title: Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture.
abstract: OBJECTIVE
  Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control.
  
  
  METHODS
  CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers.
  
  
  RESULTS
  The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state.
  
  
  CONCLUSIONS
  Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps.
authors.affiliation: Healthcare Engineering Research Group, Centre for Biological Engineering, Wolfson School of Mechanical and Manufacturing Engineering, Loughborough University, Loughborough, Leicestershire, UK.
authors.forename: Katie E
authors.initials: KE
authors.lastname: Glen
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Workman
authors.forename: Forhad
authors.initials: F
authors.lastname: Ahmed
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Ratcliffe
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Stacey
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Thomas
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D016923:Cell Death / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D004912:Erythrocytes / Q000378:metabolism / Q000502:physiology*; D005312:Fetal Blood / Q000378:metabolism / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism*
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D051880:Receptors, Notch; C064045:delta protein; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
keywords: bio-process; erythrocytes; ex vivo; hematopoiesis; manufacture; metabolism