Towards the Mass Manufacture of Blood Platelets

Blood donation will at some point in the next decade or two be replaced with the mass manufacture of blood, produced to order and as needed. It will be far more efficient than the present system of donations and stockpiles, but there is still a great deal of work to be accomplished in order to reach this goal. The review here covers just a fraction of the scope of work, focused on the technical details of the production of platelets and their predecessor cells. Currently this is being carried out somewhat in advance of any ability to scale up to a far larger pace of production, but that will come with time. As the paper shows, there is already a considerable variety and sophistication in the equipment used to generate platelets outside the body.

Platelets (PLTs) fulfill essential functions in primary hemostasis and wound healing and maintain immunological properties, but also play a role in inflammation and cancer. In vivo, PLTs are formed by demarcation and cytoplasmatic shedding from one large precursor cell known as a megakaryocyte (MK). MKs reside within the bone marrow where they differentiate from hematopoietic stem cells. During their maturation they migrate to the sinusoids vessels, and extend protusions (proplatelets; proPLTs) through the vessel pores. The shear stress within the sinusoidal lumen supports the release of PLTs into the blood stream. Understanding the basic biology of thrombopoiesis and its physiological mechanisms is fundamental to efficiently mimic PLT production in vitro, an approach that is gaining importance for future transfusion and regenerative medicine. The demand for PLT transfusion is constantly rising. While the majority of PLT transfusions is provided to patients with reduced PLT counts after chemotherapy or hematopoietic progenitor cell transplantation, other clinical causes may require urgent PLT transfusion.

To achieve clinical numbers of in vitro generated MKs and PLTs, current next-generation strategies employ fluidic biomimetic reactors recapitulating the natural bone marrow environment. In 2006 researchers demonstrated PLT differentiation from human cord blood-derived CD34+ progenitor cells in a three-phase culture system. The first two differentiation phases were based on static cultures using hTERT human stroma cell as feeders. However, the final maturation of MKs and PLTs occurred in a suspension culture system. Another new feature introduced by this study was the co-culture of MKs in combination with human umbilical vein endothelial cells (HUVEC), since endothelial cells are known to fulfil stimulatory functions on proPLT formation.

In 2009, researchers published the first 3D PLT bioreactor built from a modular perfusion system. The device contained a central producer cell disc covered by a layer of pre-expanded CD34+ progenitor cells, while medium and gas flow occurred in separate spaces above and below this cell layer. This setup allowed the harvest of PLTs from the lower medium space over 30 days. Later, they further improved this bioreactor prototype. They increased PLT production by regulating the oxygen supply and inducing controlled shear stress with help of a continuous medium flow though the cell scaffold. These first approaches demonstrated the feasibility to produce PLTs not only in suspension cultures but also in continuous perfusion systems which can significantly facilitate the upscaling of PLT production.

In 2011 researchers established a 3D model one step ahead to a close technical analogue of the bone marrow microenvironment by the application of silk protein biomaterial. To simulate the natural niche, growth factor-coated silk microtubes (mimicking sinusoidal vessels) were embedded in modules filled with type I collagen gel. MKs were differentiated from CD34+ cells and seeded between the collagen gel and each microtube. They migrated towards the microtube and released proPLTs into the constitutive flow of media within the microtubes. However, only 7% of MKs exhibited proPLT production. In 2015 researchers presented a follow-up of this prototype, equipped with an additional silk sponge encompassing the microtube to better mimic the stiffness of the sinusoidal vessel surrounding. Moreover, they improved the entrapment of growth factors and extracellular matrix components, and seeded HUVEC into the lumen of the silk microtubes. These new features led to a threefold increase in numbers of released PLTs.

Step by step, bioreactor bioengineering for efficient PLT production became increasingly complex. In 2014, researchers presented the first PLT bioreactor-on-a-chip that, despite its small size, considered a broad spectrum of parameters to recapitulate the bone marrow microenvironment. To mimic the stiffness of the natural bone marrow, MKs were seeded in hydrogels such as alginate. To improve MK trapping, extracellular matrix proteins were added into the surrounding media, or used to coat the membrane separating the MK chamber from the lower flow chamber. ProPLT formation was stimulated with help of endothelial cell contacts, and PLT release was optimized using controlled hemodynamic vascular shear stress. In 2016 researchers developed a 'microfluidic model of the PLT generating organ', constituted by a single-flow chip, in which MKs derived from human cord blood (hCB) CD34+ cells were constitutively perfused and captured by thousands of vWF (von Willebrand factor)-coated micropillars to release PLTs into the media flow. This setup enabled a high throughput of millions of MKs.

In summary, it is currently possible to efficiently differentiate MKs from induced pluripotent stem cells (iPSCs), but they show a restricted capacity to produce PLTs in vitro. Physiologically, one MK produces thousands of PLTs into the circulation. In contrast, the protocols available only allow the production of up to hundreds PLTs per MK. This delays the possibility for clinical application of in vitro produced PLTs. Yields of PLT production may profit in the future from the harmonization of MK expansion and differentiation culture systems towards a synchronized PLT formation and release. The application of shear stress in the designed bioreactor aimed to provide a physical cue to induce a synchronized proPLT formation, extension, and PLT release. However, it remains highly desirable to identify biological or chemical signals that might support this process.



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