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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 819-819
Author(s):  
Xin Guan ◽  
Lan Wang ◽  
Yu Zhang ◽  
Bin Shen ◽  
Zhihua Ren ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Pilot Scale ◽  
Cell Number ◽  
Human Platelets ◽  
Scale Production ◽  
Culture Bottle ◽  
Cd34 Cells ◽  
Pilot Scale Production

Abstract 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. Disclosures Ren: Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2888-2888
Author(s):  
Ana Frias ◽  
Christopher D. Porada ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Cell Number ◽  
Human Cord Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

Ex Vivo Large Scale Generation of Human Platelets from Cord Blood CD34+ Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1892-1892
Author(s):  
Takuya Matsunaga ◽  
Ikuta Tanaka ◽  
Masayoshi Kobune ◽  
Yutaka Kawano ◽  
Maki Tanaka ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Large Scale ◽  
Culture Medium ◽  
Ex Vivo ◽  
Aggregation Assay ◽  
Three Phase ◽  
Cd34 Cells ◽  
Phase Culture

Abstract To obtain a large quantity of platelets (PLTs) from cord blood stem cells (CBSC) in vitro, we employed three-phase culture system. We first expanded CBSC on a monolayer of human telomerase catalytic subunit gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and thrombopoietin (TPO) for 14 days (1st phase), and then cultured them to differentiate into megakaryocytes for another 14 days with refreshing medium which contain interleukin-11 (IL-11) in addition to original cytokine cocktail (2nd phase). Subsequently, we transferred the cells to a liquid culture medium containing SCF, FL, TPO and IL-11, and cultured them for 5 days (3rd phase) to recover PLTs in the culture medium. The quantity of PLTs recovered from one CB unit (5 x 106 CD34+ cells) was calculated to be 10.5 units (2 x 1011 PLTs). These CB-derived PLTs exhibited quite similar feature as those from peripheral blood in morphology as revealed by electron micrograph and in functions as revealed by aggregation assay and by FACS detecting expression of P-selectin and activated glycoprotein IIb-IIIa antigens upon fibrinogen/ADP stimulation. Thus our three-phase culture system was considered to be useful for large scale generation of PLTs from CB for clinical usage.

Mesenchymal Stem Cells Promote the Engraftment of Cord Blood CD34+ Cells but Do Not Accelarate Platelet Recovery NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1267-1267
Author(s):  
Yvette van Hensbergen ◽  
Helen de Boer ◽  
Manon C. Slot ◽  
Laurus F. Schipper ◽  
Anneke Brand ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Human Platelets ◽  
Scid Mice ◽  
Post Transplantation ◽  
Platelet Recovery ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Aim: Delayed platelet reconstitution in the peripheral blood (PB) remains a problem in transplantation with umbilical cord blood (CB)-derived stem cells. Previously, we have shown that transplantation with ex-vivo expanded CB CD34+ cells (CD34exp) with thrombopoietin for 10 days, results in an accelerated platelet reconstitution in NOD/SCID mice. It has been shown that mesenchymal stem cells (MSC) are able to enhance the overall engraftment when co-transplanted with CB CD34+ cells. Therefore, we investigated whether co-transplantation of MSC with CD34+ cells or CD34exp cells may have an additive effect in shortening the time to platelet recovery and on the total number of platelets in the PB at 6 weeks after transplantation. Methods: To evaluate the time to platelet recovery and the total number of platelets at 6 weeks after transplantation, we used 4 groups of irradiated NOD/SCID mice, divided according to the transplant received: 1) CD34+ 2) MSC+CD34+ 3) CD34exp 4) MSC+CD34exp. Human platelet recovery was measured twice a week for the first three weeks and once a week thereafter, using an assay that reliably detects 1x106plt/L. The percentage of human CD45+ cells in the bone marrow (BM) was evaluated at 6 weeks after transplantation. Results: In accordance with previous experiments, platelet recovery started earlier in mice transplanted with CD34exp cells compared to CD34+ cells (Table 1). Co-transplantation of MSC with CD34+ cells did not result in an accelerated platelet recovery during the first 2 weeks after transplantation, as was observed for expanded cells. However, co-transplantation of MSC did enhance the number of platelets at 6 weeks after transplantation (454.2±264.5 plt/μ l for MSC+CD34+ vs. 101.9±78.4 plt/μ l for CD34+). MSC had no affect on either the time to platelet recovery nor the total number of human platelets at 6 weeks after transplantation when co-transplanted with CD34exp cells. To assess the overall efficacy of the MSC on the engraftment of human CB cells, we evaluated the percentage of human CD45+ cells in the BM of the NOD/SCID mice at 6 weeks after transplantation. In mice transplanted with MSC+CD34+, the percentage of human CD45+ cells was higher compared to controls transplanted with CD34+ cells only (30.4% for MSC+CD34+ vs. 17.8% for CD34+). No further engraftment enhancing effect of MSC was observed following transplantation of CD34exp cells only (32.1% for CD34exp vs. 35.7% for MSC+CD34exp). Conclusion: Our results show that transplantation with CD34exp cells results in an accelerated platelet recovery in NOD/SCID mice, an effect that can not be achieved by co-transplantation of MSC+CD34+ cells. However, at 6 weeks after transplantation co-transplantation with MSC+CD34+ cells results in a higher number of platelets in the PB. In addition, the level of engraftment of human CD45+ cells in the BM of NOD/SCID mice is increased by co-transplantation of MSC+CD34+ cells. In contrast, MSC did not affect the time to platelet recovery, the number of human platelets at 6 weeks after transplantation, or the engraftment of human CD45+ cells in the BM when co-transplanted with CD34exp. Table 1: % of mice with ≥ 1x106 platelets/L in the PB Days post transplantation 6 9 13 16 CD34+ 0% 20% 67% 100% MSC+CD34+ 20% 0% 80% 100% CD34exp 83% 100% 100% 100% MSC+CD34exp 60% 100% 100% 100%

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 5044-5051 ◽  
Author(s):  
Isabelle I. Salles ◽  
Tim Thijs ◽  
Christine Brunaud ◽  
Simon F. De Meyer ◽  
Johan Thys ◽  
...  
Keyword(s):  
Cord Blood ◽  
Flow Model ◽  
Ex Vivo ◽  
Human Platelets ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34+ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 × 103/μL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibα (GPIbα) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

Ex Vivo Expansion of Human Umbilical Cord Blood CD34+ Cells in a Collagen Bead—Containing 3-Dimensional Culture System

2003 ◽  
Vol 78 (2) ◽  
pp. 126-132 ◽  
Author(s):  
Han-Soo Kim ◽  
Jong Baeck Lim ◽  
Yoo Hong Min ◽  
Seung Tae Lee ◽  
Chuhl Joo Lyu ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
3 Dimensional ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Encapsulated feeder cells within alginate beads for ex vivo expansion of cord blood-derived CD34+ cells

2016 ◽  
Vol 4 (10) ◽  
pp. 1441-1453 ◽  
Author(s):  
Xiuwei Pan ◽  
Qiong Sun ◽  
Haibo Cai ◽  
Yun Gao ◽  
Wensong Tan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Alginate Beads ◽  
Ex Vivo Expansion ◽  
Cell Culture System ◽  
Feeder Cells ◽  
Cd34 Cells

A co-culture system based on encapsulated feeder cells within alginate beads was developed through optimizing the detailed aspects of the cell culture system to expand CD34-positive (CD34+) cells ex vivo.

Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4411-4411
Author(s):  
Stephen E Fischer ◽  
Yiwei Ma ◽  
Caitlin Smith ◽  
Anirudhasingh Sodha ◽  
Yukang Zhao
Keyword(s):  
Bone Marrow ◽  
Tissue Culture ◽  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Phenotype ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 4411 Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo. In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation. Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells. Disclosures: No relevant conflicts of interest to declare.

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