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Unified microcarrier process for human pluripotent stem cell expansion, and cardiomyocyte differentiation and purification in a 500 mL bioreactor
e22
23rd Annual ISCT Meeting
GTG-banding assay confirmed the stability of eMSCs karyotype during long-term culturing (up to P10). After 48h incubation period in serum-free medium eMSCs secreted following proteins: IL-1ra (74,6 ± 9,5 pg/mL), IL-6 (29.8 ± 8.3 pg/mL), IL-8 (138.5 ± 33.3 pg/mL), IL-10 (9.6 ± 5.5 pg/mL), IFNγ (55.9 ± 3.8 pg/mL), VEGF (92.2 ± 19.8 pg/mL), GM-CSF (133.2 ± 5.1 pg/ mL), FGF-2 (17.8 ± 4.3 pg/mL), IP-10 (39.9 ± 3.3 pg/mL) and MCP-1 (41.1 ± 6.7 pg/mL). Thus, obtained endometrial MSCs meet minimal ISCT criteria for MSCs. They produce a range of cytokines, chemokines and growth factors which make them a perspective object for the use in the regenerative medicine application.
LB44 EXOSOMES FOR REGENERATIVE MEDICINE: MANUFACTURING CHALLENGES AND POTENTIAL STRATEGIES Ivan Wall1,2, Ivano Colao1, Ana Valinhas1, Nicola Goddard1, Dan Bracewell1, Randolph Corteling3 1 Department of Biochemical Engineering, UCL, London, United Kingdom, 2 BK21 Department of Nanobiomedical Science, Dankook University, Cheonan, Republic of Korea, 3ReNeuron Ltd, Bridgend, United Kingdom Exosomes are emerging as a novel class of therapeutics that confer many of the regenerative functions originally ascribed to the cells that produce them. From their emergence as possible biomarkers of diseases such as cancer and infection they are rapidly gathering momentum as candidate regenerative medicines. The potential market for exosomes is huge, spanning a range of clinical indications that stem cells have been expected to be utilized for. Overwhelmingly positive pre-clinical data on the effectiveness of exosomes has underpinned this excitement, yet there remain some substantial manufacturing challenges that relate to scale of production and reliability of measurement and characterization strategies. In particular, challenges of purifying exosomes at scale whilst preserving their therapeutic attributes need to be overcome to ensure products can be made consistently. We have created, in collaboration with industry partners, new strategies to address the upstream cell culture processes needed to generate exosomes and the downstream purification methods that can be applied to purify them at scale. These will be presented, along with a discussion of current challenges for the manufacture of cellderived exosomes.
LB45 UNIFIED MICROCARRIER PROCESS FOR HUMAN PLURIPOTENT STEM CELL EXPANSION, AND CARDIOMYOCYTE DIFFERENTIATION AND PURIFICATION IN A 500 ML BIOREACTOR Steve Oh1, Allen Chen1, Sherwin Ting1, Heming Wei2, Shaul Reuveny1 1 Bioprocessing Technology Institute, Singapore, Singapore, 2National Heart Centre, Singapore, Singapore Human pluripotent stem cells (hPSCs) are a renewable source for generating cardiomyocytes in treating myocardial infarction. We have developed an integrated three stage cardiomyocyte (CM) production process that comprises of hPSC expansion, differentiation, and purification using microcarriers (MC) in a 500 mL controlled bioreactor. Up to 18 fold increase in hPSC expansion was achieved, 3.66 × 106 cells/mL in 7 days with maintenance of high levels of pluripotency. Cardiac differentiation of hPSC expanded on MCs can be done by a simple change from growth medium to cardiac induction medium, producing up to 1.08 × 106 CM/mL with 48.1% cells expressing cardiac marker, troponin-T (cTnT), after 10 days in cultures. A subsequent CM purification phase was developed using a basal medium that comprised of 5 mM lactate. Presence of glucose or absence of lactate could result in lower CM production. The optimal time to initiate the treatment was at day 10 post differentiation. Earlier time points would result in lower CM concentrations while later time of initiation offered no significant benefits in CM production. The optimal treatment duration was 5 days and this has resulted in generating higher CM yields of 1.34 × 106 CM/mL (1.24 fold) with 72.5% (1.51 fold) cardiac troponin-T positive cells. The robustness of this platform was also demonstrated with an induced pluripotent cell line, producing 1.33 × 106 CM/mL with an 83.1% expression of cardiac troponin-T after 23 days of culture. Structure characterization showed extensive striated sarcomeres confirming cardiac ontogeny, and patch clamp assays
showed ventricular (80%) and atrial (14%) cells types were present in the CM population.
LB46 MUSCULAR TROPHISM AND VOLUNTARY CONTRACTILE CAPACITY IMPROVEMENT AFTER INTRAMUSCULAR IMPLANT OF AUTOLOGOUS MUSCULAR PROGENITOR CELLS IN SCI PATIENTS M. Teresita Moviglia-Brandolino, Gustavo A. Moviglia, Samanta C. Piccone, Gustavo A. Albanese, Damian Couto Centro de Investigacion en Ingenieria de Tegidos y Terapia Celular, Universidad Maimonides, Buenos Aires, Argentina Introduction: Between June 2013 and December 2015 ten previously chronic and complete SCI patients were part of a clinical trial consisted of autologous neural progenitor cells implant (each one received 2–3 implants, one each 12 months) and intensive rehabilitation program. During 2015 10/10 patients showed Electromyography recovery in muscles that were previously affected by the lesion. Despite this, muscular atrophy caused by previously denervation persisted. Looking forward to improve muscular trophism and contractile capacity we performed intramuscular cell implant in muscles that show electromyography recovery. Method: In January 2016, 10 chronic and complete SCI patients who recovered electromyography register after neural progenitor cell implant received intramuscular cell implant. The implant consisted in a co culture of autologous muscular progenitor cells and effector T lymphocytes. Muscles that recover electromyography activity in response to voluntary order were selected. Using ultrasound guide cells were injected in between muscle fibers. Each muscle received 6–8 implants with 6 weeks in between. All patients continued with the rehabilitation program. Results: All implants were well tolerated. Implanted muscles showed: volume increase, improvement in contractile capacity (strength and range of motion). Significant changes in ultrasound and histology were also observed. The first changes were observed after three weeks; they persisted and improved after consecutive muscular implants. Conclusion: In previously denervated muscles, even though muscular electrical activity associated with voluntary order has been recovery, improving muscular conditions allows to achieve functional response (improvement in trophism and contractile function). To achieve this goal, intramuscular cell implant seems to be effective. The amount of muscular cell that need to be implanted depends on the muscular volume and the severity of the atrophy.
LB47 MAKING MULTIPLE THERAPEUTIC CELL PRODUCTS FROM A CGMP-COMPLIANT IPSC LINE Xianmin Zeng1,2,3, Ying Pei3, Helen Cifuentes1, Thelma Garcia1, Deepak Lamba1, Mahendra Rao2 1 Buck Institute, Novato, California, United States, 2NxCell Inc, Novato, California, United States, 3XCell Science Inc, Novato, California, United States Induced pluripotent stem cell (iPSC) can differentiate into multiple phenotypes, and indeed multiple protocols for such differentiation have been established. Although the efficiency of different protocols is variable, most protocols work with most lines with some tweaking. To confirm this hypothesis and to make a clinically compliant line available for independent evaluation, we have taken the current Good Manufacture Practice (cGMP)-compliant iPSC line developed by the NIH, and used it to develop cellular products that are currently being considered for therapy. We focused on retinal cells, MSC, NSC, dopaminergic neurons and astrocytes. We obtained a WCB from the NIH and prepared a stock at passage 20. After testing quality we used it to generate multiple products using standard protocols. To confirm that these protocols could also be used for other iPSC lines we obtained a second clinically compliant line and tested the reproducibility of our methodology. Our results confirmed that the same protocols could be used with minimal modifications with multiple qualified lines. We believe that our demonstration that multiple products can be made from the same WCB bank and the same protocols can be used with multiple lines offers a path to a cost effective strategy for developing cellular products.
23rd Annual ISCT Meeting
GTG-banding assay confirmed the stability of eMSCs karyotype during long-term culturing (up to P10). After 48h incubation period in serum-free medium eMSCs secreted following proteins: IL-1ra (74,6 ± 9,5 pg/mL), IL-6 (29.8 ± 8.3 pg/mL), IL-8 (138.5 ± 33.3 pg/mL), IL-10 (9.6 ± 5.5 pg/mL), IFNγ (55.9 ± 3.8 pg/mL), VEGF (92.2 ± 19.8 pg/mL), GM-CSF (133.2 ± 5.1 pg/ mL), FGF-2 (17.8 ± 4.3 pg/mL), IP-10 (39.9 ± 3.3 pg/mL) and MCP-1 (41.1 ± 6.7 pg/mL). Thus, obtained endometrial MSCs meet minimal ISCT criteria for MSCs. They produce a range of cytokines, chemokines and growth factors which make them a perspective object for the use in the regenerative medicine application.
LB44 EXOSOMES FOR REGENERATIVE MEDICINE: MANUFACTURING CHALLENGES AND POTENTIAL STRATEGIES Ivan Wall1,2, Ivano Colao1, Ana Valinhas1, Nicola Goddard1, Dan Bracewell1, Randolph Corteling3 1 Department of Biochemical Engineering, UCL, London, United Kingdom, 2 BK21 Department of Nanobiomedical Science, Dankook University, Cheonan, Republic of Korea, 3ReNeuron Ltd, Bridgend, United Kingdom Exosomes are emerging as a novel class of therapeutics that confer many of the regenerative functions originally ascribed to the cells that produce them. From their emergence as possible biomarkers of diseases such as cancer and infection they are rapidly gathering momentum as candidate regenerative medicines. The potential market for exosomes is huge, spanning a range of clinical indications that stem cells have been expected to be utilized for. Overwhelmingly positive pre-clinical data on the effectiveness of exosomes has underpinned this excitement, yet there remain some substantial manufacturing challenges that relate to scale of production and reliability of measurement and characterization strategies. In particular, challenges of purifying exosomes at scale whilst preserving their therapeutic attributes need to be overcome to ensure products can be made consistently. We have created, in collaboration with industry partners, new strategies to address the upstream cell culture processes needed to generate exosomes and the downstream purification methods that can be applied to purify them at scale. These will be presented, along with a discussion of current challenges for the manufacture of cellderived exosomes.
LB45 UNIFIED MICROCARRIER PROCESS FOR HUMAN PLURIPOTENT STEM CELL EXPANSION, AND CARDIOMYOCYTE DIFFERENTIATION AND PURIFICATION IN A 500 ML BIOREACTOR Steve Oh1, Allen Chen1, Sherwin Ting1, Heming Wei2, Shaul Reuveny1 1 Bioprocessing Technology Institute, Singapore, Singapore, 2National Heart Centre, Singapore, Singapore Human pluripotent stem cells (hPSCs) are a renewable source for generating cardiomyocytes in treating myocardial infarction. We have developed an integrated three stage cardiomyocyte (CM) production process that comprises of hPSC expansion, differentiation, and purification using microcarriers (MC) in a 500 mL controlled bioreactor. Up to 18 fold increase in hPSC expansion was achieved, 3.66 × 106 cells/mL in 7 days with maintenance of high levels of pluripotency. Cardiac differentiation of hPSC expanded on MCs can be done by a simple change from growth medium to cardiac induction medium, producing up to 1.08 × 106 CM/mL with 48.1% cells expressing cardiac marker, troponin-T (cTnT), after 10 days in cultures. A subsequent CM purification phase was developed using a basal medium that comprised of 5 mM lactate. Presence of glucose or absence of lactate could result in lower CM production. The optimal time to initiate the treatment was at day 10 post differentiation. Earlier time points would result in lower CM concentrations while later time of initiation offered no significant benefits in CM production. The optimal treatment duration was 5 days and this has resulted in generating higher CM yields of 1.34 × 106 CM/mL (1.24 fold) with 72.5% (1.51 fold) cardiac troponin-T positive cells. The robustness of this platform was also demonstrated with an induced pluripotent cell line, producing 1.33 × 106 CM/mL with an 83.1% expression of cardiac troponin-T after 23 days of culture. Structure characterization showed extensive striated sarcomeres confirming cardiac ontogeny, and patch clamp assays
showed ventricular (80%) and atrial (14%) cells types were present in the CM population.
LB46 MUSCULAR TROPHISM AND VOLUNTARY CONTRACTILE CAPACITY IMPROVEMENT AFTER INTRAMUSCULAR IMPLANT OF AUTOLOGOUS MUSCULAR PROGENITOR CELLS IN SCI PATIENTS M. Teresita Moviglia-Brandolino, Gustavo A. Moviglia, Samanta C. Piccone, Gustavo A. Albanese, Damian Couto Centro de Investigacion en Ingenieria de Tegidos y Terapia Celular, Universidad Maimonides, Buenos Aires, Argentina Introduction: Between June 2013 and December 2015 ten previously chronic and complete SCI patients were part of a clinical trial consisted of autologous neural progenitor cells implant (each one received 2–3 implants, one each 12 months) and intensive rehabilitation program. During 2015 10/10 patients showed Electromyography recovery in muscles that were previously affected by the lesion. Despite this, muscular atrophy caused by previously denervation persisted. Looking forward to improve muscular trophism and contractile capacity we performed intramuscular cell implant in muscles that show electromyography recovery. Method: In January 2016, 10 chronic and complete SCI patients who recovered electromyography register after neural progenitor cell implant received intramuscular cell implant. The implant consisted in a co culture of autologous muscular progenitor cells and effector T lymphocytes. Muscles that recover electromyography activity in response to voluntary order were selected. Using ultrasound guide cells were injected in between muscle fibers. Each muscle received 6–8 implants with 6 weeks in between. All patients continued with the rehabilitation program. Results: All implants were well tolerated. Implanted muscles showed: volume increase, improvement in contractile capacity (strength and range of motion). Significant changes in ultrasound and histology were also observed. The first changes were observed after three weeks; they persisted and improved after consecutive muscular implants. Conclusion: In previously denervated muscles, even though muscular electrical activity associated with voluntary order has been recovery, improving muscular conditions allows to achieve functional response (improvement in trophism and contractile function). To achieve this goal, intramuscular cell implant seems to be effective. The amount of muscular cell that need to be implanted depends on the muscular volume and the severity of the atrophy.
LB47 MAKING MULTIPLE THERAPEUTIC CELL PRODUCTS FROM A CGMP-COMPLIANT IPSC LINE Xianmin Zeng1,2,3, Ying Pei3, Helen Cifuentes1, Thelma Garcia1, Deepak Lamba1, Mahendra Rao2 1 Buck Institute, Novato, California, United States, 2NxCell Inc, Novato, California, United States, 3XCell Science Inc, Novato, California, United States Induced pluripotent stem cell (iPSC) can differentiate into multiple phenotypes, and indeed multiple protocols for such differentiation have been established. Although the efficiency of different protocols is variable, most protocols work with most lines with some tweaking. To confirm this hypothesis and to make a clinically compliant line available for independent evaluation, we have taken the current Good Manufacture Practice (cGMP)-compliant iPSC line developed by the NIH, and used it to develop cellular products that are currently being considered for therapy. We focused on retinal cells, MSC, NSC, dopaminergic neurons and astrocytes. We obtained a WCB from the NIH and prepared a stock at passage 20. After testing quality we used it to generate multiple products using standard protocols. To confirm that these protocols could also be used for other iPSC lines we obtained a second clinically compliant line and tested the reproducibility of our methodology. Our results confirmed that the same protocols could be used with minimal modifications with multiple qualified lines. We believe that our demonstration that multiple products can be made from the same WCB bank and the same protocols can be used with multiple lines offers a path to a cost effective strategy for developing cellular products.