Understanding mechanotype of cord blood CD34+ undergoing in vitro differentiation into red blood cells. Application to development of novel label-free purification techniques.

Every year the World Health Organisation reports a shortage of safe blood for transfusion. Consequently there is an urgent need for alternative sources of red blood cells (RBC). One of the possible options is to manufacture RBC from stem cells. Potentially transfusable RBCs are already produced in vitro using CD34+ cells from umbilical cord blood. Those hematopoietic stem cells undergo 21 days of differentiation, recapitulating distinct stages of in vivo erythropoiesis. Because the differentiation protocol is not 100% efficient, the end product is a heterogeneous cell mixture containing fully-functional red blood cells (enucleated cells) and cells that remain at earlier developmental stages (nucleated cells) as well as free-floating nuclei expelled during the enucleation process. Enucleated cells need to be purified from potentially harmful nucleated cells, if intended for the clinical use. Industry viable purification technologies require cost-effective, automated and scalable approaches. Traditionally, target cells separation is performed by FACS (Fluorescent Activated Cell Sorting) and MACS (Magnetic Activated Cell Sorting). Both techniques require the addition of modifying agents such as antibodies and DNA stains that are costly and require separate quality-control processes. We propose to develop a label-free technique that would allow separation based on cells’ endogenous mechanical properties such as size and deformability. In this research, for the first time, novel high-throughput technique called real-time deformability cytometry (RT-DC) was used to assess changing cell mechanotype of CD34+ undergoing the differentiation process. Additionally enucleated and nucleated cells and nuclei were characterised to identify traits that could serve as a basis for separation in label-free systems.