Pilot-Scale Production of Megakaryocytes/Platelets from Cord Blood CD34⁺ Cells in a Bottle Turning Device Culture System

Due to a donor-platelet shortage, ex vivo generated megakaryocytes/platelets have emerged as an effective substitute to alleviate thrombocytopenia. We have previously reported a "two-stage culture system" for producing megakaryocytes/platelets from CD34⁺ cellsand demonstrated their safety and efficacy in myeloablative murine and non-human primate models. Here, we present pilot-scale production of megakaryocytes/platelets in a bottle turning device culture system that significantly increases the yield of functional megakaryocytes/platelets.

Enrichedcord blood CD34⁺ cells were first cultured in modified IMDM basal medium (serum-free) with the addition of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), and interleukin (IL)-3 in a T-25 flask. After 6 days of expansion, cells were transferred into a 2 L culture bottle with 600 ml medium in a bottle turning device at the density of 1.5×10⁵/mL for additional 7 days. The culture medium was modified IMDM supplemented with SCF, TPO, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). At various time points, cell numbers were counted with a hemocytometer and expression of CD34, CD41a, and CD42b surface markers was monitored by flow cytometry. Resulting megakaryocytes were further confirmed by morphological examination and DNA ploidy analysis. Expression of relevant oncogenes was measured by quantitative real-time polymerase chain reaction (qPCR) for evaluating potential tumorigenicity. Moreover, these megakaryocytes were transplanted into sub-lethally irradiated NOD/SCID mice (1×10⁶ CD41a⁺/CD42b⁺ megakaryocytes per mouse) to assess human platelet-releasing potential and function in vivo.

The initial cell number was 1×10⁶ with CD34 purity of 93.4% ±2.8%. For 6-day culture, the absolute number of total cells and CD34⁺ cells reached 9.3×10⁷±1.8×10⁷ and 4.0×10⁷±5.6×10⁶, respectively, which were calculated into a 92.9±18.1 and 42.8±5.2-fold increase, respectively. For additional 7-day culture in the turning bottles, 2.2×10¹⁰±3.2×10⁹ cells were obtained with the proportions of CD41a⁺ and CD42b⁺ cells at 80.1%±7.4% and 63.6%±5.9%, respectively. Approximately 2.0×10⁴ megakaryocytes/platelets were generated from each input CD34⁺ cell. These megakaryocytes were morphologically discernible as they were much larger than starting CD34⁺ cells with apparent lobular nuclei and numerous α-granules. DNA content analysis revealed that about 25.4%±1.3% of megakaryocytes exhibited a polyploidy level of >8N. Moreover, about 10% megakaryocytes have formed pro-platelet fragments and released platelets in cultures. Compared to initial CD34⁺ cells, generated megakaryocytes showed similar levels of expression of proto-oncogenes including c-Myb, N-Ras, and K-Ras whereas c-Myc, bmi-1, cyclin B, and hTERT expression was decreased, strongly suggesting that ex vivogenerated megakaryocytes are unlikely tumorigenic. Releasing of human platelets by injected megakaryocytes were detected in mouse peripheral blood with a percentage of 0.5%±0.1% as early as 30 min following transplantation, after which the percentage of human platelets increased rapidly with a peak level of 3.2%±0.6% at about 4-6 h post-transplantation. The half-life of released platelets in mice was about 10 h and about 23±6 human platelets were released per megakaryocyte in the transplanted mice. Furthermore, these platelets were activated, expressing CD62P after stimulation by ADP, suggesting that released human platelets in mice are functional.

Combined, we have established a bottle turning device culture system for pilot-scale production of megakaryocytes/platelets ex vivo. One unit of cord blood (80 ml) with 2×10⁶ CD34⁺ cells can generate 4×10¹⁰ megakaryocytes/platelets that are sufficient for treating 130 patients (with an average weight of 60 kg and infusion cell number 5×10⁶cells/kg). In conclusion, our new culture system is capable of manufacturing GMP-grade megakaryocytes/platelets sufficient for various clinical applications.