Spatiotemporal Mapping of Erythroid, Stromal, and Osteogenic Niche Formation to Support Physiologic Red Cell Production in a Three-Dimensional Hollow Fibre Perfusion Bioreactor

摘要: Current in vitro human erythroid culture platforms require abnormally high cytokine supplementation and use lower cell density ( 107/mL) when perfused with cytokine-free media long-term culture. In order to study the role of this manufactured HFBR microenvironment on spatiotemporal physiologic erythropoiesis, we now extend our previous reports by implementing 5-fold less concentrations than those used typical ex vivo erythropoietic cultures. Herein, show a >107 red harvest from over 28-days spontaneous expansion stromal cells, maintenance progenitor pools, formation niches defined areas within structure differential situ production 23 growth factors varying time. The 5.25 mL scaffold was inoculated 108 CBMNCs HFs were rapidly (20 mL/h) serum-free StemSpan medium gradually supplemented gradient decreasing SCF (50 - 0 ng/mL) increasing EPO (0 0.3 U/mL) 28 days maintain cells whilst inducing erythropoiesis. Quantitative confocal microscopy analyses sections demonstrated that DAPI+ maintained (>107/mL), viability (>80%), while more 107 enucleated filtered through 28-day Inside HFBR, hematopoietic (total 3.1∙106 CD34+ 5.5∙106 CKIT+ MNCs) expanded across various stages maturation (28-day total increase 1.2∙107 EPOR+, 1.8∙107 CD71+, 2.3∙107 CD235a+ MNCs); CD235a+mature phenotypes enriched 10-fold HF filtrate days. Stromal differentiated during mesenchymal stem marker Stro-1 (2.2∙107 cells), pre-osteoblast osterix (OSx; 1.6∙107 mature osteoblast osteopontin (OPN; 0.5∙107 cells). Expression collagen-1, fibronectin, laminin-2 detected microscopy, enzyme-linked immunoassays multilineal, unsupplemented profiles including interleukins produced primarily day 0-12 (IL-6, IL-10, IL-21) as well colony stimulating which increased 20-28 (G-CSF, GM-CSF, EGF, VEGF, Ang-2, PDGF, FGF-β). Using novel computational have developed, DAPI+MNCs found self-associate into expanding 50-500µm clusters throughout local 10-20 fold, representing niche-like areas. At 14 28, MNCs formed clustered far expressed hypoxic (HIF1a, PIMO), stromal, markers (Stro-1, OSx, laminin-2, VCAM-1, CD45, EPOR: >1400µm HFs). 3-fold MNC near comprised (CD45, CD34, CKIT, CD235a, CD71: Our data suggested dense inoculation platform using enabled simultaneous differentiation erythroid, osteogenic lineages, generation an inductive environment. This environment multilineal progenitors, erythrocytes, generated support situ, interactive could be quantitatively mapped zones. developed may represent physiologically-relevant system erythropoiesis potentially provide for translational protocols. Disclosures No relevant conflicts interest declare.