Two-compartment microfluidic bioreactor with functionalized PEG hydrogels to promote platelet production in culture

Intellectual Merit Generating multiple units of 3-6 x 1011 platelets in culture will require advances in hematopoietic stem and progenitor cell (HSPC) expansion, megakaryocytic cell (Mk) production and maturation, and platelet release and recovery. Platelet production in vitro has received much less attention than HSPC expansion or Mk production. Several groups have produced small quantities of platelets with functional activity similar to that of freshly harvested platelets, but the yields are very low. Many studies showed yields of less than one platelet per Mk, as compared to several thousand platelets in vivo. Recent reports have shed new light on platelet biogenesis. Proplatelet formation (PPF) requires Mk adhesion to selected extracellular matrix (ECM) ligands, but is inhibited by the formation of organized focal adhesions and stress fibers. The Mk bone marrow niche – from which proplatelets are extended between endothelial cells into the sinusoids – is a very soft tissue, so compliant surfaces are likely to increase PPF. Shear from blood flow is important for proplatelet and platelet release. Inspired by the Mk bone marrow sinusoid niche, this project will employ a two-compartment microfluidic bioreactor that incorporates compliant poly-(ethylene glycol) (PEG) hydrogels functionalized with ECM ligands and a porous membrane between compartments to mimic Mk PPF across the sinusoidal endothelium. The microbioreactor system will be used to investigate effects of shear stress, hydrogel stiffness, and ECM ligand type(s) and density, as well as interactions between these variables, on PPF and platelet production. Increasing gradients of pH and chemokines across the membrane will be investigated to further increase PPF. The perfusion system will allow platelets to be harvested as they are released, which is important because platelet quality deteriorates during storage at room temperature and even faster in culture at 37°C. The quality of culture-derived platelet will be verified by evaluating platelet activation in response to agonists. Possible clinical-scale systems based on the microbioreactor include modified hollow-fiber bioreactors and spiral-wound membrane cartridges.