Currently, platelet transfusions, possessing profound clinical importance in the clotting of blood and healing of wounds, are entirely derived from human volunteer donors. This approach is limited by a 5-day shelf life, the potential risk of contamination, and differences in donor/recipient immunology. In vivo, platelets are formed when bone marrow megakaryocytes (Mks) extend long, cytoplasmic projections, called proplatelets (proPLTs), into the sinusoids where shear forces accelerate proPLT elongation and release platelets into circulation. Additionally, platelet formation can occur from trapped Mks in the lung capillary bed. Developing a clinically relevant ex vivo platelet production process is limited by (1) expansion and differentiation of hematopoietic steam and progenitor cells (HSPCs) into Mks and (2) generation of platelet-like-particles (PLPs) from mature Mks. We and others have made progress in addressing these challenges yet major limitations remain to deliver a donor-independent process for ex vivo platelet production. In the first part of this work, we aimed to further understand ex vivo PLP production from Mks through the engineering of novel microfluidic bioreactors that mimic in vivo physiological conditions in the bone marrow and lung. Leveraging computational fluid dynamics (CFD) modeling to help guide and understand the hydrodynamics of the systems, we developed uniform-shear-rate bioreactors (USRBs) that permit real-time visualization of the proPLT formation process and the rapid-release of individual PLPs, which has been observed in vivo, but not previously reported for platelet bioreactors. We showed that modulating shear forces and flow patterns had an immediate and significant impact on PLP generation. By identifying particularly effective operating conditions within a physiologically relevant environment, these USRB bioreactors provide a useful tool for the study and analysis of proPLT/PLP formation to further our understanding of PLP release. Critical advancements are needed to improve scalability and increase Mk culture productivity. In the second part of this work, we evaluated Mk production from mobilized peripheral blood CD34+ cells cultured on a commercially available gas-permeable silicone rubber membrane, which provides efficient gas exchange. This technology has been used to accelerate the expansion of other cell types, such as T-cells, for cell-based therapies and demonstrated scalability. Additionally, we investigated the use of fed-batch media dilution schemes since this cell-culture technique was shown to be beneficial for HSPC expansion. Our new culture process improved Mk yields by over two-fold while retaining Mk potential to make proPLTs and generate PLPs. Finally, we aimed to improve PLP potential through pharmacological inhibition of the Rho GTPases: RhoA, Cdc42 and Rac1. These targets are regulators of the actin cytoskeleton and have been implicated in the polyploidization and proPLT formation of Mks. Our goal in this study was to enhance ploidy levels and proPLT formation thus increasing PLP yields but overall, we saw limited improvements. The results further underline the lack of understanding driving Mk maturation and PLP generation and highlight the need of more Mk mechanistic studies of the pathways that regulate the fate of late-stage Mks.

Creator

• Martinez, Andres Felipe

DOI

• https://doi.org/10.21985/n2-xpf2-hx98

Subject

• Bioengineering
• Chemical engineering
• Cellular biology

Language

• en

Alternate Identifier

• etdadmin_upload_657405
• http://dissertations.umi.com/northwestern:14571

Keyword

• Applied sciences
• Biological sciences
• Megakaryocyte
• Cell therapies
• Computational fluid dynamics
• Microfluidic bioreactor

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright