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Protocol for the direct culture of thawed umbilical cord tissue
e20
23rd Annual ISCT Meeting
Conventionally, manufacturing of cell therapy products is performed by manual operations and quality is usually controlled by testing of final products. However, the manufacture of high quality cell therapy products with minimum out-ofspecifications and lower production cost requires the next generation of cell therapy manufacturing with fully automated system. This will enable consistent high quality production from highly variable raw materials and processes through non-invasive online monitoring providing quality control during manufacture. We performed a proof of concept study for automated cell therapy manufacturing and in-process quality monitoring through in-depth analyses during a 10 passage culture of CGT-RCiB10, a research-grade hiPSC established by Cell and Gene Therapy Catapult from a GMP pre-seed lot. Both manual and automated cultures using Essential-8/Vitronectin-N/Accutase were completed. At each passage, daily microscope imaging and analysis of spent medium (pH, DO, DCO2, glucose, lactic acid, glutamine, glutamic acid, and 95 metabolite panels by liquid chromatography-tandem mass spectrometry (LC-MS/ MS)) were performed. At P0, P5 and P10, detailed cellular characterization, including gene expression profiling by TaqMan®Scorecard, cell surface marker and transcription factor analysis by flow cytometry, karyotyping (G-banding and CGH) and directed differentiation assays, were completed. Multivariate analysis was performed and the results indicate that the cells grown in the automated system for 10 passages maintained pluripotency, high viability, normal karyotype, and three germ layer differentiation potential, and highly reproducible and more consistent data was obtained by automated culture compared to manual culture. Integrated analysis of metabolic profiles quantified by LC-MS/MS and in-line sensors and numeric features of cell morphology is a powerful approach for better understanding of the process, and can be used for noninvasive online quality monitoring for automated cell therapy manufacturing. Based on this learning, Tokyo Electron is currently developing GMPcompliant automated systems equipped with in-process monitoring which enable scale-up production and differentiation of clinical grade hPSCs. LB37 PROTOCOL FOR THE DIRECT CULTURE OF THAWED UMBILICAL CORD TISSUE Julieta Henao1,2, Carlos Alberto Isaza1,2, Jainer Aranzazu1 1 Stem cell Laboratory, CeMaB, Pereira, Risaralda, Colombia, 2Laboratorio de genética médica y células madre, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia After direct cryopreservation of umbilical cord tissue, the recovery of viable and enough mesenchymal stem cells should be ensured to allow appropriate expansion and effective utilization. In the different protocols for the separation and subsequent culture of umbilical cord (CU) stem cells, enzymatic digestion is used; this causes a chemical stress for the tissue and damages inherent to the freezing are added, which can affect cellular recovery, causes unwanted cellular changes, as well as early cell senescence and quiescence. We evaluated in 10 thawed samples the result of direct culture of the umbilical cord fragments seeded without previous use of collagenase: at the end of the first pass (P1) of a total of 5 cm of CU, 10 × 106 viable cells positive were obtained for CD73, CD90, CD105 and CD45 and CD31 negative. This CU cryopreservation method and its direct post-freeze culture reduces time, costs and improves vital mesenchymal stem cells in suitable amounts for use in different therapies. LB38 EFFECTS OF HYPOXIC CULTURE SYSTEM ON CANINE STEM CELL PLASTICITY Jienny Lee1, Jeong Su Byeon1, Sunray Lee2, Hyun Sook Park2, In-Soo Cho1, Sang-Ho Cha1 1 Viral Disease Division, Animal and Plant Quarantine Agency, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea, 2Cefobio, Seoul, Republic of Korea Mesenchymal stem cells (MSCs) have the ability to differentiate into multilineage cells, which confers great promise for use in regenerative medicine. General cell culture systems supply oxygen content of approximately 21%. Oxygen concentration is an important component of stem cell “niche”, where it plays a fundamental role in maintaining the stem cell plasticity and proliferation. In this study, canine MSCs (cMSCs) were isolated from adipose tissue. The cells represented stemness markers (Oct4, Sox2, and Nanog) and differentiation potentials into the mesoderm at early passages. The aim of this study was to evaluate the effects of hypoxic culture condition on the stemness expression and differentiation potentials into mesoderm for the optimal culturing of MSCs. We
observed that the proliferation of the cMSCs meaningfully increased when cultured under hypoxic culture condition than in normoxic culture condition, during 7 consecutive passages. We also examined the multipotency of hypoxic conditioned cMSCs using quantitative real-time RT-PCR. As results, we found that the expression levels of stemness genes such as Oct-4, Nanog, and Sox-2 were increased in hypoxic culture condition when compared to the normoxic culture condition. We also found that hypoxic culture condition significantly expressed cell survival-related genes such as glucose transporter, TERT, and telomere holoenzyme complex than seen under the normoxic culture condition. Also, expressions of the chondrogenic differentiation potential-related genes (aggrecan and collagen type-II alpha I) of cMSCs under hypoxic culture condition were found to be significantly higher than that of cMSCs under normoxic culture condition. Dogs are a promising biomedical model suitable for evaluation of novel therapies, such as those employing stem cells in spontaneous disease and experimental settings. Therefore, studies in dogs should help in optimization of growth and differentiation of cMSCs, to facilitate future stem cell-based repair and regenerative therapies. Taken together, these results suggest that hypoxic culture condition has the ability to induce proliferation of MSCs and augment their chondrogenic potential. This study suggests that cell proliferation of c-MSC under hypoxic culture condition could be beneficial, when considering these cells for cell therapies of canine bone-related diseases.
LB39 OPTIMAL CULTURING CONDITION FOR BOVINE MESENCHYMAL STEM CELLS Jienny Lee, Mi Jeong Park, Jeong Su Byeon, Na-Yeon Gu, In-Soo Cho, Sang-Ho Cha Viral Disease Division, Animal and Plant Quarantine Agency, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea Mesenchymal stem cells (MSCs) have the ability to differentiate into several cell types; these characteristics have implications for cell therapy and biotechnology fields. It has been reported that the bovine stem cells isolated from various tissues such as adipose, bone marrow, umbilical cord blood, amniotic fluid, and liver. However, bovine tongue epithelium-derived MSCs have not been fully studied. In this study, we tried isolation and characterization of bovine tongue epithelium-derived MSCs by investigating phenotype morphology, proliferating properties, and stemness. Also, to select the optimal culture condition for cell growth, 3 conditions were tested for in vitro culture of MSCs by examining passage 6, including IMDM/F12 medium, DMEM-low glucose/F12 medium, and DMEM-low glucose medium, supplemented with 1% penicillin/streptomycin and 10% FBS. In this study, bovine tongue epithelium-derived cells were isolated by collagenase digestion method. For characterization of MSCs, cell surface markers were analyzed by flow cytometry (FACS) and quantitative real-time RTPCR (qRT-PCR) at 4 passages (P4). FACS analysis revealed that the cells were strongly positive for CD44, and negative for CD45. In regards to qRT-PCR, the cells positively expressed CD29, CD44, CD90, and CD105, but negative for CD45, and MHC-II. Also, the cells presented stemness markers such as Oct3/ 4, Sox-2, and Nanog. Among the 3 different culture conditions tested (IMDM/ F12 medium, DMEM-low glucose/F12 medium, and DMEM-low glucose medium), the cells cultured in DMEM-low glucose medium demonstrated the highest cumulative population doubling level and stemness. Unlike embryonic stem cells, in vitro cultured MSCs easily undergo cellular senescence and lose their stemness. Therefore, it is very important to optimize the in vitro culture condition of MSC.
LB40 A CGMP SENDAI VIRAL REPROGRAMMING KIT FOR GENERATION OF CLINICAL-GRADE IPSC Chad C. MacArthur, Uma Lakshmipathy Cell Biology, Thermo Fisher Scientific, Carlsbad, California, United States The induced pluripotent stem cell (iPSC) field has grown immensely in the past ten years, starting with basic research, and now moving more toward clinical applications. As iPSC move towards the clinic, it is vital that high quality cells are used. These cells should be free of reprogramming transgenes, free of adventitious agents, genetically stable, and functionally pluripotent. Typical reprogramming workflows often include animal origin components, such as fetal bovine serum (FBS) or bovine serum albumin (BSA), which can be potential sources of adventitious agents. IPSC that are generated in a xeno-free,
23rd Annual ISCT Meeting
Conventionally, manufacturing of cell therapy products is performed by manual operations and quality is usually controlled by testing of final products. However, the manufacture of high quality cell therapy products with minimum out-ofspecifications and lower production cost requires the next generation of cell therapy manufacturing with fully automated system. This will enable consistent high quality production from highly variable raw materials and processes through non-invasive online monitoring providing quality control during manufacture. We performed a proof of concept study for automated cell therapy manufacturing and in-process quality monitoring through in-depth analyses during a 10 passage culture of CGT-RCiB10, a research-grade hiPSC established by Cell and Gene Therapy Catapult from a GMP pre-seed lot. Both manual and automated cultures using Essential-8/Vitronectin-N/Accutase were completed. At each passage, daily microscope imaging and analysis of spent medium (pH, DO, DCO2, glucose, lactic acid, glutamine, glutamic acid, and 95 metabolite panels by liquid chromatography-tandem mass spectrometry (LC-MS/ MS)) were performed. At P0, P5 and P10, detailed cellular characterization, including gene expression profiling by TaqMan®Scorecard, cell surface marker and transcription factor analysis by flow cytometry, karyotyping (G-banding and CGH) and directed differentiation assays, were completed. Multivariate analysis was performed and the results indicate that the cells grown in the automated system for 10 passages maintained pluripotency, high viability, normal karyotype, and three germ layer differentiation potential, and highly reproducible and more consistent data was obtained by automated culture compared to manual culture. Integrated analysis of metabolic profiles quantified by LC-MS/MS and in-line sensors and numeric features of cell morphology is a powerful approach for better understanding of the process, and can be used for noninvasive online quality monitoring for automated cell therapy manufacturing. Based on this learning, Tokyo Electron is currently developing GMPcompliant automated systems equipped with in-process monitoring which enable scale-up production and differentiation of clinical grade hPSCs. LB37 PROTOCOL FOR THE DIRECT CULTURE OF THAWED UMBILICAL CORD TISSUE Julieta Henao1,2, Carlos Alberto Isaza1,2, Jainer Aranzazu1 1 Stem cell Laboratory, CeMaB, Pereira, Risaralda, Colombia, 2Laboratorio de genética médica y células madre, Universidad Tecnológica de Pereira, Pereira, Risaralda, Colombia After direct cryopreservation of umbilical cord tissue, the recovery of viable and enough mesenchymal stem cells should be ensured to allow appropriate expansion and effective utilization. In the different protocols for the separation and subsequent culture of umbilical cord (CU) stem cells, enzymatic digestion is used; this causes a chemical stress for the tissue and damages inherent to the freezing are added, which can affect cellular recovery, causes unwanted cellular changes, as well as early cell senescence and quiescence. We evaluated in 10 thawed samples the result of direct culture of the umbilical cord fragments seeded without previous use of collagenase: at the end of the first pass (P1) of a total of 5 cm of CU, 10 × 106 viable cells positive were obtained for CD73, CD90, CD105 and CD45 and CD31 negative. This CU cryopreservation method and its direct post-freeze culture reduces time, costs and improves vital mesenchymal stem cells in suitable amounts for use in different therapies. LB38 EFFECTS OF HYPOXIC CULTURE SYSTEM ON CANINE STEM CELL PLASTICITY Jienny Lee1, Jeong Su Byeon1, Sunray Lee2, Hyun Sook Park2, In-Soo Cho1, Sang-Ho Cha1 1 Viral Disease Division, Animal and Plant Quarantine Agency, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea, 2Cefobio, Seoul, Republic of Korea Mesenchymal stem cells (MSCs) have the ability to differentiate into multilineage cells, which confers great promise for use in regenerative medicine. General cell culture systems supply oxygen content of approximately 21%. Oxygen concentration is an important component of stem cell “niche”, where it plays a fundamental role in maintaining the stem cell plasticity and proliferation. In this study, canine MSCs (cMSCs) were isolated from adipose tissue. The cells represented stemness markers (Oct4, Sox2, and Nanog) and differentiation potentials into the mesoderm at early passages. The aim of this study was to evaluate the effects of hypoxic culture condition on the stemness expression and differentiation potentials into mesoderm for the optimal culturing of MSCs. We
observed that the proliferation of the cMSCs meaningfully increased when cultured under hypoxic culture condition than in normoxic culture condition, during 7 consecutive passages. We also examined the multipotency of hypoxic conditioned cMSCs using quantitative real-time RT-PCR. As results, we found that the expression levels of stemness genes such as Oct-4, Nanog, and Sox-2 were increased in hypoxic culture condition when compared to the normoxic culture condition. We also found that hypoxic culture condition significantly expressed cell survival-related genes such as glucose transporter, TERT, and telomere holoenzyme complex than seen under the normoxic culture condition. Also, expressions of the chondrogenic differentiation potential-related genes (aggrecan and collagen type-II alpha I) of cMSCs under hypoxic culture condition were found to be significantly higher than that of cMSCs under normoxic culture condition. Dogs are a promising biomedical model suitable for evaluation of novel therapies, such as those employing stem cells in spontaneous disease and experimental settings. Therefore, studies in dogs should help in optimization of growth and differentiation of cMSCs, to facilitate future stem cell-based repair and regenerative therapies. Taken together, these results suggest that hypoxic culture condition has the ability to induce proliferation of MSCs and augment their chondrogenic potential. This study suggests that cell proliferation of c-MSC under hypoxic culture condition could be beneficial, when considering these cells for cell therapies of canine bone-related diseases.
LB39 OPTIMAL CULTURING CONDITION FOR BOVINE MESENCHYMAL STEM CELLS Jienny Lee, Mi Jeong Park, Jeong Su Byeon, Na-Yeon Gu, In-Soo Cho, Sang-Ho Cha Viral Disease Division, Animal and Plant Quarantine Agency, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea Mesenchymal stem cells (MSCs) have the ability to differentiate into several cell types; these characteristics have implications for cell therapy and biotechnology fields. It has been reported that the bovine stem cells isolated from various tissues such as adipose, bone marrow, umbilical cord blood, amniotic fluid, and liver. However, bovine tongue epithelium-derived MSCs have not been fully studied. In this study, we tried isolation and characterization of bovine tongue epithelium-derived MSCs by investigating phenotype morphology, proliferating properties, and stemness. Also, to select the optimal culture condition for cell growth, 3 conditions were tested for in vitro culture of MSCs by examining passage 6, including IMDM/F12 medium, DMEM-low glucose/F12 medium, and DMEM-low glucose medium, supplemented with 1% penicillin/streptomycin and 10% FBS. In this study, bovine tongue epithelium-derived cells were isolated by collagenase digestion method. For characterization of MSCs, cell surface markers were analyzed by flow cytometry (FACS) and quantitative real-time RTPCR (qRT-PCR) at 4 passages (P4). FACS analysis revealed that the cells were strongly positive for CD44, and negative for CD45. In regards to qRT-PCR, the cells positively expressed CD29, CD44, CD90, and CD105, but negative for CD45, and MHC-II. Also, the cells presented stemness markers such as Oct3/ 4, Sox-2, and Nanog. Among the 3 different culture conditions tested (IMDM/ F12 medium, DMEM-low glucose/F12 medium, and DMEM-low glucose medium), the cells cultured in DMEM-low glucose medium demonstrated the highest cumulative population doubling level and stemness. Unlike embryonic stem cells, in vitro cultured MSCs easily undergo cellular senescence and lose their stemness. Therefore, it is very important to optimize the in vitro culture condition of MSC.
LB40 A CGMP SENDAI VIRAL REPROGRAMMING KIT FOR GENERATION OF CLINICAL-GRADE IPSC Chad C. MacArthur, Uma Lakshmipathy Cell Biology, Thermo Fisher Scientific, Carlsbad, California, United States The induced pluripotent stem cell (iPSC) field has grown immensely in the past ten years, starting with basic research, and now moving more toward clinical applications. As iPSC move towards the clinic, it is vital that high quality cells are used. These cells should be free of reprogramming transgenes, free of adventitious agents, genetically stable, and functionally pluripotent. Typical reprogramming workflows often include animal origin components, such as fetal bovine serum (FBS) or bovine serum albumin (BSA), which can be potential sources of adventitious agents. IPSC that are generated in a xeno-free,