In Vitro Platelet Production: Promoting Megakaryocyte Terminal Differentiation, Demonstrating Platelet Functionality, and Developing an Automated Process for Platelet Harvest

Platelets (PLTs) produced in vitro from megakaryocytes (MKs) derived from hematopoietic stem cells (HSCs) could potentially overcome some of the limitations of donated PLTs, including contamination and recipient alloimmunization. Although significant advances have been made with respect to generating large quantities of MKs from HSCs in culture, improvements in MK maturation, including proplatelet (proPLT) formation (PPF) and PLT release, as well as in the functionality of generated PLTs, are needed for in vitro PLT production to become clinically relevant. Currently, the number of PLTs generated per MK in vitro pales in comparison to the thousands generated per MK in vivo, and in-vitro -derived PLTs often display signs of pre-activation. The aims of this work were therefore twofold: to promote in-vitro-derived MK terminal differentiation and to improve in-vitro-derived PLT functionality and recovery.;With respect to the first goal, we sought to identify factors involved in MK terminal differentiation, then develop a tunable culture surface to control these factors for improved MK PPF. We specifically explored the role of adhesion in MK polyploidization and PPF and examined changes in PPF when MKs were cultured on soft, ligand-functionalized hydrogels designed to limit cell spreading. Adhesion was found to be essential for initiation of primary MK PPF, but did not affect primary MK polyploidization.;For the second goal, we aimed to first generate functional PLTs in vitro, then develop an efficient process for harvesting PLTs soon after generation in order to maintain PLT functionality. After switching to a different serum-free medium, subjecting proPLT-forming MKs to overnight agitation, and employing a non-enzymatic cell dissociation solution during PLT harvest, we demonstrated in vitro production of functional PLTs. We subsequently adapted a commercially-available, spinning-membrane filtration device to separate in-vitro-derived PLTs and MKs without inducing PLT pre-activation. Recovered MKs exhibited PPF and shed PLTs after reseeding in culture, suggesting that the process could be used to harvest PLTs at multiple time points during culture to maximize functional PLT yield.