Cytotherapy最新文献

{"title":"The Future of Distributed Manufacturing: Rapid Production and Release of CAR-T on a Microfluidic Chip","authors":"T.I. Pasa ,&nbsp;A. Dietz ,&nbsp;K. Loutherback","doi":"10.1016/j.jcyt.2025.03.070","DOIUrl":"10.1016/j.jcyt.2025.03.070","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Chimeric Antigen Receptor-T (CAR-T) cell therapy is a powerful new tool in the fight against cancer, with approved products for treatment of cancer and promising results in non-malignant diseases. While CAR-T has shown clinical utility, its complex manufacturing process results in high costs, with the price of a single dose greater than $350k. The manufacturing process relies on centralized production in specialized cGMP clean rooms using highly trained personnel. Significant overhead and logistics are required to handle multiple patient samples at once and ensure transportation of patient material between treatment and manufacturing sites.</div><div>We have developed a microfluidic chip-based approach that can integrate all critical process elements of CAR-T manufacturing (cell isolation, washing, culture, characterization and release testing) into a closed system. Critical design features allow cost-effective optimization and validation of new protocols from research scale to clinical production. We demonstrate the utility of this approach by making an antiCD19 CAR-T product.</div></div><div><h3>Methodology</h3><div>A microfluidic device was created with 310k microwells with 30 μm diameter and 100 μm depth. AntiCD19 CAR-T cells were produced by capturing CD3 cells into microwells from PBMCs using positive immunomagnetic isolation. Cells were labeled with CD3 and CD45 surface markers to characterize purity and total cell loading. Following characterization, perfusion culture was performed with T cell culture media and an anti-CD19 lentiviral vector (LVV) that also transduces GFP for 18 hours. After culture, the chip was imaged to characterize percentage of transduced cells by GFP expression. Export was performed by inverting the chip to sediment cells from wells and flushing to a collection vial. The entire process takes less than 24 hours.</div></div><div><h3>Results</h3><div>In three experiments, an average of 3.7e6 cells were loaded with 99.8% CD3 purity. After 18 hours of culture with LVV, 57% of cells were CAR+ as measured by GFP expression. Images of the chip before and after export indicate 2.61e6 cells were exported with post collection viability averaging 90%.</div></div><div><h3>Conclusion</h3><div>We have demonstrated an integrated, closed CAR-T manufacturing process capable of producing millions of cells in less than 24 hours. Our approach is scalable to produce larger doses has the potential to significantly reduce costs in a format that is easy to scale from concept to clinic.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S43"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Protocol and Machine Learning Algorithms for the Derivation of Autologous Human iPSC on an Automated Platform to Enable Clinical Semi-autonomous Manufacturing","authors":"W. Ansari ,&nbsp;T. Gorba ,&nbsp;H. Tran ,&nbsp;C. Pivaroff ,&nbsp;S. McCann ,&nbsp;N. Maloney ,&nbsp;C. Johnson ,&nbsp;D. Barken ,&nbsp;M. Pesavento ,&nbsp;E. Xu ,&nbsp;U. Nguyen ,&nbsp;A. Bratt-Leal ,&nbsp;K. Raineri","doi":"10.1016/j.jcyt.2025.03.072","DOIUrl":"10.1016/j.jcyt.2025.03.072","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>A robotics platform and control algorithms to enable reproducible manufacture of autologous induced pluripotent stem cell (iPSC) derived cell therapies is in development for intended clinical application, starting with dopaminergic neuron precursor cells for Parkinson's disease. Unlike allogeneic therapies, autologous therapies minimize the risk of rejection and eliminate the need for immune suppression. Current manufacturing technologies and instruments are unsuitable for production of autologous iPSCs for use in cell therapy applications. Here we report establishment of a repeatable automated workflow and in process control algorithms leveraging a robotics platform to produce clonal iPSC cultures comparable to those derived manually.</div></div><div><h3>Methodology</h3><div>Fibroblasts from three different donors were reprogrammed using a non-integrating method and cultured on the robotics platform. Automated weeding of residual fibroblasts around emerging iPSC colonies was performed, followed by automated positive selection and transfer to a new culture vessel. The platform also conducted automated feeding and passaging of the clonally derived iPSC lines.</div></div><div><h3>Results</h3><div>The iPSCs generated via the automated process showed high viability and expression of pluripotent cell identity markers Tra1-81 and Oct3/4 by flow cytometry and were comparable to manually derived control iPSC cultures. Deep learning models were trained from images captured and annotated by stem cell experts and later integrated to autonomously guide key process decisions, enabling semi-autonomous operation (Figures 1-3).</div></div><div><h3>Conclusion</h3><div>The automated iPSC generation process establishes a foundation for a semi-autonomous parallel process of autologous clinical iPSC production. This approach enhances throughput, ensures better traceability, and improves lot-to-lot consistency by mitigating human operator variability with respect to skill, judgement, and fatigue.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S44-S45"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of HLA-DR+CD1c-CD14-XCR1+ dendritic cells expressing CAR from induced pluripotent cells as an alternative treatment for cancer","authors":"S. Couto ,&nbsp;P. do Amaral Costa Ribeiro ,&nbsp;L. Catharino ,&nbsp;A. Ralph ,&nbsp;V. Rocha ,&nbsp;T. Gremen ,&nbsp;R.N. Ramos","doi":"10.1016/j.jcyt.2025.03.073","DOIUrl":"10.1016/j.jcyt.2025.03.073","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Immunotherapy based on T cells expressing CARs has been shown to be a promising strategy for treating hematological tumors. However, similar results have not been reported for solid tumors. Therefore, other immune cells have been explored to express CARs for immunotherapy applications. Dendritic cells are a heterogeneous cell population specialized in antigen presentation, with great migratory capacity, representing good candidates for solid tumor therapies. Due to their scarcity in peripheral blood and low proliferative potential, an \"off-the-shelf\" source such as induced pluripotent stem cells is needed to enable their use in cell therapy. Thus, our aim is to develop iPSC-DCs expressing CAR as an alternative treatment for solid tumors.</div></div><div><h3>Methodology</h3><div>Electroporation of the piggyBac transposon system was utilized to insert a CAR into an iPSC line. Differentiation of CAR-iPSCs was carried out following an established step-by-step protocol: (A) embryoid body formation, (B) generation of hematopoietic stem-like cells and (C) production of immune myeloid-like cells. Subsequently, differentiation into myeloid immune cells was induced using GM-CSF and IL-4 after 30 days. Flow cytometry was employed to analyze the phenotype of the differentiated cells. Co-culture of CAR-iPSC-DC cells with T cells was performed to observe induction of T cell proliferation.</div></div><div><h3>Results</h3><div>We have achieved an initial transfection efficiency of 2%. Sequential cell sorting increased the purity of CAR-expressing cells to 95%, with stable expression maintained for over 40 days. We obtained differentiated cells with typical myeloid immune cell morphology, including a few macrophage-like cells, with large cytoplasmic vacuoles and numerous cells with dendrite projections, closely resembling DC morphology. Moreover, cells presented a HLA-DR+CD64lowXCR1high phenotype, similar to cDC1 found in PB. Preliminary data showed that these cDC1-like cells are able to induce CD3+ T cell proliferation in vitro, similar to that observed with moDCs, reinforcing their similarity with PB DCs.</div></div><div><h3>Conclusion</h3><div>This study highlights the feasibility of using iPSC-derived DCs as an \"off-the-shelf\" cell source for cancer immunotherapies. The observed cDC1-like phenotype and the ability of these cells to stimulate CD3+ T cell proliferation in vitro provide a strong foundation for further investigation into their functionality and antitumor potential. The next steps include in vitro and in vivo analysis of cDC1-like function and their antitumor effects.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S45-S46"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Accelerating sterility testing: Symcel calScreener+ achieves results in <3 days with direct inoculation in non-destructive and continuous monitoring system","authors":"W. Paulander","doi":"10.1016/j.jcyt.2025.03.034","DOIUrl":"10.1016/j.jcyt.2025.03.034","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Sterility testing is a critical quality control process for cell and gene therapy products (CGTPs), where rapid and reliable detection of microbial contaminants is essential to meet regulatory standards and ensure patient safety. Traditional sterility testing methods, requiring up to 14 days, delay batch release and increase costs. The Symcel calScreener+ utilizes isothermal microcalorimetry to enable faster, phenotypic detection of microbial contaminants in a non-destructive and product-matrix-independent manner. This study evaluates the calScreener+ 3-day sterility test for its accuracy, reliability, detection times, and robustness in addressing challenges such as small sample volumes and complex matrices with high eukaryotic cell concentrations.</div></div><div><h3>Methodology</h3><div>The calScreener+ monitors metabolic activity in product samples as low as 0.05 ml to detect microbial growth. Validation studies assessed its Limit of Detection (LOD), detection times, comparability, and specificity range across 20 microbial strains relevant to regulatory guidelines. The evaluation used TSB and FTM media at dual temperatures (25°C and 35°C) and included key microorganisms such as <em>Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus brasiliensis</em>, and <em>Cutibacterium acnes</em>. Robustness and ruggedness were assessed under varying conditions, including temperature fluctuations and growth inhibitors. Statistical analyses confirmed reliability and reproducibility.</div></div><div><h3>Results</h3><div>The calScreener+ demonstrated an LOD of &lt;5 CFU and delivered results within 3 days, even in complex matrices with high eukaryotic cell concentrations (10^6–10^8 cells/ml). Detection times included <em>S. aureus, P. aeruginosa</em>, and <em>C. albicans</em> within 24 hours; <em>A. brasiliensis</em> within 30 hours; and <em>C. acnes</em> within 60 hours. It effectively detected a broad range of microorganisms relevant to sterility testing, including critical contaminants such as <em>Burkholderia</em> complex and <em>Penicillium</em> species, with robust performance under varying conditions.</div></div><div><h3>Conclusion</h3><div>The Symcel calScreener+ offers a sensitive and rapid solution for sterility testing in CGTPs, enabling phenotypic detection of microbial contaminants within days instead of weeks. Its small sample volume minimizes material use, ideal for limited batch sizes. By eliminating enrichment steps and reducing detection times, the calScreener+ streamlines workflows and enhances operational efficiency, positioning it as a transformative tool for pharmaceutical quality control.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S24"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888113","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Multifunctional Microfluidic Device for Tailoring Analytical Assay Development for Multiproduct Cell Therapy Products (CTPs)","authors":"A. Nisar ,&nbsp;S. Singh ,&nbsp;P. Gururaj Joshi ,&nbsp;K.R. Pandit ,&nbsp;G. Narula ,&nbsp;U. Mohanty ,&nbsp;J. Krishnan","doi":"10.1016/j.jcyt.2025.03.022","DOIUrl":"10.1016/j.jcyt.2025.03.022","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>The rising demand for cell therapies necessitates innovative tools to streamline analytical development &amp; QC assays. We propose a microfluidic device to transform analytical workflows by handling small samples, reducing reagents, &amp; automating processes.</div></div><div><h3>Methodology</h3><div>This device minimizes reagent requirements while maintaining high throughput, enabling cost-efficient and scalable processes. Its microfluidic architecture precisely manipulates &amp; separates T cells from whole blood samples, reducing sample volume significantly compared to conventional methods. Beyond T cell isolation, the device integrates a multifunctional platform capable of performing analytical assays, including ELISA, effectively replacing traditional 96-well plates. This integration enhances its utility by enabling real-time cytokine profiling, immunophenotyping, high-sensitivity biomarker detection, single-cell analysis &amp; multiplexed assays on a single platform.</div></div><div><h3>Results</h3><div>The design of this microfluidic device was developed after thorough review of existing literature &amp; iterative modifications. Key considerations included device dimensions, operational sequences, particle size compatibility, reagent material compatibility, hydrophobicity, and detection sensitivity. The final configuration comprises two pairs of hexagonal channels connected via one inlet and two outlets, utilizing a herringbone structure to optimize cell capture &amp; retention. This structure disrupts flow, increasing turbulence &amp; enhancing cell interaction with channel surfaces. The device leverages microfluidic principles, manipulating fluid behavior using microliter to picoliter volumes with channel dimensions in the micrometer range. It adheres to the Hele-Shaw flow property, defined as Stokes Flow between two parallel plates separated by a narrow gap, ensuring laminar flows with low Reynolds numbers. Fabricated from advanced polymers like PDMS and PMMA, the device ensures durability, biocompatibility, and seamless integration with automated workflows. It supports parallel processing of multiple samples, reducing assay time while maintaining precision and reproducibility. The system is compatible with existing laboratory tools like flow cytometry &amp; imaging systems, enabling effortless adoption in clinical and industrial settings.</div></div><div><h3>Conclusion</h3><div>By combining versatility, scalability, and cost-efficiency, this microfluidic system represents a robust platform for CAR T analytical development, poised to advance the manufacturing and QC landscapes of CGT.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S18"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acute respiratory distress vs healthy lung environments differently affect mesenchymal stromal cell extracellular vesicle miRNAs","authors":"Sara Rolandsson Enes ,&nbsp;Irakli Dzneladze ,&nbsp;Thomas H. Hampton ,&nbsp;Samuel L. Neff ,&nbsp;Lori Asarian ,&nbsp;Jayita Barua ,&nbsp;Tobias Tertel ,&nbsp;Bernd Giebel ,&nbsp;Nicolas Pereyra ,&nbsp;David H. McKenna ,&nbsp;Pingzhao Hu ,&nbsp;Erica Acton ,&nbsp;Alix Ashare ,&nbsp;Kathleen D. Liu ,&nbsp;Anna D. Krasnodembskaya ,&nbsp;Karen English ,&nbsp;Bruce A. Stanton ,&nbsp;Patricia R.M. Rocco ,&nbsp;Michael A. Matthay ,&nbsp;Claudia C. dos Santos ,&nbsp;Daniel J. Weiss","doi":"10.1016/j.jcyt.2025.01.006","DOIUrl":"10.1016/j.jcyt.2025.01.006","url":null,"abstract":"<div><div>The acute respiratory distress syndrome (ARDS) inflammatory environment alters mesenchymal stromal cell (MSC) gene and protein expression but effects on microRNA (miRNA) content of MSC-extracellular vesicle (EVs) remain unknown. To assess this, sequencing analysis of EV-miRNAs prepared from human bone marrow-derived MSCs (hMSCs) exposed <em>ex vivo</em> to bronchoalveolar lavage fluid (BALF) from ARDS patients or healthy volunteers (HV) identified a number of differentially expressed miRNAs. Discriminant, differential expression, and functional enrichment analyses identified 14 miRNAs significantly changed following ARDS versus HV BALF exposure. Network analysis showed 4 (miR-760, miR-3175, miR-885-3p, and miR-766-3p) of the 14 EV-miRNAs formed a regulatory “hub”, suggesting co-targeting of specific gene pathways. <em>In silico</em> prediction identified a number of pathways important in lung injury. Two miRNAs involved in regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), miRNA-145-5p and miRNA-138-5p, were also significantly increased in ARDS BALF-exposed hMSCs EVs. Functionally, EVs from hMSCs exposed to either ARDS or HV BALF had differential effects on CFTR Cl^- secretion by cultured primary human bronchial epithelial cells, an effect predicted to reduce mucociliary clearance. The potential clinical impact of these finding highlights the need for further studies assessing the role of hMSC-EV miRNAs in regulating lung inflammation and mucociliary clearance.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages 581-596"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143411368","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of CD141+vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells","authors":"Y. Son ,&nbsp;G. Park ,&nbsp;D. Hwang ,&nbsp;S. Yim ,&nbsp;H. Hong","doi":"10.1016/j.jcyt.2025.03.025","DOIUrl":"10.1016/j.jcyt.2025.03.025","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Critical limb ischemia (CLI) is a condition characterized by insufficient blood flow to the lower limbs, resulting in severe ischemia and potentially leading to amputation. This study aims to identify novel vasculogenic precursor cells (VPCs) in human bone marrow and evaluate their efficacy in combination with bone marrow-derived mesenchymal stem cells (BM-MSCs) for the treatment of CLI</div></div><div><h3>Methodology</h3><div>VPCs and BM-MSCs from bone marrow were cultured <em>ex vivo</em> and comparatively characterized, whose therapeutic effects on neovascularization and long-term tissue regeneration were investigated after singular or a combination transplantation in a mouse CLI model</div></div><div><h3>Results</h3><div>VPCs, expressing high levels of hepatocyte growth factor and c-MET, were identified from human bone marrow aspirates. These cells exhibited strong vasculogenic capacity <em>in vitro</em> but possessed a cellular phenotype distinct from those of previously reported endothelial precursor cells in circulation or cord blood. They also expressed most surface markers of BM-MSCs and demonstrated multipotent differentiation ability. Screening of 376 surface markers revealed that VPCs uniquely display CD141 (thrombomodulin). CD141⁺VPCs are present in BM aspirates as a rare population and can be expanded <em>ex vivo</em> with a population doubling time of approximately 20 h, generating an elaborate vascular network even under angiogenic factor-deficient conditions and recruiting BM-MSCs to the network as pericyte-like cells. Intramuscular transplantation of a combination of human CD141⁺VPCs and BM-MSCs at a ratio of 2:1 resulted in limb salvage, blood flow recovery, and regeneration of large vessels in the femoral artery-removed CLI model, with an efficacy superior to that of singular transplantation. Importantly, large arteries and arterioles in dual cell transplantation expressed human CD31 in the intima and human α-smooth muscle actin in media layer at 4 and 12 weeks, indicating their lineage commitment to endothelial cells and vascular smooth muscle, respectively, <em>in vivo</em>.</div></div><div><h3>Conclusion</h3><div>Dual cell therapeutics comprising human CD141+VPCs and BM-MSCs could be developed for clinical trial to cure human PAD.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S19-S20"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancing Allogenic Cell Therapy with Automated iPSC Processing and Engineering: Benefits of a Closed Modular Approach.","authors":"R.R. Somasagara ,&nbsp;O. Cohen ,&nbsp;Y. Kim ,&nbsp;P. Chaluvappa ,&nbsp;L. Bailey Steinitz ,&nbsp;V. Chandra ,&nbsp;C.T. Dargitz ,&nbsp;A. Chandrasekaran ,&nbsp;N. Ravinder","doi":"10.1016/j.jcyt.2025.03.077","DOIUrl":"10.1016/j.jcyt.2025.03.077","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have emerged as a promising platform for next-generation immunotherapy, offering a homogeneous, scalable and versatile approach for consistent large-scale manufacturing of off the shelf allogeneic therapies. This approach involves multiple steps including iPSC culturing and banking followed by gene editing and differentiation to tumor specific iNK cells to enhance cytotoxicity and tumor-targeting specificity, while minimizing risks of graft-versus-host disease. Building closed modular automated workflows will help minimize risks associated with manual processes associated with these methods. Through this work we built closed automated cell harvest, gene delivery and editing protocols that can enable iNK-based cell therapy manufacturing.</div></div><div><h3>Methodology</h3><div>In our current workflow, we cultured and expanded iPSCs up to a billion cells in a 10-layer cell factory system for master cell bank preparation. iPSC manual process and harvest in 10-layer cell factory system is very labor intensive and prone to contamination. Utilizing CTS Rotea counterflow centrifugation system minimized human intervention at multiple stages, including removal of media, washing of cells, addition of cell detachment media, collecting, concentrating iPSCs and delivering cells to collecting bags. Using this protocol, we processed the harvesting of entire iPSC culture in 10-layer cell factory system in a single batch to create a master bank for cell therapy development. We then successfully used these iPSC banks and carried out CRISPR-based non-viral gene editing using Neon NxT or CTS Xenon closed automated electroporation system for gene delivery, GMP grade CTS™ StemFlex media and high fidelity CRISPR-Cas9 system.</div></div><div><h3>Results</h3><div>The iPSCs processed using this method retain pluripotency characteristics with good viability and expansion rate. The CRISPR-based non-viral gene editing results shows successful generation of engineered CAR-iPSC with reproducible KI efficiency of up to 15%. We established the optimal target gene and promoter combination to stabilize transgene expression during the differentiation of iPSCs to iNK cells.</div></div><div><h3>Conclusion</h3><div>With methods developed through this work, we successfully generated potent CAR iNK cells. Together the workflows described here utilizing the clean room compliant closed automated cell processing and gene delivery platforms and the GMP compatibility iPSC and NK media systems enable clinical scale iNK cell therapy manufacturing.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S47-S48"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888221","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Scalable Production of Hematopoietic Stem Cells and Microglia from iPSCs Using Stirred Tank Bioreactors for Consistent Cell Therapy Manufacturing","authors":"J. Cotovio ,&nbsp;P. van Loenen ,&nbsp;S. Braam ,&nbsp;M. Argenziano","doi":"10.1016/j.jcyt.2025.03.040","DOIUrl":"10.1016/j.jcyt.2025.03.040","url":null,"abstract":"<div><h3>Background and Aims</h3><div>Hematopoietic stem cells (HSCs) and microglia are vital for blood, immune, and central nervous system (CNS) development, offering significant potential for cell therapy and neurodegenerative disease treatment. However, producing these cells from human induced pluripotent stem cells (hiPSCs) with consistent quality and scalability remains challenging due to complex differentiation processes. Ncardia has developed robust protocols enabling large-batch, scalable differentiation of HSCs and microglia using stirred tank bioreactors.</div></div><div><h3>Methodology</h3><div>We successfully transitioned small-scale 2D adherent differentiation into 250mL stirred tank bioreactors, aiming to scale up to 1L and 3L systems. A Design of Experiments (DoE) approach was used to optimize critical process parameters (CPPs), including dissolved oxygen, agitation speed, and inoculation density. Parallel protocol testing and in-process monitoring, supported by data-driven analysis, enabled rapid optimization and high reproducibility.</div></div><div><h3>Results</h3><div>Our protocols generated high-quality HSCs and microglia with key functional properties. hiPSC-derived HSCs displayed typical morphology, expressed essential markers (&gt;90% CD34, CD45, CD43), and differentiated into multiple immune cell lineages such as NK cells, macrophages, and microglia, reinforcing their cell therapy potential. Similarly, Ncyte® Microglia expressed core markers (&gt;80% IBA1, TMEM119, CX3CR1) and exhibited functional capabilities like phagocytosis and cytokine secretion in response to inflammatory stimuli. Their ability to co-culture with neurons further enhances their applications in drug discovery and toxicity testing.</div></div><div><h3>Conclusion</h3><div>By integrating expertise in stem cell biology with advanced bioreactor technology, Ncardia enables scalable, high-quality production of HSCs and microglia. This innovation accelerates cell therapy development while supporting regenerative medicine, immunotherapy, and neurodegenerative disease research.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S27"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of very small embryonic-like stem cells (VSELs) in the peripheral blood of rabbits subjected to induced retinal vein occlusion as a proposed early detection tool for the disease","authors":"E. Gounari ,&nbsp;A. Komnenou ,&nbsp;E. Kofidou ,&nbsp;V. Karampatakis ,&nbsp;G. Koliakos","doi":"10.1016/j.jcyt.2025.03.041","DOIUrl":"10.1016/j.jcyt.2025.03.041","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Very Small Embryonic-like Stem Cells (VSELs) have been successfully detected in human peripheral blood following mobilization under stressful conditions (Bhartiya et al.2016). The aim of the present study is the detection of VSELs in a pharmaceutically induced animal model and the correlation of their number with pro-inflammatory cytokine levels andanimals’ clinical evaluation.</div></div><div><h3>Methodology</h3><div>14 New Zealand rabbits were divided into group I-RVO and group II-control. For the RVO induction group I received intravitreal injections of PD0325901 at a dose of 0.1mL/eye. Successful RVO induction was confirmed through clinical evaluation, histological examinations, quantification of secreted cytokines in the vitreous fluid, and molecular analysis of retinal tissues according to our group's recently published results (Gounari et al.2022).</div><div>Peripheral blood was collected at days 0,7,14 and 36 while VSELs isolated and counted as we have previously described (Gounari et al.2018).</div></div><div><h3>Results</h3><div>Counting of VSELs showed a statistically significant increase in the number of VSELs in the first days compared to the control group (*P=0.03), which was markedly reduced after 2 weeks which proves the direct mobilization of VSELs into the peripheral blood. H&amp;E staining of cell smears in RVO-induced animal group revealed very small sized cells with high nucleo-cytoplasmic ratio able to form embryonic bodies (EBs) in hanging-drop cultures. The positive alkaline phosphatase staining, in combination with the overexpression of the Oct3/4, Nanog and Sox-2 transcription factors confirmed the existence of undifferentiated cells with embryonic like features as a response to damage. Secreted levels of TNF-α, IL-6 and IL-8 measured in plasma samples are related to the numbers of detected-VSELs.</div></div><div><h3>Conclusion</h3><div>We here present a method for VSELs isolation and quantification from RVO-induced animals’ blood. Further research is needed in order to develop an optimized protocol based on VSELs detection able to be applied for the early diagnosis of RVO or other retinal vascular diseases.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S27-S28"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}