Platelets are anucleate cell crucial for hemostasis, immune responses, and tissue regeneration that are produced by megakaryocytes (MKs). Clinical platelet transfusions are vital for treating thrombocytopenia and other conditions, such as bleeding. However, maintaining a continuous supply is challenging due to platelets' short shelf life and potential adverse reactions. Therefore, in vitro platelet production from stem cells presents a promising alternative, offering the possibility of universal compatibility. Yet, the production time for platelet concentrates remains inefficient (20-30 days). Cryopreservation of MKs could provide an immediate source for in vitro platelet production, eliminating the need for extended expansion and differentiation periods. This research investigates varied cryopreservation techniques on megakaryocytes and their progenitors derived from peripheral blood CD34+ hematopoietic stem and progenitor cells. Different cryoprotectants like dimethyl sulfoxide (DMSO) and ethylene glycol (EG), as well as sugars such as trehalose and sucrose were tested and compared to 10% DMSO standardized protocol. Survival rates, expansion (Trypan blue exclusion counting), and surface markers expression (CD34, CD41 and CD42b, by flow cytometry) were evaluated to gauge cell recovery post-cryopreservation, along with functionality assessed by observing proplatelet formation.
Overall, MK progenitors were resistant to cryopreservation stress maintaining high viability (71.5%) and preserving their capacity for expansion and differentiation similar to fresh cells compared to mature MK. Progenitors retained their ability to produce proplatelets, unlike mature MKs. The combination of trehalose with DMSO impeded MK progenitors’ expansion, while with EG showed promising results as DMSO alternative. While starting in vitro platelet production from thawed MK progenitors seems feasible, cryopreservation of mature megakaryocytes warrants further investigation. Future optimizations should explore alternative cryoprotectants and techniques such as vitrification to enhance post-thaw viability and functionality, particularly focusing on functional platelet production.