RESUMEN
We previously proposed a microfluidic bioreactor with glass-Si-glass layers to evaluate the effect of the fluid force on platelet (PLT) production and fabricated a three-dimensional (3D) microchannel by combining grayscale photolithography and deep reactive ion etching. However, a challenge remains in observing the detailed process of PLT production owing to the low visibility of the microfluidic bioreactor. In this paper, we present a transparent microfluidic bioreactor made of cyclo-olefin polymer (COP) with which to observe the process of platelet-like particle (PLP) production under a bright-field, which allows us to obtain image data at a high sampling rate. We succeeded in fabricating the COP microfluidic bioreactor with a 3D microchannel. We investigated the bonding strength of COP-COP layers and confirmed the effectiveness of the microfluidic bioreactor. Results of on-chip PLP production using immortalized megakaryocyte cell lines (imMKCLs) derived from human-induced pluripotent stem cells show that the average total number of produced PLPs per imMKCL was 17.6 PLPs/imMKCL, which is comparable to that of our previous glass-Si-glass microfluidic bioreactor (17.4 PLPs/imMKCL). We succeeded in observing PLP production under a bright-field using the presented microfluidic bioreactor and confirmed that PLP fragmented in a narrow area of proplatelet-like protrusions.
RESUMEN
The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.
