Cytotherapy最新文献

{"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":"Integrin α4 and CCR5 on IFNγ-activated MSC determine biodistribution to critical sites for aGVHD prophylaxis in mice","authors":"E. Foppiani ,&nbsp;A.J. Burnham ,&nbsp;L. Daley-Bauer ,&nbsp;E. Horwitz","doi":"10.1016/j.jcyt.2025.03.009","DOIUrl":"10.1016/j.jcyt.2025.03.009","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>Mesenchymal stromal cells (MSC) activated with interferon-γ (γMSC) exhibit potent immunosuppressive properties, yet nonactivated MSC remain the focus of clinical trials. Extensive mouse studies show that γMSC can prevent acute Graft versus Host Disease (GVHD) after hematopoietic cell transplantation, but the molecular mechanisms affecting their distribution post-IV infusion and the sites of activity remain unclear. This study aims to identify the site(s) of activity and elucidate the molecular and cellular mechanisms governing γMSC trafficking.</div></div><div><h3>Methodology</h3><div>A mouse model of fully MHC mismatched hematopoietic cell transplantation (HCT) was used. Human γMSC were infused 24 hours post-HCT, and their tissue localization was assessed. CRISPR/Cas9 was used to modify γMSC, and in vitro analyses conducted using IncuCyte or fluorescence microscopy. Macrophages depletion with clodronate was performed to study their role in trafficking and GVHD prevention.</div></div><div><h3>Results</h3><div>We observed that γMSC were enriched in gut-associated lymphoid tissue (GALT) 16 hours post-IV infusion, critical site for the development of aGVHD. <em>In vitro</em>, γMSC exhibited significantly greater adhesion to spleen endothelial cells compared to other SLO endothelial cells, and integrin α4 was crucial for this interaction, as α4 knockdown reduced binding by 94%. We identified CCL3, CCL4, and CCL5 as key chemokines upregulated in murine activated T cell conditioned media and blocking these chemokines collectively abolished γMSC migration. γMSC expressed CCR5 knockdown in γMSC reduced migration by 90%. <em>In vivo</em>, knockdown of α4 or CCR5 in γMSC impaired their trafficking to GALT and significantly the prophylactic effects against GVHD, although T cell suppression by γMSC was retained. Furthermore, depletion of monocytes/macrophages by liposomal clodronate did not affect γMSC trafficking to the spleen but abolished the GVHD-suppressive activity of γMSC, indicating that macrophages are not required for γMSC trafficking, but essential for their therapeutic function.</div></div><div><h3>Conclusion</h3><div>Our study reveals that the coordinated expression of integrin α4 and CCR5 on γMSC governs their trafficking to GALT, where they exert their immunosuppressive effect and significantly reduce GVHD in a mouse model. These findings highlight the importance of GALT localization in γMSC efficacy, offering insights that could inform strategies to enhance the therapeutic potential of γMSC in GVHD prevention.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Page S11"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888175","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":"Using Artificial Intelligence to Improve Cell Therapy Assays: Automated Quantitative Image Analysis of Cells on Matrices","authors":"A.M. Bornschlegl ,&nbsp;A. Dietz","doi":"10.1016/j.jcyt.2025.03.082","DOIUrl":"10.1016/j.jcyt.2025.03.082","url":null,"abstract":"<div><h3>Background &amp; Aim</h3><div>As the field of cell therapy continues to advance, the combination of cells and directed delivery methods (such as three-dimensional scaffolds, cell printing etc.) continues to grow. These technologies require methods to accurately determine cell numbers and viability to enhance process optimization and develop appropriate release tests. Current methods have limited dynamic range and require substantial manual effort to produce results. Here we describe a simple fluorescent imaging-based method for counting live and dead cells in scaffold cultures that is consistent, automated, and quantitative.</div></div><div><h3>Methodology</h3><div>First, we optimized labware to support and control the samples providing uniform imaging fields with standard methods for cell number (Hoechst 33342) and viability (Propidium Iodide). We then used a traditional, nondynamic image quantitation software (Gen5, Winooski, VT) across a range of cell concentrations and compared it to the ability of a trained artificial intelligence software (Aiforia, Helsinki, Finland) to determine both cell counts and cell viability in image analysis. Using Aiforia, the live cell counts were highly correlative to seeding concentration (p=0.0007; r2=0.96) across all tested ranges whereas Gen5 showed no correlation (p=0.6; r2=0.09). Dead cell counts measured by the two methods were correlated to each other (p=0.004; r2=0.90) indicating that both systems were equally capable using Propidium Iodide based detection.</div></div><div><h3>Results</h3><div>After completing proper training of the AI system, it provided a clear improvement in data accuracy from its ability to recognize cells amidst highly dynamic backgrounds typical of scaffold culture images.</div></div><div><h3>Conclusion</h3><div>We believe that cell therapy will significantly benefit from AI based approaches.</div></div>","PeriodicalId":50597,"journal":{"name":"Cytotherapy","volume":"27 5","pages":"Pages S49-S50"},"PeriodicalIF":3.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888226","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":"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}