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Bioengineering approaches to blood cell production
namely 3D structure, topography, local stiffness, and physical constraints, as a guiding cue for controlling ex vivo survival, migration, differentiation and maturation of dif- ferent cell types.90 In 2D cultures, cells are seeded in liquid medium in a monolayer in the presence of molecules and soluble factors diluted directly in the medium, but the complexity of 3D tissues is completely lost. Currently, the best approach to try to model the 3D complexity of native human tissues ex vivo is to exploit biomaterials. Indeed, 3D environments made of fibers/nanofibers, solid scaffolds, and hydrogels have demonstrated the possibility to enhance the culture area and to induce better expansion of HSC during long-term cultures. We now discuss different approaches that have been undertaken to support erythro- poiesis and megakaryopoiesis ex vivo.
Erythropoiesis and erythrocyte production in three-dimensional cultures
The study of erythropoiesis in three dimensions remains marginal. Housler et al. customized a 3D com- partmental hollow fiber perfused bioreactor.91 The system was composed of a network of three independent bun- dles of capillaries, two of which were used for counter- current medium perfusion and the third for oxygen and carbon dioxide transport. The bioreactor enabled expan- sion of erythrocyte progenitors and enucleation of ery- throblasts. In an effort to try to maximize enucleation and recapitulate erythroblastic islands ex vivo, Lee et al. seeded late erythroblasts derived from umbilical cord blood- derived stem cells in different macroporous scaffolds and demonstrated the impact of the pore size on cell viability. Interestingly, they found clusters of mature erythroblasts, reminiscent of erythroblastic islands in the bone marrow, which in turn increased the maturation status and enucle- ation rate.92 Fauzi et al. confirmed increased cell viability and proliferation, proposing a 3D alginate hydrogel asso- ciated with a rotating wall vessel system cultured with murine embryonic stem cells. This system demonstrated that early exposure to stem cell factor guides the differen- tiation of cells toward the erythroid lineage and allows a single-step culture for the production of definitive ery- throcytes in 21 days.93 Allenby et al. further demonstrated the importance of combining the 3D architecture with flow, developing a perfused 3D hollow fiber bioreactor.94 Four ceramic hollow fibers were encased in a 3D polyurethane porous scaffold incorporated in a perfusion system that provided normoxic and hypoxic zones as in the bone marrow environment. Specific ports in the cir- cuit enabled medium and egressed cell sampling for extra- cellular metabolic, protein, and cell analysis. The wall shear rate generated inside the system was estimated to be similar to that in murine bone marrow vasculature. The bone marrow-like environment of this bioreactor enabled cells to be seeded at high density to obtain con- tinuous erythropoiesis. In addition to large-scale produc- tion these tools are candidates as models to study normal and abnormal erythropoiesis and for drug screening. Recently, a 3D model of erythropoiesis, made with polyurethane, was proposed to study erythroid failure in myelodysplastic syndromes.95 To mimic the erythroblas- tic islands and enucleation process, primary bone marrow cells from healthy subjects and myelodysplastic patients were seeded in the 3D scaffold, which had a pore size and distribution close to that of bone marrow architec- ture. The 3D culture enabled continuous expansion and
complete maturation of erythrocytes over 4 weeks. Most importantly, culture of CD34+ cells in 3D scaffolds facili- tated the greatest expansion and maturation of erythroid cells, including generation of erythroblastic islands and enucleated erythrocytes.
Megakaryopoiesis and platelet production in three-dimensional cultures
As already discussed, megakaryopoiesis is critically influenced by the mechanics and biochemical composi- tion of bone marrow. In an attempt to mimic such a struc- ture in vitro, Currao et al. produced 3D hyaluronan hydro- gels functionalized with extracellular matrix components by photo-crosslinking.96 When cultured inside such a structure, megakaryocytes demonstrated the ability to form platelets into a collagen type IV enriched environ- ment, while this function was almost abrogated in the presence of collagen type I. The 3D culture in hydrogel also showed, for the first time, the impact of physical con- straints on megakaryopoiesis and mechano-transduction pathways. In pullulan-dextran 3D hydrogel megakary- ocytes were larger and had increased ploidy and expres- sion of lineage-specific transcription factors.97 Methylcellulose hydrogels showed that viability and mat- uration are directly linked to stiffness of the environment. Indeed, a stiff environment led to decreased survival and growth of megakaryocyte progenitors, while in a less stiff environment, megakaryocytes were more mature in terms of ploidy and morphology of the demarcation membrane system, which closely resembled that of bone marrow megakaryocytes. After recovery and transfer into the liq- uid medium, proplatelet production increased two-fold, due to the activation of mechano-transduction pathways and to a different actomyosin rearrangement.98 Subsequently, Abbonante et al. demonstrated that the acti- vation of TRPV4 (transient receptor potential cation chan- nel subfamily V member 4), a membrane mechano-sensi- tive cation channel, regulates mechano-transduction path- ways that, in turn, control thrombopoiesis on soft sub- strates.23 Specifically, human megakaryocytes cultured on soft silk scaffolds (≤10 MPa) showed increased activation of TRPV4, leading to calcium influx and increased platelet production as a consequence of β1 integrin activation and internalization and of Akt phosphorylation.
By putting together all the pieces of information, scien- tists have been trying to develop flow bioreactors that mimic blood flow and allow better oxygenation and distri- bution of cytokines and nutrients during the 3D culture. The first platelet bioreactor was presented by Sullenbarger and colleagues. It was composed of a polycarbonate cham- ber with three disks of woven polyester or colloidal crystal hydrogel with medium flowing under and over the disks but not through them, thus limiting shear stress.99 The cul- ture of CD34+ cells in this bioreactor allowed a long-term production of platelets and a higher number of collected platelets. Interestingly, when the oxygen concentration within the device was set at a low level (5%), HSC expan- sion was increased and platelet production was decreased. Contrariwise, culture at high oxygen tension (20%) increased the production of platelets but lowered HSC expansion.100,101 A different combination of oxygen tensions at the beginning and at the end of the culture increased both HSC expansion and the final yield of platelets. More recently, Shepherd et al. developed a flow bioreactor based on a two-layer collagen scaffold.102 The porous, structurally
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