We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.

IntroductionThe Notch signaling pathway guides cell-fate decisions in multiple developmental processes. 1,2 Intercellular communications that control the developmental fate of multipotent cells are mediated by the Notch family of transmembrane receptors in several invertebrate and vertebrate developmental systems. The Notch proteins are single-pass receptors that are activated by the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. 3 To date, 4 human Notch genes have been identified and all are expressed on hematopoietic cells. 2 Also, 5 human Notch ligands, Delta-like1/3/4, and Jagged1/2 were identified, and all were shown to bind to Notch-1, Notch-2, and Notch-4. 4,5 Notch receptors are matured in the secretory pathway and presented at the cell surface as heterodimeric molecules. Interaction with ligands leads to 2 additional proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. NICD enters the nucleus, where it interacts with the DNA-binding protein, CSL (C-promoter binding factor [CBF]-1, Suppressor of Hairless, LAG-1; also known as recombination signal-binding protein Jk [RBP-Jk]). 6 In the absence of NICD, CSL represses transcription through interactions with a corepressor complex, containing a histone deacetylase. Upon entering the nucleus, NICD displaces the corepressor complex from CSL and replaces it with a transcriptional activation complex that includes NICD, Mastermind (MAML-1), the histone acetyltransferase p300, and, possibly, PCAF p300/CBP (cyclic AMP response element-binding protein [CREB]-binding protein)-associated factor. Notch signaling, thus, converts CSL from a repressor to an activator, leading to the transcription of target genes. The target genes include members of the Hes and HRT/ HERP/Hey families of transcriptional repressors; therefore, Notch signaling is often viewed as a transcription cascade.The classical view holds that Notch signaling controls the balance between the progenitor pool and its differentiating progeny and thus is involved in the maintenance of stem cell fate. 1,2 In fact, a number of studies have provided evidence that ligand-induced Notch signaling favored hematopoietic stem cell (HSC) selfrenewal, increased the numbers of progenitors, and promoted HSC survival. Moreover, Notch signaling may be instructive for differentiation toward a particular fate. It plays a crucial role in the hematopoietic system, especially in the regulation of the T-cell lymphoid lineage commitment 7-9 and in late stages of B-cell development. 10 Because of the key role of Notch signaling in supporting early T-cell differentiation, it was generally established that Notch concomitantly negatively regulates myeloid lineage development. The megakaryocytic and erythroid lineages are extremely linked, because they share a common bipotent progenitor called the MEP (MK/erythroid progenitor). The role of Notch in megakaryocytic and erythroid development remains a matter of debate. While some data report that Notch signaling...