Characterization of Hematopoietic Cell Expansion, Oxygen Uptake, and Glycolysis in a Controlled, Stirred-Tank Bioreactor System

Collins, Paul C.; Nielsen, Lars K.; Patel, Sanjay; Papoutsakis, E. T.; Miller, William M.

doi:10.1021/bp980032e

Cited by 71 publications

(50 citation statements)

References 33 publications

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“…Collins et al achieved a high density of cells (6.0 · 10 6 cells/mL) in a stirred tank bioreactor; however, the generated RBCs were not viable. 11 Boehm et al cultivated erythroblasts at low agitation speeds; however, the cell density was low, and cells were not cultured in a 3D system. 12 In our method, terminal erythroid maturation was achieved by 3D culture in a spinner flask; however, erythroid cells were extremely weak owing to their exposure to shear stress, and some cells leaked from the microcarriers.…”

Section: Discussionmentioning

confidence: 99%

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

Lee

Han

Choi

et al. 2015

Tissue Engineering Part A

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Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34 + cells over 15 days and then allowed to form tight aggregates at a minimum density of 1 · 10 7 cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 mm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

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Section: Discussionmentioning

confidence: 99%

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

Lee

Han

Choi

et al. 2015

Tissue Engineering Part A

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“…Based on reports that addition of fresh media or cytokines (Collins et al, 1998b;McNiece et al, 2000b;Paquette et al, 2002) increases expansion of cultured HPC, and increasing culture duration results in a more mature cell population (Paquette et al, 2002), we developed a robust dilution feeding strategy over 15 days for ex vivo expansion of neutrophils. Following magnetic isolation of CD34 þ cells from UCB, cultures were initiated at a density of 1 Â 10 4 CD34…”

Section: Ex Vivo Expansion Of Neutrophils From Ucb Cd34 R Cellsmentioning

confidence: 99%

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Timmins

Palfreyman

Marturana

et al. 2009

Biotech & Bioengineering

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Dose-intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum-free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800-fold expansion in cell number was achieved for UCB, and up to 4,000-fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10-fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave-type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients.

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“…Nonetheless, many attempts have been made to scale up HSC cultures over the past twenty years, aiming at increasing the number of useful stem cells obtained from limited quantities collected, for cell therapy and transplantation following chemotherapy. A wide range of bioreactors have been tested: airlift bioreactors [71], spinner flasks [69,71,[88][89][90][91][92] and stirred bioreactors [93,94], hollow-fibers [71], fixed-bed reactors with microcarriers [91,95,96] and rotating wall vessel reactors [97,98]. The data have been compared [76,98], with best results (total cell expansion up to 435-fold, 32-fold expansion of CD34 + cells and 21-fold expansion of colony forming unit-granulocyte/macrophages (CFU-GM)) for stirred and rotating wall vessel reactors, far beyond the yields in static flasks.…”

Section: State-of-the Art In Large-scale Blood Productionmentioning

confidence: 99%

Large‐scale production of red blood cells from stem cells: What are the technical challenges ahead?

Rousseau

Giarratana

Douay

2013

Biotechnology Journal

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Blood-transfusion centers regularly face the challenge of donor blood shortages, especially for rare blood groups. The possibility of producing universal red blood cells from stem cells industrially has become a possible alternative since the successful injection of blood generated in vitro into a human being in 2011. Although there remains many biological and regulatory issues concerning the efficacy and safety of this new product, the major challenge today for future clinical applications is switching from the current limited 2-dimensional production techniques to large-scale 3-dimensional bioreactors. In addition to requiring technological breakthroughs, the whole process also has to become at least five-fold more cost-efficient to match the current prices of high-quality blood products. The current review sums up the main biological advances of the past decade, outlines the key biotechnological challenges for the large-scale cost-effective production of red blood cells, proposes solutions based on strategies used in the bioindustry and presents the state-of-the-art of large-scale blood production.

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Characterization of Hematopoietic Cell Expansion, Oxygen Uptake, and Glycolysis in a Controlled, Stirred-Tank Bioreactor System

Cited by 71 publications

References 33 publications

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Large‐scale production of red blood cells from stem cells: What are the technical challenges ahead?

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