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 plateletreleasing and tissue-homing ability in vivo. A yield of ~ 2.5 × 10 4 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.

In vitro Generation of Megakaryocytes and Platelets

“…Based on this, new cytokines (such as IL-11) and new chemical compounds (such as Y27632 and DAC) have been identified, a new 3D culture system (such as roller-bottle culture system), and new stromal cells (such as hTERT-transduced stroma) have been introduced to the culture conditions. These optimizations have significantly improved the differentiation efficacy, with 2 × 10 4 MKs obtained from 1 seeded CD34 + HSC, compared with the previous 5 MKs per HSC (Norol et al, 1998;Guan et al, 2020), and 1.26 × 10 11 -1.68 × 10 11 platelets generated from one CB unit, which equal to 2.5-3.4 units of donorderived platelets (Matsunaga et al, 2006). However, only a limited number of CD34 + HSCs, about 3-5 × 10 6 , could be extracted from one unit of CB, and these cells are difficult to obtain and culture.…”

“…Compared to those at 37 • C, the differentiation cultures maintained at 39 • C promoted the proliferation and differentiation efficiency of HSCs, accelerated MK maturation by 3-4 days, and improved platelets output by more than 15-fold (Proulx et al, 2004). Later, the 3D culture method, specifically the roller-bottle cell culture system, was also found to be able to improve the efficiency of platelet generation from CB HSCs by a few folds, compared with the routinely used static culture condition, either in a small research scale (Yang et al, 2016) or in a Good Manufacturing Practice (GMP) standard culture (Guan et al, 2020).…”

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

“…A nature-inspired system for functional MK and platelet production using silk-based vascular tubes was already introduced a decade ago [ 37 ]. With a more old-fashioned approach, roller-bottles were recently utilized to significantly enhance MK propagation from cord blood-derived CD34 + HSPCs by about 1.8-fold to 2.5 × 10 4 MKs per initial CD34 + HSPC compared to static culture conditions; the number of platelets obtained was not disclosed in this study [ 40 ]. In our study, platelet release from MKs was supported by culturing on an orbital shaker during the final stage III.…”

In Vitro Human Haematopoietic Stem Cell Expansion and Differentiation

“…More recently, clinical-grade protocols for megakaryocyte generation from UCB-derived HSPCs have been developed using human albumin and a defined cocktail of cytokines and supplements [ 138 ]. Finally, 3D culture methods (roller-bottle cell culture system) have been able to further improve the efficiency of megakaryocyte and platelet generation from UCB-derived HSPCs [ 139 , 140 ].…”