic stem cell niche: what are we trying to replicate? Journal of Chemical Technology and Biotechnology 83: 421-443. doi: 10.1002/jctb.1856

Peng JC, Hyde C, Pai S, O’Sullivan BJ, Nielsen LK, Thomas R (2006) Monocyte-derived DC primed with TLR agonists secrete IL-12p70 in a CD40-dependent manner under hyperthermic conditions. Journal of Immunotherapy 29:606-615.

Peng JC, Abu Bakar S, Richardson MM, Jonsson JJ, Frazer IH, Nielsen LK, Morahan G, Thomas R (2006) IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS. Immunology and Cell Biology 84: 227-232. doi:10.1111/j.1440-1711.2006.01419.x

Peng JC, Thomas R, Nielsen LK (2005) Generation and maturation of DC for clinical application under serum-free conditions. Journal of Immunotherapy 28:599-609.

Project contacts

Lead investigator Professor Lars Nielsen
Research group AIBN Systems & Synthetic Biology
Contact email


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