(338f) Enhancing Ex Vivo Platelet Production through Shear Forces within Defined Bioreactors

Authors: 
Martinez, A. F., Northwestern University
Jenkins, K. A., Northwestern University
McMahon, R. D., Northwestern University
Miller, W. M., Northwestern University
Currently, platelet transfusions possessing profound clinical importance are entirely derived from human volunteer donors. However, this approach is limited by a 5-day shelf life and differences in donor/recipient histocompatibility. In vivo, platelets are formed when bone marrow megakaryocytes (Mks) extend long, cytoplasmic projections, called proplatelets (PPL), into the sinusoid where shear forces accelerate PPL elongation and release platelets into circulation. Methods of in vitro platelet production have yielded less than 10 platelets/megakaryocyte (plts/Mk), compared to >1000 plts/Mk in vivo. There is a need for a clinically relevant process for platelet production, but much is unknown about what initiates and regulates PPL formation and how to maximize platelet release. We are interested in the generation of ex-vivo platelets through the optimization of Mk maturation, following in the production of functional platelets through a microfluidic bioreactor that utilizes shear forces on Mks to generate platelets. Microfluidic devices have emerged as a valuable tool for cell culture studies. There are numerous advantages to utilizing a microfluidic platform, including a low input cell requirement, the ability to screen multiple conditions in parallel, compatibility with time-lapse imaging, and tight control of microenvironment conditions. In addition, device fabrication is straightforward and inexpensive using soft photolithography.

Through the use of computational fluid dynamic simulations and microfluidic device fabrication, a design â?? test â?? build methodology is used to present a dual-flow microfluidic bioreactor system in which aspects of the bone marrow niche are recapitulated to both study the platelet formation process and enhance in vitro yields. Experimental studies are conducted to validate the simulations in terms of streamline profiles and flow patterns with and without cell capture. Microenvironment characteristics include shear profiles and extracellular matrix (ECM) protein coatings. Furthermore, the design of the bioreactor generates uniform shear profiles on Mks and PPLs and allows for exploration of a wide range of physiological shear rates. Our results indicate that our bioreactor design produces 21 ± 3 plts/Mk, and we are currently working to further increase production. Bioreactor-derived platelets are shown to be functional and retain characteristics similar to those of fresh blood platelets. Characterization of the collected platelets from the bioreactor includes surface markers expression, activation in the presence of the thrombin agonist, and morphological/cytoskeletal changes before and after activation. With this microfluidic reactor and further experimental plans, we aim to understand the factors required for initiation of PPL formation to improve in vitro platelet yields.