A 3D biomimetic model for in vitro studies of pancreatic cancer.
A great challenge when conducting ex vivo studies of leukaemia is the construction of an appropriate experimental platform that would recapitulate the bone marrow (BM) environment. Such a 3D scaffold system has been previously developed in our group [1]. Additionally to the BM architectural characteristics, parameters such as oxygen and glucose concentration are crucial as their value could differ between patients as well as within the same patient at different stages of treatment, consequently affecting the resistance of leukaemia to chemotherapy. The effect of oxidative and glucose stress-at levels close to human physiologic ones-on the proliferation and metabolic evolution of an AML model system (K-562 cell line) in conventional 2D cultures as well as in 3D scaffolds were studied. We observed that the K-562 cell line can proliferate and remain alive for 2 weeks in medium with glucose close to physiological levels both in 20 and 5% O2. We report interesting differences on the cellular response to the environmental, i.e., oxidative and/or nutritional stress stimuli in 2D and 3D. Higher adaptation to oxidative stress under non-starving conditions is observed in the 3D system. The glucose level in the medium has more impact on the cellular proliferation in the 3D compared to the 2D system. These differences can be of significant importance both when applying chemotherapy in vitro and also when constructing mathematical tools for optimisation of disease treatment.
No abstract
Current in vitro human erythroid culture platforms require abnormally high cytokine supplementation and use lower cell density (<106/mL) compared with that present in bone marrow during physiologic erythropoiesis. These in vitro culture conditions limit extracellular interactions, are not dynamic, and exhaust stem and progenitor cell pools, thereby limiting culture longevity. We have developed a three-dimensional hollow fibre perfusion bioreactor (HFBR) comprised of a collagen-coated polyurethane scaffold which surrounded ceramic hollow fibres (HFs) and expanded dense inocula of human umbilical cord blood (CB) mononuclear cells (MNCs; >107/mL) when perfused with cytokine-free media in 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 cytokine concentrations than those used in typical ex vivo erythropoietic cultures. Herein, we show a >107 red cell harvest from the HFBR culture over 28-days with spontaneous expansion of stromal cells, maintenance of erythroid progenitor pools, and formation of stromal and erythroid cell niches in defined areas within the HFBR structure with differential in situ production of 23 growth factors varying over time. The 5.25 mL HFBR scaffold was inoculated with 108 CBMNCs and HFs were rapidly perfused (20 mL/h) with serum-free StemSpan medium gradually supplemented with a cytokine gradient of decreasing SCF (50 - 0 ng/mL) and increasing EPO (0 - 0.3 U/mL) over 28 days in order to maintain progenitor cells whilst inducing erythropoiesis. Quantitative confocal microscopy analyses of HFBR sections demonstrated that DAPI+ CBMNCs maintained high cell density (>107/mL), and high viability (>80%), while more than 107 enucleated cells were filtered through HFs over the 28-day culture. Inside the HFBR, hematopoietic progenitor cells were maintained (total of 3.1∙106 CD34+ and 5.5∙106 CKIT+ MNCs) while erythroid cells were expanded across various stages of maturation (28-day total increase of 1.2∙107 EPOR+, 1.8∙107 CD71+, and 2.3∙107 CD235a+ MNCs); CD235a+mature red cell phenotypes were enriched 10-fold in the HF filtrate over 28 days. Stromal cells expanded and differentiated during the 28-day HFBR culture with a total increase of mesenchymal stem cell marker Stro-1 (2.2∙107 cells), pre-osteoblast marker osterix (OSx; 1.6∙107 cells), and mature osteoblast marker osteopontin (OPN; 0.5∙107 cells). Expression of human collagen-1, fibronectin, and laminin-2 was detected by microscopy, while enzyme-linked immunoassays on HFBR filtrate detected 23 multilineal, unsupplemented cytokine profiles including interleukins produced primarily from day 0-12 (IL-6, IL-10, IL-21) as well as colony stimulating factors and stromal growth factors which increased in production from day 20-28 (G-CSF, GM-CSF, EGF, VEGF, Ang-2, PDGF, FGF-β). Using a novel confocal microscopy computational analyses that we have developed, DAPI+MNCs were found to self-associate into expanding 50-500µm clusters throughout the 28-day culture which increased local cell density 10-20 fold, representing niche-like areas. At day 14 and 28, MNCs formed clustered niches far from HFs which expressed hypoxic (HIF1a, PIMO), stromal, and erythroid markers (Stro-1, OSx, collagen-1, laminin-2, VCAM-1, CD45, EPOR: >1400µm from HFs). At day 28, 3-fold more MNC clusters formed near HFs and were comprised of hematopoietic progenitor and erythroid phenotypes (CD45, CD34, CKIT, CD235a, CD71: <700µm from HFs). Our data suggested that the dense inoculation of CBMNCs in a serum-free HFBR platform using physiologic concentrations of SCF and EPO enabled the long-term simultaneous differentiation of human erythroid, stromal, and osteogenic lineages, and the generation of an ex vivo erythroid inductive environment. This environment maintained multilineal progenitors, enabled harvest of mature erythrocytes, generated cytokine support in situ, and formed interactive cell niches which could be quantitatively mapped in spatiotemporal zones. The HFBR we have developed may represent a more physiologically-relevant culture system to study ex vivo erythropoiesis and could potentially provide a platform for translational cell expansion protocols. Disclosures No relevant conflicts of interest to declare.
Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.
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