Cytotherapy最新文献

{"title":"Single Extracellular Vesicle Profiling: Harnessing NanoFlow Cytometry for Quantitative Surface Marker Analysis","authors":"M. Cramer ,&nbsp;S. Lenzini ,&nbsp;E. Zakhem ,&nbsp;J.A. Rowley","doi":"10.1016/j.jcyt.2025.03.042","DOIUrl":"10.1016/j.jcyt.2025.03.042","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Heterogeneity of extracellular vesicle (EV) populations complicates their study, as traditional bulk analysis methods fail to capture individual differences in size, content, and surface markers. Single-vesicle analysis offers a more precise approach, enabling detailed characterization of EV subpopulations. In this study, we developed protocols for calibrated NanoFlow cytometry to quantify single-vesicle surface marker expression and applied them to purified MSC-EVs from three different tissue sources.</div></div><div><h3>Methodology</h3><div>We generated highly purified MSC-EVs through RoosterBio established production and purification protocols. EV tetraspanin identity markers (CD81 and CD63) and MSC identity marker (CD73) were selected for single-vesicle analysis. For each antibody, optimization steps were performed to identify the optimal staining protocol. Beads with known equivalent reference fluorophore (ERF) values were obtained from NanoFCM. Data was acquired on the NanoFCM Flow NanoAnalyzer and single-vesicle analysis methods were established to quantify the number of CD molecules per particle. The lower limit of detection was determined by background fluorescence. We then applied this analysis to purified EVs from bone marrow (BM), umbilical cord (UC), and adipose (AD) MSC-EVs.</div></div><div><h3>Results</h3><div>Antibody titration was used to select a dilution of 800X for samples at 1 × 10¹⁰ P/mL. A standard curve was generated from ERF beads to calibrate arbitrary fluorescence intensity to units of Alexa Fluor™ 488. The antibody-specific limit of detection for positive expression was between 4 – 10 molecules per particle. Across all tissue sources, particles were 50 – 70% CD81+, 40 – 60% CD63+, and 15 – 35% CD73+. Single-vesicle analysis suggested CD81 and CD63 were enriched on AD-EVs (40 CD81, 53 CD63 per particle) compared to UC-EVs (33 CD81, 44 CD63 per particle) and BM-EVs (30 CD81, 41 CD63 per particle). CD73 was preferentially expressed on larger EVs and showed ∼20 CD73 per particle for all tissue sources.</div></div><div><h3>Conclusion</h3><div>We successfully developed single-vesicle quantitation of EV surface markers by calibrated NanoFlow cytometry. Application of this novel analysis not only showed retention of CD81, CD63, and CD73 in our highly purified EVs, but also discerned tissue- and marker-dependent differences in EV properties. This high-resolution characterization of EVs can advance our understanding of EV diversity, aid interpretation of EV bioactivity, and support development of methods to enrich specific EV subtypes.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S28"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887592","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":"Characterization of engineered CAR-Treg cells to induce immune tolerance in liver transplanted patients","authors":"M. Martinez-Llordella ,&nbsp;R. Kenefeck ,&nbsp;S. Seshadri ,&nbsp;F. Mehtar ,&nbsp;A. Voitovich ,&nbsp;C. Burke ,&nbsp;J. Wang-Jairaj ,&nbsp;L. Baylor-Curtis ,&nbsp;A. Lesniak ,&nbsp;A. Demetris ,&nbsp;N. Belmonte ,&nbsp;A. Sanchez-Fueyo ,&nbsp;L. Devey","doi":"10.1016/j.jcyt.2025.03.068","DOIUrl":"10.1016/j.jcyt.2025.03.068","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Regulatory T (Treg) cells play a critical role in maintaining immune tolerance and controlling inflammatory responses. Adoptive cell therapy with Tregs has been an exciting prospect for the modulation of immune responses in the clinic, indicating the feasibility and safety of this approach in multiple autoimmune and transplantation settings. However, the implementation of engineering tools to confer antigen-specificity and phenotypic stability is expected to enhance the effectiveness observed from previous Treg products.</div></div><div><h3>Methodology</h3><div>LIBERATE is a first-in-human Phase I/IIa clinical trial designed to evaluate the safety and activity of autologous CAR-Tregs in promoting liver transplantation tolerance in HLA-A2-negative patients who have received a graft from an HLA-A2-positive donor. A proprietary GMP manufacturing process has been developed to engineer recipient-derived QEL-001 cell products that express an anti-HLA-A2 targeted CAR, with a FOXP3 phenotype lock, and a safety switch. An extensive characterization of QEL-001 CAR-Treg products has demonstrated the maintenance of phenotypic and functional characteristics of unmodified Tregs.</div></div><div><h3>Results</h3><div>A safety cohort consisting of three patients found QEL-001 to be well tolerated within the protocol-defined dose range, with no serious adverse events or dose-limiting toxicities. No treatment related adverse events were reported during the 4-week post-dose follow-up. Analysis of blood samples by FACS and scRNAseq showed persistence of CAR-Tregs in circulation for up to 12 months post-infusion and maintenance of canonical markers of stable and effective Tregs. Liver biopsies collected post-infusion demonstrated phenotypic stability and evidence of graft trafficking, leading to substantial enrichment of CAR-Tregs in the liver compared to the periphery.</div><div>An expansion cohort is currently being conducted with the addition of lymphodepletion by low-dose rATG previous to QEL-001 infusion. Active ATG was cleared from circulation prior cell infusion and mediated a significant reduction in T-cell frequencies. QEL-001 CAR-Treg engraftment levels in circulation was improved compared to the safety cohort who did not receive rATG conditioning, and the Treg/Teff ratio increased favoring immunoregulation.</div></div><div><h3>Conclusion</h3><div>This study provides pioneering data to evaluate the phenotype and homeostasis of engineered Tregs in the clinic, supporting further investigation of these therapies in a broad range of inflammatory, autoimmune and transplantation applications.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S41-S42"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887601","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":"Aims and Scope","authors":"","doi":"10.1016/S1465-3249(25)00604-8","DOIUrl":"10.1016/S1465-3249(25)00604-8","url":null,"abstract":"","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page A1"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887516","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":"Introduction from President","authors":"","doi":"10.1016/S1465-3249(25)00610-3","DOIUrl":"10.1016/S1465-3249(25)00610-3","url":null,"abstract":"","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S1"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887520","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":"Non-viral Delivery of CRISPRa Tools for Scalable Directed Differentiation of Induced Pluripotent Stem Cells","authors":"S. Perez ,&nbsp;M. Braam ,&nbsp;O. Awonusi ,&nbsp;M. Jayawardena ,&nbsp;R. Esse ,&nbsp;M. Lopes Tiburcio ,&nbsp;S. Krishnan ,&nbsp;E. Zucchelli ,&nbsp;V. Di Cerbo","doi":"10.1016/j.jcyt.2025.03.055","DOIUrl":"10.1016/j.jcyt.2025.03.055","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Induced pluripotent stem cells (iPSCs) have transformative potential for advanced cell therapies, enabling the development of allogeneic, off-the-shelf products to enhance manufacturing cost-efficiency and accessibility. However, conventional differentiation protocols relying on cocktails of growth factors, cytokines, and media supplements are lengthy, yield suboptimal efficiencies, and produce undesired cell types or residual pluripotent stem cell impurities. These limitations compromise both manufacturing efficiency and product quality, representing key bottlenecks for commercialisation.</div></div><div><h3>Methodology</h3><div>To address these challenges, we developed an innovative CRISPR-mediated transcriptional activation (CRISPRa) system combined with non-viral delivery to establish a scalable forward programming workflow. Our approach leverages delivery of catalytically dead Cas9-VPR mRNA and four CRISPR guide RNA pools targeting known critical haematopoietic transcription factors (GATA2, TAL1, ETV2, and LMO2), driving differentiation toward haemato-endothelial progenitors.</div></div><div><h3>Results</h3><div>Optimisation of RNA payload delivery achieved up to 90% transfection efficiency with over 80% cell viability. At 72 hours post-transfection, iPSCs demonstrated robust expression of the four target transcription factors and reductions in pluripotency markers expression. To support scalable biomanufacturing, we then integrated iPSCs aggregates formation with CRISPRa delivery and expression, facilitating the transition from 2D cultures to 3D systems. This adaptation preserved comparable target transcription factors activation and induced the expression of a selection of 14 downstream haematopoietic genes, including CD31, CD34, CD43, and CDH5. Moreover, a 25–75% reduction of pluripotency markers expression was observed, alongside significant morphological changes within 72h (figure below). Finally, we optimised lipid nanoparticle-based delivery for CRISPRa complexes instead of electroporation, achieving high activation efficiency of target genes within a workflow amenable to scalable stir-tank bioreactors.</div></div><div><h3>Conclusion</h3><div>Integration of these cutting-edge molecular engineering tools into scalable processes has the potential to unlock the next-generation of iPSC-derived manufacturing. Our results demonstrate an innovative approach to enable the generation of a scalable, CRISPRa-mediated differentiation platform, addressing critical inefficiencies in current workflows and supporting enhancements of allogeneic therapies manufacturing capacity.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S35"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143886716","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":"SUCCESSFUL REDIRECTION OF TCR SPECIFICITY AGAINST CRYPTIC LEUKEMIA ANTIGENS USING NON-VIRAL TCR ENGINEERING","authors":"C. Rulleau ,&nbsp;M. Aubin ,&nbsp;A. Loiselle ,&nbsp;G. Boudreau ,&nbsp;B. Haley ,&nbsp;J. Delisle","doi":"10.1016/j.jcyt.2025.03.015","DOIUrl":"10.1016/j.jcyt.2025.03.015","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Acute myeloid leukemia (AML) remains highly lethal despite significant treatment advances, with a high relapse rate and an urgent need for novel therapies. The discovery and characterization of new antigenic targets capable of eliciting therapeutic responses remain critical to treating more patients. Leukemia cells can express tumor-specific antigens (TSA) absent in healthy tissues. Proteogenomic analysis have identified several MHC Class I-associated peptides eluted from AML cells that are derived from extra-exomic regions of the genome and selectively expressed in AML. These cryptic antigens present as promising targets for immunotherapy, prompting us to characterize T-cell responses against them and develop a T-cell receptor (TCR) transgenic strategy for clinical application.</div></div><div><h3>Methodology</h3><div>Using synthesized peptides, we generated cryptic TSA-specific CD8⁺ T cell lines by co-culturing healthy donor T cells with autologous dendritic cells presenting the peptide of interest. Antigen-specific T cells were identified via cytokine secretion assays and fluorescent MHC-peptide multimers. Sorted multimer-positive T cells underwent RNA 5’RACE PCR to amplify their TCR alpha and beta chains. These were cloned into a nanoplasmid and inserted <em>in situ</em> into the TRAC locus of the endogenous TCR via single-step TCR replacement using CRISPR-Cas9, ensuring deletion of native TCR chains. Antigenic redirection was confirmed through cytokine secretion after exposure to cryptic TSA peptide and cytotoxicity assay.</div></div><div><h3>Results</h3><div>After establishing that cryptic TSAs were recognized by T cells in healthy donors' peripheral blood, we demonstrated that at least 60% (6/10) of tested peptides were antigenic. T-cell responses were highly oligoclonal, and as few clonotypes were present among antigen-specific cells, alpha and beta chain pairing following 5’RACE PCR was facilitated. Assessment of CD3 and TCR expression using flow cytometry confirmed successful TCR replacement. Functional assays, including ELISpot, cytokine secretion, and cytotoxicity assays, confirmed that these engineered TCRs could redirect T cells' antigenic specificity towards the recognition of cryptic leukemia antigens.</div></div><div><h3>Conclusion</h3><div><em>Ex vivo</em> expansion of cryptic antigen-specific T cells enables TCR identification that can be used to devise new T-cell immunotherapies through TCR engineering. This strategy allows the generation of large numbers of AML-specific, reactive T lymphocytes towards cryptic TSA, offering promising new therapeutic avenues.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S14-S15"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887855","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 NOVEL ENGINEERED SKIN HEALS FULL THICKNESS BURN WOUNDS: A CASE REPORT","authors":"S. Akbarzadeh ,&nbsp;C. Arellano ,&nbsp;I. Banakh ,&nbsp;M.M. Rahman ,&nbsp;C.H. Lo ,&nbsp;W. Teague ,&nbsp;M. Bertinetti ,&nbsp;H. Cleland","doi":"10.1016/j.jcyt.2025.03.048","DOIUrl":"10.1016/j.jcyt.2025.03.048","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Timely wound closure in massive burns remains an unmet clinical need. Engineered skin substitutes can provide an alternative treatment to split skin grafting. However, since the development of Cultured Epithelial Autograft (Green's method), almost 50 years ago, limited cell and tissue therapies for burns have been introduced to clinics. The aim of this study was to show, as a proof of concept, that engineered skin grafts can substitute native skin grafts to close large burn wounds permanently.</div></div><div><h3>Methodology</h3><div>Here we describe engineering a dermal/epidermal composite skin graft in the treatment of a child suffering from burns &gt; 60% total body surface area. A 5cm² biopsy was collected from the child. Dermal and epidermal stem and progenitor cells were isolated and expanded according to good manufacturing practice (GMP) guidelines. A hydrogel, combined with autologous fibroblasts, was prepared in-house to act as a niche for the stem and progenitor interfollicular stem and progenitors <em>in vitro</em> and <em>in vivo</em>. Over 500 cm² engineered skin was produced, clear of endotoxin, mycoplasma, bacteria and yeast pathogens. The engineered skin was grafted on a full thickness wound on right arm and right foot, temporised with NovoSorb ® Biodegradable Temporising Matrix (BTM). Grafts were analysed histologically. Cosmetic and scar outcome were measured over 12 months.</div></div><div><h3>Results</h3><div>Engineered skin graft take was estimated as 95% on day 14 post grafting without any need for native skin grafting. Histological analysis of the grafts, 2 weeks and 6 weeks post grafting, confirmed persistence of a complete and continuous, although slightly hyperproliferative, epidermis. The survival of the stem and progenitor keratinocytes in engineered skin was confirmed by immunofluorescence using Keratin 5, and Integrin beta 1 specific antibodies. Grafts were fully vascularised. Engineered graft scar quality, measured by POSAS, has remained superior to the gold standard native skin grafting at 6- and 12-months post grafting.</div></div><div><h3>Conclusion</h3><div>This study is a proof of concept for the application of this novel engineered skin as a definitive treatment option in paediatric burn injuries. This technology will reduce the need for traditional skin grafting in children with limited donor skin.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S31"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887996","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":"Machine learning-driven optimization of hepatocyte functional maturation from hPSC","authors":"C. Elabd ,&nbsp;Y. Ovadia ,&nbsp;V. Juneja Shah ,&nbsp;F. Li","doi":"10.1016/j.jcyt.2025.03.051","DOIUrl":"10.1016/j.jcyt.2025.03.051","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Producing large quantities of functionally mature hepatocytes remains a significant challenge for clinical applications, including liver failure treatment and preclinical drug testing. Differentiating human pluripotent stem cells (hPSCs) into hepatocyte-like cells (HLCs) offers a promising solution, with numerous protocols developed for this purpose. However, these methods typically yield immature cells lacking the full repertoire of functionalities characteristic of primary human hepatocytes.</div><div>We developed machine learning (ML) algorithms capable of predicting hepatocyte maturation directly from live-cell, label-free phase contrast (PC) images. These algorithms were successfully deployed to analyze phenotypic screening data, enabling the optimization of differentiation protocols to produce HLCs with improved functional maturation.</div></div><div><h3>Methodology</h3><div>hPSC differentiation into HLCs was performed following an 18-day protocol described in Ang et al. 2018. ML models were trained on paired PC and immunofluorescence (IF) images of six markers involved in metabolism, detoxification, and protein synthesis. Models took solely PC images as input and produced a multi-dimensional hepatocyte maturation score as output.</div><div>Validated ML models were used to rank and select hits in phenotypic screening experiments where differentiating hPSC-derived HLCs were exposed to small molecules and biologics at varying concentrations, durations, and combinations in iterative cycles. The top-ranking hits were compared to controls in a follow-up validation experiment using gold standard in vitro assays of hepatocyte function – CYP3A4 enzymatic activity, albumin secretion, and urea secretion. A mixed-effect model was used to assess statistical significance.</div></div><div><h3>Results</h3><div>ML-predicted hepatocyte maturation scores showed strong correlations (Pearson r &gt; 0.97) across a panel of IF markers. Leveraging these ML predictions in live cultures, we rapidly and cost-effectively screened for combinations of molecules that significantly enhanced hepatocyte maturation. This led to generational improvements in protocols in a matter of months, resulting in statistically significant increases in CYP3A4 activity (+55%), albumin secretion (+40%), and urea secretion (+130%) over the baseline.</div></div><div><h3>Conclusion</h3><div>Our approach highlights a novel, ML-driven approach to optimizing differentiation protocols for improved functional maturation. More generally, this method holds promise for applications in upstream process development as well as downstream quality control.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S32"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143887999","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":"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}