Good Manufacturing Practice-Grade of Megakaryocytes Produced by a Novel Ex Vivo Culturing Platform.

Abstract

Ex vivo (EV)‐derived megakaryocytes (MKs) have shown great promise as a substitute for platelets in transfusion medicine to alleviate a severe shortage of donor‐platelets. Challenges remain that include poor efficiency, a limited scale of production, and undefined short‐term storage conditions of EV‐derived MKs. This study aims to develop a high‐efficiency system for large‐scale production of Good Manufacturing Practice (GMP)‐grade MKs and determine the short‐term storage condition for the MKs. A roller‐bottle culture system was introduced to produce GMP‐grade MKs from small‐molecule/cytokine cocktail expanded hematopoietic stem cells. Various buffer systems and temperatures for the short‐term storage of MKs were assessed by cell viability, biomarker expression, and DNA ploidy levels. MKs stored for 24 hours were transplanted into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to confirm their platelet‐releasing and tissue‐homing ability in vivo. A yield of ~ 2.5 × 104 CD41a+/CD42b+ MKs with purity of ~ 80% was achieved from one original cord blood CD34+ cell. Compared with the static culture, the roller‐bottle culture system significantly enhanced megakaryopoiesis, as shown by the cell size, DNA ploidy, and megakaryopoiesis‐related gene expression. The optimal storage condition for the MKs was defined as normal saline with 10% human serum albumin at 22℃. Stored MKs were capable of rapidly producing functional platelets and largely distributing in the lungs of NOD/SCID mice. The novel development of efficient production and storage system for GMP‐grade MKs represents a significant step toward application of these MKs in the clinic.